A type 2 Ferrier rearrangement-based synthesis of d-myo-inositol 1,4,5-trisphosphate

Article abstract of DOI:10.1016/j.tetasy.2009.03.006

The synthesis of d-myo-inositol 1,4,5-trisphosphate (InsP₃) from methyl α-d-glucopyranose, via a type 2 Ferrier rearrangement is reported. A key intermediate in this synthesis possesses orthogonal protecting groups at the 1-, 4- and 5-position, making it a versatile starting point for the synthesis of unnatural InsP₃ derivatives. Biological evaluation of the synthetic InsP₃ demonstrates that this compound evokes selective Ca²⁺ release via activation of InsP₃ receptors.

Full text of DOI:10.1016/j.tetasy.2009.03.006

Tetrahedron: Asymmetry 20 (2009) 857–866
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
A type 2 Ferrier rearrangement-based synthesis of D-myo-inositol
1,4,5-trisphosphate
Neil S. Keddie a, Geert Bultynck b, Tomas Luyten b, Alexandra M. Z. Slawin a, Stuart J. Conway c,
*
a EaStCHEM and School of Chemistry, Centre for Biomolecular Sciences, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK
b Laboratory of Molecular Signalling, Campus Gasthuisberg O/N1, K.U. Leuven, Herestraat 49, Bus 802, B-3000 Leuven, Belgium
c Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of
D
-myo-inositol 1,4,5-trisphosphate (InsP3) from methyl
a
-
D
-glucopyranose, via a type 2
Received 4 February 2009
Accepted 10 March 2009
Available online 1 April 2009
Ferrier rearrangement is reported. A key intermediate in this synthesis possesses orthogonal protecting
groups at the 1-, 4- and 5-position, making it a versatile starting point for the synthesis of unnatural InsP3
derivatives. Biological evaluation of the synthetic InsP3 demonstrates that this compound evokes selec-
tive Ca2+ release via activation of InsP3 receptors.
Dedicated to Professor George Fleet on the
occasion of his 65th birthday
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
derivatives that have orthogonal protecting groups attached to at
least three of their oxygen atoms.
D
-myo-Inositol 1,4,5-trisphosphate 15 (InsP3) is a ubiquitous
As myo-inositol is meso, syntheses that begin from this cheap
and readily available starting material have to overcome the issue
of enantioselectivity at some point in the route. With the notable
exception of work reported by Miller and co-workers,17–25 synthe-
ses to single enantiomers of inositol derivatives have largely
depended on the resolution of diastereomers.2,3 One alternative
to this approach is the use of chiral pool materials to carry stereo-
chemistry through the synthesis. The most useful chiral pool mate-
rial for the synthesis of myo-inositol derivatives is glucose. The
type 2 Ferrier rearrangement26,27 is an elegant method for the con-
version of glucopyranosides into inositol derivatives and there are
a number of examples of inositol polyphosphate and phosphatidyl-
inositol polyphosphate derivatives being synthesised using this
approach.28–43 Of these syntheses, only two have focussed on the
synthesis of InsP3,28,29 and two have focussed on the synthesis of
unnatural InsP3 derivatives.35,39 However, in all of the above syn-
theses of InsP3, the protecting groups employed do not provide
suitable orthogonality for the synthesis of the unnatural InsP3
derivatives in which we are interested. Herein we report a syn-
thetic route, based on the type 2 Ferrier rearrangement, which
encompasses orthogonal protecting groups at the 1-, 4- and 5-po-
sition and will potentially enable selective elaboration of the
1-,2-,4-,5- and 6-positions of the inositol ring. To exemplify our
strategy, we have synthesised InsP3 and demonstrated that this
material releases Ca2+ in a permeabilised cell 45Ca2+ flux assay, in
a manner that is consistent with selective activation of InsP3Rs.
intracellular Ca2+-releasing second messenger that mediates a
wide range of cellular functions.1 InsP3 is produced within cells
in response to GPCR activation by extracellular stimuli and goes
on to activate its own intracellular receptors (InsP3Rs). Activation
of InsP3Rs leads to a rise in intracellular Ca2+ concentration that
mediates fundamental processes such as fertilisation, behaviour,
learning and memory. It is now clear that the InsP3 signalling
cascade resides downstream of most important endogenous chem-
ical transmitters including acetylcholine, adrenaline, dopamine,
glutamate and 5-HT, in addition to some tyrosine kinase receptors.
InsP3 is therefore involved in the biological function and hence in
dysfunction of these transmitters and the processes that they
mediate.1
The biochemical importance of InsP3 has prompted many syn-
theses of both the naturally occurring molecule and numerous
unnatural derivatives. Many of these compounds have proved
invaluable tools in the study of the InsP3Rs.2,3 Especially useful
compounds include the metabolically stable phosphorothioates,2,3
the adenophostins,4–11 photoactivated InsP3 derivatives12,13 and
membrane permeant InsP3 derivatives.12,14,15 Despite the numer-
ous InsP3 derivatives that have been reported, the synthesis of this
class of compounds remains challenging. We are interested in the
development of selective probes for Ca2+ signalling, and in particu-
lar InsP3Rs.14,16 Therefore, the development of expeditious and ver-
satile synthetic routes, which allow the generation of a variety of
InsP3 derivatives from a common intermediate, is highly desirable.
In order to achieve this goal, it is necessary to synthesise inositol
2. Results and discussion
The synthesis of InsP3 15 commenced from methyl
pyranose 1 (Scheme 1), which was selectively protected at the
a-D-gluco-
* Corresponding author. Tel.: +44 (0) 1865 285109; fax: +44 (0) 1865 285002.
E-mail address: stuart.conway@chem.ox.ac.uk (S.J. Conway).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.03.006

858
N. S. Keddie et al. / Tetrahedron: Asymmetry 20 (2009) 857–866
OH
O
MPM
O
MPM
O
i
ii
O
O
HO
HO
O
O
HO
HO
HO
HO
BnO
OCH3
OCH3
OCH3
1
2
3
iii
OAc
OH
O
MPM
O
v
vi
iv
O
O
O
O
PMBO
TIPSO
PMBO
TIPSO
PMBO
TIPSO
O
TIPSO
BnO
BnO
BnO
OCH3
BnO
OCH3
OCH3
OCH3
7
5
4
6
Scheme 1. The synthesis of the Ferrier rearrangement substrate 7. Reagents and conditions: (i) 4-OMe-PhCH(OMe)2, amberlyst-15, DMF, 200 mbar, 80 °C, 58%; (ii) nBu2SnO,
TBABr, BnBr, MeCN, reflux, 54%; (iii) Et3N, TIPSOTf, CH2Cl2, rt, 92%; (iv) DIBAL-H, CH2Cl2, rt, 88%; (v) Dess–Martin Periodinane, CH2Cl2, rt, 82%; (vi) K2CO3, Ac2O, DMAP, MeCN,
reflux, 65%.
4- and 6-position to give the anisylidene acetal 2. Treatment of 2
with di-n-butyl tin oxide and benzyl bromide in the presence of
tetrabutylammonium bromide and 3 Å molecular sieves effected
benzylation, with 2:1 selectivity in favour of the desired 2-O-ben-
zyl compound 3 over the unwanted regioisomer, and a 54%
isolated yield of the desired compound 3. TIPS protection of the
3-position hydroxyl group was achieved by treatment of 3 with
TIPS triflate in the presence of triethylamine, affording 4 in excel-
lent yield. DIBAL-H mediated cleavage of the anisylidene acetal 4
proved to be regioselective, giving an excellent yield of the de-
sired compound, with the PMB group attached to the 4-position
of 5. High yields were obtained in this step only when the quench
and work-up procedures were conducted at lower temperature.
The 6-position hydroxyl group was oxidised to the corresponding
aldehyde 6 using Dess–Martin periodinane. This compound 6 was
stable and subjecting it to silica gel column chromatography was
found to produce better results in the formation of the enol ace-
tate and subsequent Ferrier rearrangement. The aldehyde 6 was
converted to the enol acetate by treatment with potassium
carbonate and acetic anhydride, affording the substrate for the
Ferrier rearrangement 7.
Small amounts of isomeric products were also observed, but not
fully characterised.
The relative stereochemistry of the inosose ring was confirmed
by obtaining an X-ray crystal structure of 8 (Fig. 1). It can be seen
that the 2-position hydroxyl group occupies the axial position,
which is required for the myo-configuration of the inositol ring
to be obtained, following reduction of the 6-position ketone.
The two main metals that have been used to promote the type 2
Ferrier rearrangement of glucose derivatives, in order to give inos-
ose products, are palladium and mercury. Given that palladium is
less toxic than mercury and has been employed in substoichiomet-
ric quantities, we first investigated the use of palladium to effect
the desired rearrangement. However, when 7 was treated with
palladium(II) chloride a large number of inseparable isomeric
products were observed. This result is not in accordance with the
data reported by Takahashi et al.;28,29 however, we attribute the
differences observed to the influence of the TIPS group, which is
present in our material but not in that of Takahashi. Gratifyingly,
on treatment with mercury(II) acetate, the enol acetate 7 was ob-
served to undergo the type 2 Ferrier rearrangement to afford
mainly the desired product 8, albeit in a low yield (Scheme 2).
Figure 1. A stick representation of the X-ray crystal structure of compound 8. The
relative stereochemistry of the 1–5 position oxygen atoms can be seen, with the 2-
position hydroxyl group sitting in an axial position. Key: carbon, green; oxygen, red;
silicon, tan.
The reduction of the 6-position ketone 8 to the inositol 9 was
achieved by treatment with tetramethylammonium triacetoxy
borohydride (Scheme 3). The stereoselectivity in this reduction is
likely as a result of the axial 2-position hydroxyl group co-ordinat-
ing to the reducing agent, leading to hydride delivery from the top
face and therefore the equatorial hydroxyl group at the 6-positon
being obtained.44 The protection of the 2- and 6-position hydroxyl
groups of 9 was required, and benzyl ethers would be the obvious
choice of protecting group. It was known from previous work that
use of standard conditions, employing sodium hydride and benzyl
bromide, would lead to acetate group migration. Hence, the use of
benzyl trichloroacetimidate in the presence of a Brønsted or Lewis
acid was investigated. Use of trifluoromethanesulfonic acid caused
degradation of the starting material 9, whereas triphenylcarbeni-
um tetrafluoroborate failed to effect the desired reaction. Use of
scandium triflate also failed to catalyse benzyl protection, in a
OAc
HO
OAc
i
O
PMBO
TIPSO
O
BnO
OPMB
OTIPS
BnO
OCH3
7
8
Scheme 2. The type 2 Ferrier rearrangement to afford the inosose 8. Reagents and
conditions: (i) (a) Hg(OAc)2, acetone/H2O (3:2); (b) NaCl(aq), 35%.

N. S. Keddie et al. / Tetrahedron: Asymmetry 20 (2009) 857–866
859
HO
HO
BOMO
BnO
BOMO
BnO
OAc
OAc
OAc
OH
i
ii
iii
O
OH
OPMB
OTIPS
OBOM
OPMB
OBOM
OPMB
OTIPS
BnO
BnO
OPMB
OTIPS
OTIPS
8
9
10
11
iv
HO
BOMO
BnO
OPO(O-Na+)2
OH
OPO(OBn)2
OBOM
OPO(OBn)2
OPO(OBn)2
BOMO
BnO
BOMO
BnO
OH
OH
vii
vi
v
OBOM
OH
OH
OBOM
OH
OTIPS
OPO(O-Na+)2
HO
OPO(O-Na+)2
15
14
13
12
Scheme 3. The synthesis of InsP3 15. Reagents and conditions: (i) Me4NBH(OAc)3, AcOH, MeCN, 89%; (ii) BOMCl, Hünig’s base, 85 °C, 82%; (iii) LiOH, MeOH, THF, 97%; (iv)
DDQ, CH2Cl2/H2O (18:1), rt, 95%; (v) TBAF in THF, CH2Cl2, rt, 90%; (vi) (a) (BnO)2PN(iPr)2, 1H-tetrazole (0.43 M in MeCN), CH2Cl2, rt; (b) 3-chloroperbenzoic acid, ꢀ78 °C?rt,
93%; (vii) H2, t-BuOH, H2O, Pd black, NaHCO3, 95%.
model system. The use of stronger Lewis acids was rejected, as they
were likely to degrade the inositol starting material 9. We then
turned our investigation to the use of Dudley’s benzylation condi-
tions,45 which involve the use of 2-benzyloxy-1-methylpyridinium
triflate in the presence of MgO, to transfer the benzyl group to the
inositol ring. However, we only ever detected trace amounts of the
desired compound. These results are in line with reports from
Miller,18 who also found Dudley’s conditions to be ineffective in
the benzyl protection of inositols. These results suggest that the
inositol hydroxyl groups are too hindered to be benzylated effec-
tively with this reagent.
Given the problems encountered when using benzyl groups, the
BOM group was considered as an alternative protecting group.
Optimised conditions for BOM protection were found to involve
premixing the BOMCl and Hünig’s base and then heating the reac-
tants in Hünig’s base, affording an 82% yield of the desired material
10. Deprotection of the acetate 10 afforded the alcohol 11, which
was then treated with DDQ to effect removal of the PMB group,
furnishing the diol 12. Subsequent deprotection of the TIPS group
afforded the known triol 1328 in good yield. Phosphitylation of
the triol 13, followed by mCPBA oxidation, afforded the perbenzy-
lated phosphate 14. Subsequent palladium black-catalysed
hydrogenolysis, in the presence of NaHCO3, afforded the desired
InsP3 15 as its presumed hexakis sodium salt.
to two key intermediates in the synthesis of unnatural InsP3
derivatives 12 and 16.
In order to confirm that our synthetic InsP3 exhibited the ex-
pected biological action, we investigated its InsP3-mediated Ca2+
mobilising properties in unidirectional Ca2+ fluxes from the endo-
plasmic reticulum (ER) Ca2+ stores, in permeabilised Lvec cells.
Lvec cells are a stable fibroblast cell line mainly containing type
3 and type 1 InsP3Rs. Challenging the cells with an InsP3R agonist
leads to a transient increase in the fractional Ca2+ loss. Figure 2A
shows the data from a typical experiment in permeabilised Lvec
cells. The addition of our compound to the efflux medium induced
a dose-dependent Ca2+ release from the non-mitochondrial intra-
cellular Ca2+ stores, a similar response was seen on addition of
commercial InsP3. The Ca2+ ionophore A2318746,47 is used to esti-
mate the maximal releasable Ca2+. The release provoked by our
compound or commercially available InsP3 is normalised to the
maximal releasable Ca2+ in order to obtain dose–response curves
(Fig. 2B). The EC50 values of the dose–response curves for synthetic
InsP3 and commercial InsP3 in Lvec cells show that our compound
has a similar potency in stimulating Ca2+ release to commercial
InsP3. Similar findings were observed in L15 cells, a stable cell line
heterologously overexpressing InsP3R1 (EC50 of our compound =
0.64 mM versus EC50 of commercial InsP3 = 0.91 mM) (data not
shown).
The above order of protecting group removal (Scheme 3) lends
itself to the synthesis of 4-position-modified InsP3 derivatives. As
after phosphitylation of the 1- and 5-position hydroxyl groups of
12, the TIPS group could potentially be removed to leave the 4-po-
sition hydroxyl group free and ready for elaboration. With a view
to the future synthesis of 5-position-modified InsP3 derivatives,
we investigated an alternative order of protecting group removal
(Scheme 4). It was demonstrated that the TIPS group of compound
11 could be removed in the presence of the PMB group, using
TBAF in THF. This reaction afforded the 1,4-diol 16, which could
potentially be phosphitylated to afford the fully protected precur-
sor. Removal of the PMB group would leave the 5-position hydro-
xyl group free and ready for elaboration. Therefore, the above
synthesis provides a viable route not only to InsP3 15, but also
In a second investigation, using a unidirectional semi-high
throughput Ca2+-flux assay in permeabilised DT40 and triple-
InsP3R knockout cells (TKO), we have examined whether the
Ca2+-mobilising properties of the synthetic InsP3 were dependent
on the presence of the InsP3Rs (Fig. 3). TKO cells are genetically
modified DT40 cells, in which all three InsP3R isoforms have been
genomically deleted. In this experiment, the intracellular Ca2+
stores of permeabilised DT40 and TKO cells were loaded with the
low-affinity Ca2+ dye, MagFluo4. Ca2+ was loaded in the non-mito-
chondrial Ca2+ stores to steady-state levels using ATP. After reach-
ing the plateau phase, thapsigargin (1
SERCA pumps and monitor the unidirectional Ca2+ leak from the
non-mitochondrial Ca2+ stores. Then, 3
M of either synthetic or
lM) was added to block the
l
commercial InsP3 was given. Clearly, the synthetic InsP3 and the
commercial InsP3 provoked a profound Ca2+ release from the ER
Ca2+ stores in permeabilised DT40 cells, but not in TKO cells. This
indicates that the synthetic InsP3 exclusively provokes Ca2+ release
in an InsP3R-dependent manner.
BOMO
BnO
BOMO
BnO
OH
OH
i
OBOM
OPMB
OTIPS
OBOM
OPMB
OH
3. Conclusion
11
16
In conclusion, we have reported a robust synthesis of InsP3,
Scheme 4. Selective deprotection of the acetate and TIPS of compound 11 to give
the intermediate 16. Reagents and conditions: (i) TBAF in THF, THF, rt, 89%.
confirmed the structure of
a key intermediate 8 by X-ray

860
N. S. Keddie et al. / Tetrahedron: Asymmetry 20 (2009) 857–866
Figure 2. (A) The synthetic InsP3 mobilises Ca2+ from the non-mitochondrial Ca2+ stores in permeabilised Lvec cells. A typical experiment showing the Ca2+-mobilising
properties of synthetic InsP3 in Lvec cells. A23187 shows the maximal releasable Ca2+. Data are plotted as fractional loss (%/2 min; the amount of 45Ca2+ leaving the stores in
2 min divided by the total store Ca2+ content at that time) as a function of time. The horizontal bar indicates the addition of InsP3 or A23187. Results represent the means SD
and are obtained from duplicate values. (B) The synthetic InsP3 mobilises Ca2+ in permeabilised Lvec cells. Dose response curves showing the Ca2+ release provoked by
synthetic and commercial InsP3 in permeabilised Lvec cells and normalised to the A23187-releasable Ca2+. Results represent the means SEM of at least three independent
experiments each performed in duplicate.
crystallographic analysis and demonstrated that the final com-
pound 15 releases Ca2+ in a 45Ca2+ assay, in a manner consistent
with selective InsP3R activation. More importantly, we have shown
that compound 11 may be a key intermediate in the synthesis of
both 4- and 5-position-modified InsP3 derivatives, as sequential
deprotection of the PMB group or the TIPS group, in either order,
is possible. Given that it is possible to add an orthogonal protecting
group at the 2-position prior to reduction and then a further pro-
tecting group at the 6-position, this route should prove a versatile
means of synthesising a large number of unnatural InsP3 deriva-
tives, which may prove useful tools for the study of intracellular
Ca2+ signalling.
4. Experimental
4.1. General experimental details
1H NMR spectra were recorded at 300 MHz, 400 MHz or
500 MHz, on Bruker Avance spectrometers, using deuterochloro-
form (or other indicated solvent) as reference and as internal deu-
terium lock. The chemical shift data for each signal are given as d in
units of parts per million (ppm) relative to tetramethylsilane (TMS)
where d TMS = 0.00 ppm. The multiplicity of each signal is indicated
by: s (singlet); br s (broad singlet); d (doublet); dd (doublet of dou-
blets); m (multiplet). 13C NMR spectra were recorded at 75.5 MHz
Figure 3. (A) The synthetic InsP3 mobilises Ca2+ from the non-mitochondrial Ca2+ stores in permeabilised DT40 cells. A typical experiment showing ER Ca2+ levels measured
by MagFluo4-fluorescence detection as a function of time using FlexStation 3. ATP was added to initiate Ca2+ uptake into the ER. After reaching steady-state ER Ca2+ levels,
thapsigargin (TG) was added to block SERCA Ca2+-uptake activity and 30 s later, cells were challenged with synthetic or commercial InsP3 (3 M) to provoke Ca2+ release from
l
the ER, which is observed as an immediate drop in the MagFluo4 signal. (B) The synthetic InsP3 does not mobilise Ca2+ from the non-mitochondrial Ca2+ stores in
permeabilised InsP3R-deficient TKO cells. A similar experiment to that described in (A) was performed, except that DT40 cells lacking all three InsP3R isoforms were used.
Addition of synthetic or commercial InsP3 (3 l
M) does not cause Ca2+ release from the ER in TKO cells.

N. S. Keddie et al. / Tetrahedron: Asymmetry 20 (2009) 857–866
861
or at 100 MHz using the DEPT Q pulse sequence with broadband
proton decoupling and internal deuterium lock. The chemical shift
data for each signal are given as d in units of ppm relative to TMS
where d TMS = 0.00 ppm. 1H and 13C spectra were assigned using
2D NMR experiments including COSY, HSQC, HMBC and DEPT Q.
Identical proton coupling constants (J) are averaged in each spec-
trum and reported to the nearest 0.1 Hz. 31P NMR spectra were
recorded at 121 MHz using broadband proton decoupling and
internal deuterium lock. The chemical shift data for each signal
are given as d in units of ppm relative to an external standard
of 85% H3PO4. Low and high resolution mass spectra were re-
corded on a Micromass LCT spectrometer using electrospray ion-
isation in either positive or negative polarity (ES+ or ESꢀ). Certain
samples were submitted to the National Mass Spectrometry Cen-
tre, Swansea. m/z values are reported in Daltons and followed by
their percentage abundance in parentheses. Microanalyses were
obtained on a Carlo Erber EA1110 analyser by the St Andrews
University microanalysis service. IR spectra were recorded on a
Perkin–Elmer GX FT-IR spectrometer as thin films between so-
dium chloride disks or as potassium bromide disks as indicated.
Absorption maxima are reported in wavenumbers (cmꢀ1). Melting
points were determined on a Kofler hot stage or an Electrothermal
9100 and are uncorrected. Optical rotations were measured on a
Perkin–Elmer 341 or 241 polarimeters using cells with a path
length of 1 dm. The concentration (c) is expressed in g/100 mL
101.9 (C-7), 99.9 (C-1), 80.9 (C-5), 72.9 (C-3), 71.8 (C-2), 68.9
(CH2-6), 62.4 (C-4), 55.6 (Ar OCH3) and 55.3 (OCH3); HRMS m/z
(ES+) [Found: (M+Na)+ 335.1101, C15H20O7Na requires M+,
335.1107]; m/z (ES+) 335 ([M+Na]+, 100%); Anal. Calcd for
C15H20O7: C, 57.69; H, 6.45. Found: C, 57.95; H, 6.37. The data
are in good agreement with the literature values.49,50
4.3. (+)-Methyl 2-O-benzyl-4,6-O-anisylidene-a-D-glucopyrano-
side 3 and (+)-methyl 3-O-benzyl-4,6-O-anisylidene-a-D-gluco-
pyranoside
A mixture of (+)-methyl 4,6-O-anisylidene-a-D-glucopyranoside
2 (15.0 g, 48.0 mmol), di-n-butyl tin oxide (13.15 g, 52.8 mmol),
tetrabutylammonium bromide (15.47 g, 52.8 mmol) and benzyl
bromide (24.64 g, 17.13 mL, 144.1 mmol) were suspended in aceto-
nitrile (400 mL). The suspension was heated at reflux via a Soxhlet
thimble filled with 3 Å molecular sieves for 18 h. The mixture was
cooled to rt and the volatile components were removed in vacuo
providing a colourless residue, which was partitioned between
water (250 mL) and ethyl acetate (250 mL). The layers were sepa-
rated and the aqueous layer was extracted with ethyl acetate
(3 ꢂ 250 mL). The combined organic phases were washed with
brine (200 mL), dried over magnesium sulfate, filtered and concen-
trated in vacuo. Purification by silica gel chromatography, eluting
with hexane, ethyl acetate and triethylamine (70:29:1, 65:34:1,
(equivalent to g/0.1 dm3). Specific rotations are denoted as ½a DT
ꢁ
60:39:1), yielded (+)-methyl 2-O-benzyl-4,6-O-anisylidene-a-D-
and are given in implied units of 10ꢀ1 deg cm2 gꢀ1 (T = ambient
temperature in °C). Analytical thin-layer chromatography (TLC)
was carried out on Merck Silica Gel 60 F254 pre-coated alumin-
ium-backed plates. Visualisation of the plates was achieved using
a UV lamp (kmax 254 or 365 nm) or thermal development after
dipping in either an ethanolic solution of phosphomolybdic acid
or an ethanolic solution of 4-anisaldehyde, sulfuric acid and acetic
acid. Flash column chromatography was carried out using Merck
Silica Gel 60 (240–400 mesh), under a positive pressure of com-
pressed air. Anhydrous CH2Cl2, THF, diethyl ether and hexane
were obtained using a MBRAUN GmbH MB SPS-800 solvent puri-
fication system. Anhydrous DMF was purchased from Sigma Al-
drich, UK and used without further purification. Where
appropriate and if not stated otherwise, all non-aqueous reactions
were performed under an inert atmosphere of nitrogen or argon
in flame-dried glassware, using a vacuum manifold with nitrogen
or argon passed through 4 Å molecular sieves and self-indicating
silica gel. In vacuo refers to the use of a rotary evaporator at-
tached to a diaphragm pump. Hexane refers to a mixture of hex-
anes and brine refers to a saturated aqueous solution of sodium
chloride.
glucopyranoside 3 (10.39 g, 54%) as a colourless crystalline solid:
Rf 0.45 (hexane/ethyl acetate 1:1); ½a D20
¼ þ32:0 (c 1.00, CHCl3)
ꢁ
{lit.39
½
a 2D0
ꢁ
¼ þ31:9 (c 1.30, CHCl3)}; mp 136–138 °C (from ethyl
acetate) [lit.39 mp 135–136.5 °C]; mmax (KBr disc)/cmꢀ1 3475 (s),
2919 (w), 1613 (s), 1518 (s), 1365 (m), 1250 (s), 1089 (s), 1040 (s)
and 990 (s); 1H NMR (400 MHz; CDCl3) d 7.45–7.29 (7H, m, Ar
CH), 6.92–6.86 (2H, m, H-3,5 ArOCH3 ring), 5.48 (1H, s, CH-7),
4.80 (1H, d, J 12.2, CHAHBPh), 4.71 (1H, d, J 12.2, CHAHBPh), 4.62
(1H, d, J 3.6, H-1), 4.25 (1H, dd, J 10.1, 4.7, CHeq-6), 4.15 (1H, ddd,
J 9.0, 9.0, 1.4 H-3), 3.84–3.77 (1H, m, H-5), 3.80 (3H, s, Ar OCH3),
3.69 (1H, dd, J 10.2, 10.1, CHax-6), 3.51–3.45 (2H, m, H-2 and H-4),
3.38 (1H, s, OCH3) and 2.66 (1H, d, J 1.4, OH); 13C NMR (100 MHz;
CDCl3) d 160.2 (C-4 ArOCH3 ring), 138.0 (C-1 Ph), 129.7 (C-1 ArOCH3
ring), 128.6 (Ph), 128.2 (Ph), 127.7 (C-2,6 ArOCH3 ring), 113.7 (C-3,5
ArOCH3 ring), 101.9 (C-7), 98.7 (C-1), 81.3 (CH), 79.6 (CH), 73.4
(CH2Ph), 70.3 (C-3), 69.0 (CH2-6), 62.0 (C-5), 55.4 (Ar OCH3) and
55.3 (OCH3); HRMS m/z (ES+) [Found: (M+Na)+ 425.1570.
C22H26O7Na+ requires M+, 425.1576]; m/z (ES+) 425 ([M+Na]+,
100%); Anal. Calcd for C22H26O7: C, 65.66; H, 6.51. Found: C,
65.96; H, 6.80. Further elution provided (+)-methyl 3-O-benzyl-
4,6-O-anisylidene-a-D-glucopyranoside (2.80 g, 15%) as a colour-
less amorphous solid: Rf 0.29 (hexane/ethyl acetate 1:1); ½a D20
¼
ꢁ
4.2. (+)-Methyl 4,6-O-anisylidene-
a-
D
-glucopyranoside 2
þ76:6 (c 1.00, CHCl3) {lit.39
½
a 2D0
ꢁ
¼ þ76:2 (c 1.70, CHCl3)}; mp
177–178 °C (from ethyl acetate) [lit.39 176–178 °C]; mmax (KBr
disc)/cmꢀ1 3449 (s), 1618 (m), 1517 (m), 1370 (m), 1252 (m),
1076 (s); 1H NMR (400 MHz; CDCl3) d 7.45–7.27 (7H, m, Ar CH),
6.94–6.89 (2H, m, H-3,5 ArOCH3 ring), 5.54 (1H, s, CH-7), 4.96
(1H, d, J 11.6, CHAHBPh), 4.82 (1H, d, J 3.8, H-1), 4.79 (1H, d, J 11.6
CHAHBPh), 4.29 (1 H, dd, J 9.6, 4.5, Heq-6), 3.87–3.71 (7H, m, Ar
OCH3, H-2, H-3, H-4 and H-5) 3.64 (1H, dd, J 9.6, 9.2, Hax-6), 3.46
(3H, s, OCH3) and 2.33 (1H, d, J 7.4, OH); 13C NMR (100 MHz; CDCl3)
d 160.0 (C-4 ArOCH3 ring), 138.5 (C-1 Ph), 129.9 (C-1 ArOCH3 ring),
128.4 (Ph), 128.0 (Ph), 127.7 (C-4 Ph), 127.4 (C-2,6 ArOCH3 ring),
113.6 (C-3,5 ArOCH3 ring), 101.3 (C-7), 99.9 (C-1), 81.9 (CH), 78.9
(CH), 74.8 (CH2Ph), 72.4 (CH), 69.0 (CH2-6), 62.6 (CH), 55.4 (Ar
OCH3) and 55.3 (OCH3); HRMS m/z (ES+) [Found: (M+Na)+
425.1582. C22H26O7Na requires M+, 425.1576]; m/z (ES+) 425
([M+Na]+, 100%); Anal. Calcd for C22H26O7: C, 65.66; H, 6.51. Found:
C, 65.82; H, 6.45. The data are in good agreement with the literature
values.39
Methyl 4,6-O-anisylidene- -glucopyranoside 2 was synthes-
a
-D
ised in a manner similar to that described by Hirama and
co-workers,48 affording the desired compound (46.0 g, 58%) as a
colourless solid: Rf 0.32 (ethyl acetate); ½a D20
¼ þ104:8 (c 1.00,
ꢁ
CHCl3) {lit.49
½
a 2D0
ꢁ
¼ þ104:7 (c 0.7, CHCl3)}; mp 193–195 °C (from
ethyl acetate) [lit.49 mp 195 °C]; mmax (KBr disc)/cmꢀ1 3373 (s),
2939 (s), 2868 (s), 1615 (s), 1588 (m), 1518 (s), 1459 (s), 1423
(s), 1304 (s), 1036 (s), 931 (s), 890 (m) and 809 (s); 1H NMR
(300 MHz; CDCl3) d 7.45–7.40 (2H, m, Ar ring), 6.91–6.88 (2H,
m, Ar ring), 5.49 (1H, s, CH-7), 4.79 (1H, d, J 4.0, H-1), 4.30–
4.26 (1H, m, H-5), 3.92 (1H, ddd, J 9.5, 9.2, 1.5, H-3), 3.80 (3H,
s, Ar OCH3), 3.84–3.76 (1H, m, CHeq-6), 3.73 (1H, dd, J 10.3,
10.1, CHax-6), 3.63 (1H, ddd, J 9.3, 9.2, 4.0, H-2), 3.47 (1H, dd, J
9.5, 9.2, H-4), 3.46 (1H, s, OCH3), 2.88 (1H, d, J 1.5, OH-3) and
2.39 (1H, d, J 9.3, OH-2); 13C NMR (100 MHz; CDCl3) d 160.3 (C-
4 Ar), 129.5 (C-1 Ar), 127.6 (C-2,6 Ar ring), 113.7 (C-3,5 Ar ring),

862
N. S. Keddie et al. / Tetrahedron: Asymmetry 20 (2009) 857–866
4.4. (ꢀ)-Methyl 2-O-benzyl-3-O-triisopropylsilyl-4,6-O-anisy-
lidene- -glucopyranoside 4
1.64 (1H, m, OH) and 1.20–1.05 (21H, m, TIPS); 13C NMR
(100 MHz; CDCl3) d 159.2 (C-4 ArOCH3 ring), 138.5 (C-1 Ph),
130.7 (C-1 ArOCH3 ring), 129.3 (C-2,6 ArOCH3 ring), 128.2 (Ph),
128.0 (Ph), 127.7 (C-4 Ph), 113.8 (C-3,5 ArOCH3 ring), 97.6 (C-1),
81.3 (C-2), 78.9 (C-4), 74.4 (C-3), 74.3 (CH2Ph), 72.6 (CH2PhOCH3),
70.4 (C-5), 62.0 (CH2-6), 55.2 (Ar OCH3), 54.9 (OCH3), 18.3 (CH3
TIPS) and 13.6 (CH TIPS); HRMS m/z (ES+) [Found: (M+Na)+
a-D
To a solution of (+)-methyl-2-O-benzyl-4,6-O-anisylidene-a-D-
glucopyranoside 3 (17.61 g, 43.8 mmol) in CH2Cl2 (300 mL) were
added a mixture of triethylamine (17.70 g, 24.4 mL, 175.0 mmol)
and TIPSOTf (20.10 g, 17.7 mL, 65.6 mmol) via a pressure equalised
dropping funnel over a period of 20 min. The solution was stirred
for 2.5 h at rt, turning from colourless to pale orange. The reaction
was quenched by the addition of aqueous saturated sodium bicar-
bonate solution (200 mL). The layers were separated and the aque-
ous phase was extracted with CH2Cl2 (2 ꢂ 300 mL). The combined
organic phases were washed with brine (200 mL), dried over mag-
nesium sulfate, filtered and concentrated in vacuo to give an or-
ange oil. Purification by silica gel chromatography, eluting with
hexane and diethyl ether (90:10), provided (ꢀ)-methyl 2-O-
583.3071. C31H48O7SiNa requires M+ 583.3067]; m/z (ES+) 583
,
([M+Na]+, 100%); Anal. Calcd for C31H48O7Si: C, 66.39; H, 8.63.
Found: C, 66.62; H, 8.71.
4.6. Methyl 2-O-benzyl-4-O-(4-methoxybenzyl)-3-O-triisopro-
pylsilyl-a-D-gluco-hexadialdo-1,5-pyranoside 6
A solution of (+)-methyl 2-O-benzyl-4-O-(4-methoxybenzyl)-3-
O-triisopropylsilyl- -glucopyranoside 5 (19.82 g, 35.3 mmol) in
a-D
benzyl-3-O-triisopropylsilyl-4,6-O-anisylidene-
a-
D
-glucopyranoside 4
CH2Cl2 (300 mL) was treated with Dess Martin Periodinane
(22.46 g, 53.0 mmol) in a single portion. The suspension was stir-
red at rt for 1.5 h, after which time TLC analysis indicated con-
sumption of the starting material. The suspension was diluted
with diethyl ether (200 mL) and aqueous saturated sodium bicar-
bonate solution (200 mL), then sodium thiosulfate (134.8 g) was
added. The biphasic mixture was stirred vigorously for 30 min
until two clear layers were observed. The layers were separated
and the aqueous phase was re-extracted with diethyl ether
(2 ꢂ 400 mL). The combined organic phases were then washed
with aqueous saturated sodium bicarbonate solution (300 mL)
and water (400 mL), dried over magnesium sulfate, filtered and
concentrated in vacuo providing a pale yellow oil. Purification of
the oil by silica gel chromatography, eluting with hexane and
diethyl ether (40:60), yielded methyl 2-O-benzyl-4-O-(4-methoxy-
(22.46 g, 92%) as a colourless oil: Rf 0.25 (hexane/diethyl ether
80:20); ½a 2D0
ꢁ
¼ ꢀ13:5 (c 0.97, CHCl3); mmax (KBr disc)/cmꢀ1 2942
(s), 2865 (s), 1616 (m), 1518 (s), 1375 (m), 1303 (m), 1250 (s),
1054 (s), 992 (s) and 831 (s); 1H NMR (400 MHz; CDCl3) d 7.41–
7.28 (7H, m, Ar CH), 6.89–6.87 (2H, m, H-3,5 ArOCH3 ring), 5.40
(1H, s, CH-7), 4.84 (1H, d, J 12.3, CHAHBPh), 4.60 (1H, d, J 12.3,
CHAHBPh), 4.47 (1H, d, J 3.7, H-1), 4.26 (1H, dd, J 8.9, 8.9, H-3),
4.20 (1H, dd, J 10.1, J 4.7, Heq-6), 3.81 (3H, s, Ar OCH3), 3.81–3.78
(1H, m, H-5), 3.66 (1H, dd, J 10.2, 10.1, Hax-6), 3.42–3.36 (2H, m,
H-2 and H-4), 3.33 (3H, s, OCH3) and 1.13–0.99 (21H, m, TIPS);
13C NMR (100 MHz; CDCl3) d 160.1 (C-4 ArOCH3 ring), 138.5 (C-1
Ph), 129.9 (C-1 ArOCH3 ring), 128.4 (Ph), 128.2 (Ph), 127.9 (C-4
Ph), 127.7 (C-2,6 ArOCH3 ring), 113.4 (C-3,5 ArOCH3 ring), 102.1
(C-7), 99.3 (C-1), 82.7 (CH), 80.8 (CH), 73.8 (CH2Ph), 71.8 (CH),
69.1 (CH2-6), 62.6 (CH), 55.3 (2 ꢂ OCH3), 18.2 (CH3 TIPS) and 12.8
(CH TIPS); HRMS m/z (ES+) [Found: (M+Na)+ 581.2919. C31H46O7S-
iNa requires M+, 581.2911]; m/z (ES+) 581 ([M+Na]+, 100%); Anal.
Calcd for C31H46O7Si: C, 66.63; H, 8.30. Found: C, 66.36; H, 8.65.
benzyl)-3-O-triisopropylsilyl-
a-
D-glucohexadialdo-1,5-pyranoside
6
(16.26 g, 82%) as a colourless oil: Rf 0.47 (hexane/ethyl acetate
50:50); mmax (thin film)/cmꢀ1 2943 (s), 2865 (s), 1742 (m), 1613
(m), 1515 (s), 1464 (m), 1250 (s), 1051 (s); 1H NMR (400 MHz;
CDCl3) d 9.59 (1H, d, J 0.6, CHO), 7.40–7.21 (7H, m, Ar CH), 6.90–
6.86 (2H, m, H-3,5 ArOCH3 ring), 4.82 (1H, d, J 10.8, CHAHBPhOCH3),
4.67 (1H, d, J 11.9, CHAHBPh), 4.64 (1H, d, J 3.3, H-1), 4.52 (1H, d, J
11.9, CHAHBPh), 4.51 (1H, d, J 10.8, CHAHBPhOCH3), 4.28 (1H, dd, J
8.8, 8.0, H-3), 4.16 (1H, d, J 9.9, H-5), 3.81 (3H, s, Ar OCH3), 3.49
(1H, dd, J 9.9, 8.0, H-4), 3.33 (1H, dd, J 8.8, 3.3, H-2), 3.24 (3H, s,
OCH3) and 1.18–1.02 (21H, m, TIPS); 13C NMR (100 MHz; CDCl3)
d 198.0 (CHO), 159.4 (C-4 ArOCH3 ring), 138.3 (C-1 Ph), 129.7 (C-
1 ArOCH3 ring), 129.5 (C-2,6 ArOCH3 ring), 128.3 (Ph), 128.0 (Ph),
127.8 (C-4 Ph), 113.8 (C-3,5 ArOCH3 ring), 97.9 (C-1), 82.3 (CH),
79.1 (CH), 74.9 (CH), 74.3 (CH2Ph), 74.0 (CH), 72.8 (CH2PhOCH3),
55.6 (Ar OCH3), 55.3 (OCH3), 18.3 (CH3 TIPS) and 13.4 (CH TIPS);
HRMS m/z (ES+) [Found: (M+Na)+ 581.2902. C31H46O7SiNa requires
M+, 581.2911]; m/z (ES+) 581 ([M+Na]+, 100%). This material was
used immediately in the next step.
4.5. (+)-Methyl 2-O-benzyl-4-O-(4-methoxybenzyl)-3-O-triiso-
propylsilyl-a-D-glucopyranoside 5
To a solution of (ꢀ)-methyl 2-O-benzyl-3-O-triisopropylsilyl-
4,6-O-anisylidene- -glucopyranoside 4 (22.46 g, 40.2 mmol) dis-
a
-D
solved in CH2Cl2 (400 mL) at rt was added a solution of DIBAL-H
(1.0 M, 201 mL, 201.0 mmol) in hexane dropwise, via a pressure
equalised dropping funnel, over a period of 30 min. The colourless
solution was stirred for an additional 40 min at rt. The solution was
then cooled to ꢀ10 °C and the reaction was quenched by the
sequential, dropwise, addition of water (8.04 mL), aqueous sodium
hydroxide solution (15% w/v, 8.04 mL) and a further portion of
water (20 mL). CAUTION: This quenching procedure may lead to
the evolution of hydrogen gas. A thick colourless gel formed, which
was stirred for 30 min at 0 °C then filtered through a pad of Celite.
The residue was washed with cold CH2Cl2 (3 ꢂ 500 mL); the result-
ing filtrates were combined and concentrated in vacuo to provide a
pale yellow oil. Purification of the oil by silica gel chromatography,
eluting with hexane and diethyl ether (40:60), yielded (+)-methyl
4.7. (ꢀ)-Methyl 6-O-acetyl-2-O-benzyl-4-O-(4-methoxybenzyl)-
3-O-triisopropylsilyl-a-D-glucohex-5-enopyranoside 7
To a solution of methyl 2-O-benzyl-4-O-(4-methoxybenzyl)-3-
O-triisopropylsilyl- -gluco-hexadialdo-1,5-pyranoside
2-O-benzyl-4-O-(4-methoxybenzyl)-3-O-triisopropylsilyl-
a
-
D-gluco-
a-
D
6
pyranoside 5 (19.82 g, 88%) as a thick colourless oil: Rf 0.35 (hex-
(16.26 g, 29.1 mmol) in acetonitrile (300 mL) was added acetic
anhydride (8.91 g, 6.41 mL, 87.3 mmol), potassium carbonate
(20.11 g, 145.5 mmol) and DMAP (355 mg, 2.9 mmol). The stirred
suspension was heated to 90 °C for 1 h, after which time TLC anal-
ysis showed consumption of starting material. The mixture was
cooled to RT and the volatile components were removed in vacuo
providing a yellow residue. This residue was partitioned between
water (400 mL) and CH2Cl2 (400 mL). The layers were separated
and the aqueous layer was re-extracted with CH2Cl2 (3 ꢂ
400 mL). The combined organic layers were washed with brine
ane/diethyl ether 30:70); ½a D20
¼ þ64:6 (c 1.56, CHCl3); mmax (thin
ꢁ
film)/cmꢀ1 3458 (m), 2942 (s), 2865 (s), 1613 (m), 115 (s), 1464
(m), 1250 (s), 1158 (m), 1104 (s); 1H NMR (400 MHz; CDCl3) d
7.40–7.24 (7H, m, Ar CH), 6.91–6.86 (2H, m, H-3,5 ArOCH3 ring),
4.85 (1H, d, J 10.7, CHAHBPhOCH3), 4.65 (1H, d, J 11.8, CHAHBPh),
4.56 (1H, d, J 10.7, CHAHBPhOCH3), 4.54 (1H, d, J 3.4, H-1), 4.52
(1H, d, J 11.8, CHAHBPh), 4.22 (1H, dd, J 9.1, 8.9 H-3), 3.81 (3H, s,
Ar OCH3), 3.76–3.56 (3H, m, H-5, Heq-6, Hax-6), 3.41 (1H, dd, J 9.9,
8.9, H-4), 3.33 (1H, dd, J 9.1, 3.4, H-2), 3.21 (3H, s, OCH3), 1.68–

N. S. Keddie et al. / Tetrahedron: Asymmetry 20 (2009) 857–866
863
(400 mL), dried over magnesium sulfate, filtered and concentrated
in vacuo to give a light brown oil. Purification of this oil by silica gel
chromatography, eluting with hexane and diethyl ether (75:25),
provided (ꢀ)-methyl 6-O-acetyl-2-O-benzyl-4-O-(4-methoxyben-
4.9. (+)-D-1-O-Acetyl-3-O-benzyl-5-O-(4-methoxybenzyl)-4-O-
triisopropylsilyl-myo-inositol 9
A mixture of acetic acid (36.5 mL) and acetonitrile (36.5 mL)
was cooled to ꢀ40 °C and to this was added tetramethylammo-
nium triacetoxyborohydride (8.63 g, 32.8 mmol). To the resulting
slurry was added a solution of (ꢀ)-3-O-benzyl-2-hydroxy-5-O-(4-
zyl)-3-O-triisopropylsilyl-a-D-glucohex-5-enopyranoside 7 (11.42 g,
65%) as a colourless oil: Rf 0.18 (hexane/diethyl ether 75:25);
½
a 2D0
ꢁ
¼ ꢀ8:6 (c 1.32, CHCl3); 1H NMR (400 MHz; CDCl3) d 7.43–
7.28 (7H, m, Ar CH), 7.08 (1H, d, J 1.5, CHOAc), 6.92–6.88 (2H, m,
H-3,5 ArOCH3 ring), 4.78 (1H, d, J 12.0, CHAHBPh), 4.68 (1H, d, J
3.0, H-1), 4.64 (1H, d, J 10.9, CHAHBPhOCH3), 4.61 (1H, d, J 12.0,
CHAHBPh), 4.58 (1H, d, J 10.9, CHAHBPhOCH3), 4.19 (1H, dd, J 8.0,
7.1, H-3), 3.83 (3H, s, Ar OCH3), 3.76 (1H, dd, J 7.1, 1.5, H-4), 3.46
(1H, dd, J 8.0, 3.0, H-2), 2.17 (3H, s, OCH3) and 1.10–1.03 (21 H,
m, TIPS); 13C NMR (100 MHz; CDCl3) d 167.4 (C@O), 159.2 (C-4 Ar-
OCH3 ring), 138.4 (C-1 Ph), 135.1 (C-5), 129.9 (C-1 ArOCH3 ring),
129.4 (C-2,6 ArOCH3 ring), 128.3 (Ph), 128.1 (Ph), 127.8 (C-4 Ph),
123.1 (CHOAc), 113.7 (C-3,5 ArOCH3 ring), 100.0 (C-1), 80.2 (C-2),
78.5 (C-4), 73.4 (C-3), 73.3 (CH2Ph), 72.6 (CH2PhOCH3), 56.3
(OCH3), 55.3 (Ar OCH3), 20.6 (CH3 Ac), 18.2 (CH3 TIPS) and 12.9
(CH TIPS); HRMS m/z (ES+) [Found: (M+Na)+ 623.3018. C33H48O8Si-
Na requires M+, 623.3016]; m/z (ES+) 623 ([M+Na]+, 100%); Anal.
Calcd for C33H48O8Si: C, 65.97; H, 8.05. Found: C, 65.88; H, 8.45.
methoxybenzyl)-4-O-triisopropylsilyl-6-oxocyclohexyl acetate 8
(3.85 g, 6.56 mmol) in acetonitrile (23.9 mL) via cannula and the
mixture stirred for 10 min at ꢀ40 °C. The mixture was then al-
lowed to warm to rt and was stirred for a further 18 h, by which
time a clear solution had formed. The reaction was quenched by
the addition of aqueous saturated Rochelle’s Salt solution
(100 mL). The resulting emulsion was stirred vigorously for 1 h
and then diluted with CH2Cl2 (250 mL). The layers were separated
and the aqueous phase was re-extracted with CH2Cl2 (2 ꢂ 250 mL).
The combined organic phases were washed with brine (200 mL),
dried over magnesium sulfate, filtered and concentrated in vacuo
providing a brown oil. The excess AcOH was then removed by aze-
otropic distillation with cyclohexane (3 ꢂ 100 mL), resulting in a
brown gum. Purification of the gum by silica gel chromatography,
eluting with hexane and diethyl ether (50:50, 40:60, 0:100),
yielded
(+)-D-1-O-acetyl-3-O-benzyl-5-O-(4-methoxybenzyl)-4-O-
4.8. (ꢀ)-3-O-Benzyl-2-hydroxy-5-O-(4-methoxybenzyl)-4-O-tri-
triisopropylsilyl-myo-inositol 9 (3.42 g, 89%) as a colourless oil: Rf
isopropylsilyl-6-oxocyclohexyl acetate 8
0.39 (hexane/ethyl acetate 50:50); ½a D20
¼ þ17:1 (c 1.00, CHCl3);
ꢁ
mmax (thin film)/cmꢀ1 3449 (m), 3012 (m), 2945 (m), 2867 (w),
1735 (m), 1612 (w), 1514 (m), 1215 (s), 768 (s); 1H NMR (400
MHz; CDCl3) d 7.39–7.26 (7H, m, Ar CH), 6.90–6.89 (2H, m, H-3,5
ArOCH3 ring), 4.87 (1H, d, J 11.4, CHAHBPhOCH3), 4.75 (1H, dd, J
9.8, 2.2, H-1), 4.70 (1H, d, J 11.4, CHAHBPhOCH3), 4.61–4.55 (2H,
m, CH2Ph), 4.31–4.28 (1H, m, H-2), 4.15 (1H, dd, J 8.9, 8.8, H-4),
4.05 (1H, ddd, J 9.8, 9.3, 3.1, H-6), 3.80 (3H, s, Ar OCH3), 3.41 (1H,
dd, J 8.9, 2.8, H-3), 3.26 (1H, dd, J 9.3, 8.8, H-5), 2.28 (1H, s, OH-
2), 2.14 (3H, s, COCH3), 1.64 (1H, d, J 3.1, OH-6) and 1.16–1.03
(21H, m, TIPS); 13C NMR (100 MHz; CDCl3) d 170.9 (C@O), 159.2
(COCH3), 137.6, 131.0, 129.2 (CH-2,6 ArOCH3 ring), 128.5 (CH Ph),
128.0 (CH Ph), 127.6 (CH Ph), 114.0 (CH-3,5 ArOCH3 ring), 84.2
(C-5), 81.4 (C-3), 74.9 (CH2Ph), 73.5 (C-4), 73.2 (C-1), 71.7
(CH2PhOCH3), 70.3 (C-6), 66.5 (C-2), 55.3 (OCH3), 21.1 (COCH3),
18.2 (CH3 TIPS) and 13.5 (CH TIPS); HRMS m/z (ES+) [Found:
(M+Na)+ 611.3012. C32H48O8SiNa requires M+, 611.3016]; m/z
(ES+) 611 ([M+Na]+, 100%); Anal. Calcd for C32H48O8Si: C, 65.28;
H, 8.22. Found: C, 64.99; H, 8.17.
To
methoxybenzyl)-3-O-triisopropylsilyl-
side (11.42 g, 19.0 mmol) in acetone (400 mL) and water
a
solution of (ꢀ)-methyl 6-O-acetyl-2-O-benzyl-4-O-(4-
a-D
-gluco-hex-5-enopyrano-
7
(260 mL) was added mercuric acetate (30.29 g, 95.1 mmol) in a sin-
gle portion. The cloudy solution immediately turned to a thick yel-
low suspension. The suspension was stirred for 3 h at rt, after
which time TLC analysis indicated consumption of the starting
material. The suspension was diluted with brine (130 mL), turning
the suspension to a slightly cloudy colourless solution after 15 min.
The mixture was then stirred for an additional 24 h. The mixture
was diluted with water (200 mL) and extracted with CHCl3
(400 mL). The aqueous phase was re-extracted with a further por-
tion of CHCl3 (400 mL). The combined organic layers were washed
with brine (200 mL), dried over magnesium sulfate, filtered and
concentrated in vacuo to give a thick colourless oil. Purification
of the oil by silica gel chromatography, eluting with hexane and
diethyl ether (75:25, 70:30, 60:40, 0:100), yielded a colourless
solid. Crystallisation from hexane and diethyl ether yielded (ꢀ)-
3-O-benzyl-2-hydroxy-5-O-(4-methoxybenzyl)-4-O-triisopropylsilyl-
6-oxocyclohexyl acetate 8 (3.85 g, 35%) as colourless needles: Rf 0.51
4.10. (ꢀ)-
D-1-O-Acetyl-3-O-benzyl-2,6-O-bisbenzyloxymethyl-
5-O-(4-methoxybenzyl)-4-O-triisopropylsilyl-myo-inositol 10
(hexane/ethyl acetate 50:50); ½a D20
¼ ꢀ28:9 (c 3.02, CHCl3); mp
ꢁ
102–103 °C (from hexane/diethyl ether); mmax (thin film)/cmꢀ1
3490 (s), 2943 (s), 2866 (s), 1756 (s), 1737 (s), 1611 (m), 1514
(m), 1467 (m), 1367 (m), 1242 (s), 1145 (s), 1094 (m), 1073 (m),
1027 (m) and 885 (m); 1H NMR (400 MHz; CDCl3) d 7.41–7.32
(7H, m, Ar CH), 6.90–6.86 (2H, m, H-3,5 ArOCH3 ring), 5.28–5.25
(1H, m, H-1), 4.79 (1H, d, J 9.7, CHAHBPhOCH3), 4.78 (1H, d, J
11.8, CHAHBPh), 4.68 (1H, d, J 11.8, CHAHBPh), 4.40 (1H, d, J 9.7,
CHAHBPhOCH3), 4.33 (1H, dd, J 4.2, 2.7, H-2), 4.28 (1H, dd, J 8.8,
8.6, H-4), 4.00 (1H, dd, J 8.8, 1.0, H-5), 3.83 (3H, s, OCH3), 3.78
(1H, dd, J 8.6, 2.7, H-3), 2.45 (1H, m, OH), 2.26 (2H, s, CH3) and
1.14–1.01 (21 H, m TIPS); 13C NMR (100 MHz; CDCl3) d 198.3 (C-
6 C@O), 169.6 (C@O), 159.2 (C-4 ArOCH3 ring), 137.4, 130.0,
129.6 (C-1 ArOCH3 ring), 128.6, 128.1, 127.8 (C-2,6 ArOCH3 ring),
113.5 (C-3,5 ArOCH3 ring), 83.7 (C-5), 80.9 (C-3), 74.9 (C-4), 74.9
(C-1), 72.9 (CH2Ph), 72.7 (CH2ArOCH3), 68.6 (C-2), 55.3 (OCH3),
20.6 (CH3), 18.2 (CH3 TIPS) and 13.0 (CH TIPS); HRMS m/z (ES+)
[Found: (M+Na)+ 609.2859. C32H46O8SiNa requires M+, 609.2860];
m/z (ES+) 609 ([M+Na]+, 100%); Anal. Calcd for C32H46O8Si: C,
65.50; H, 7.90. Found: C, 65.32; H, 8.29.
A mixture of benzyloxymethyl chloride (7.28 g, 6.46 mL,
46.47 mmol) and Hünig’s base (20 mL) was stirred for 1 h at rt,
forming a light brown precipitate. The precipitate was allowed to
settle, then the solution was carefully decanted from the mixture
via cannula into a solution of (+)-
D-1-O-acetyl-3-O-benzyl-5-O-
(4-methoxybenzyl)-4-O-triisopropylsilyl-myo-inositol
9
(3.42 g,
5.81 mmol) in Hünig’s base (40 mL). The resulting solution was
heated to 85 °C for 15 h, then allowed to cool to rt. TLC analysis
of the mixture indicated consumption of the starting material.
The volatile components were removed in vacuo and the resulting
brown residue was partitioned between water (250 mL) and ethyl
acetate (250 mL). The layers were separated and the aqueous
phase re-extracted with ethyl acetate (2 ꢂ 250 mL). The combined
organic layers were washed with brine (250 mL) dried over magne-
sium sulfate, filtered and concentrated in vacuo affording a brown
oil. Purification of the oil by silica gel chromatography, eluting with
hexane and diethyl ether (100:0, 98:2, 96:4, 94:6, 92:8, 80:20), fur-
nished (ꢀ)-
D-1-O-acetyl-3-O-benzyl-2,6-O-bisbenzyloxymethyl-5-O-
(4-methoxybenzyl)-4-O-triisopropylsilyl-myo-inositol 10 (3.94 g,

864
N. S. Keddie et al. / Tetrahedron: Asymmetry 20 (2009) 857–866
82%) as a colourless gum, which solidified on standing to waxy so-
(OCH3), 18.3 (CH3 TIPS) and 13.4 (CH TIPS); HRMS m/z (ES+) [Found:
(M+Na)+ 809.4079. C46H62O9SiNa requires M+, 809.4061]; m/z (ES+)
809 ([M+Na]+, 100%); Anal. Calcd for C46H62O9Si: C, 70.20; H, 7.94.
Found: C, 70.21; H, 7.74.
lid: Rf 0.46 (hexane/diethyl ether 50:50); ½a D20
¼ ꢀ40:7 (c 0.75,
ꢁ
CHCl3); mmax (KBr disc)/cmꢀ1 3415 (m), 2944 (s), 2866 (s), 1741
(s) 1615 (m), 1514 (s), 1458 (m), 1366 (m), 1247 (s), 1119 (s),
1039 (s), 739 (s) and 699 (m); 1H NMR (400 MHz; CDCl3) d 7.25–
7.15 (17H, m, Ar H), 6.77–6.73 (2H, m, H-3,5 ArOCH3 ring), 4.87
(1H, d, J 6.7, OCHAHBO), 4.81–4.65 (8H, m, H-1 and 7 ꢂ CHAHB pro-
tons), 4.59 (1H, d, J 12.3, CHAHBPh), 4.50 (1H, d, J 12.3, CHAHBPh0),
4.39 (1H, d, J 12.3, CHAHBPh), 4.38 (1H, d, J 11.1, CHAHBPh00), 4.31
(1H, dd, J 2.1, 2.1, H-2), 4.23 (1H, dd, J 9.3, 9.0, H-4), 4.14 (1H, dd,
J 9.3, 9.2, H-6), 3.71 (3H, s, Ar OCH3), 3.29 (1H, dd, J 9.2, 9.0, H-5),
3.27 (1H, dd, J 9.3, 2.1, H-3), 1.72 (3H, s, COCH3) and 0.96–0.90
(21H, m, TIPS); 13C NMR (100 MHz; CDCl3) d 170.7 (C@O), 158.7
(COCH3), 138.0, 137.8, 137.6, 131.2, 128.4, 128.3 (CH-2,6 ArOCH3
ring), 128.3, 128.2, 127.6, 127.5, 127.5, 127.4, 127.4, 113.5
(CH-3,5 ArOCH3 ring), 96.2 (OCH2O), 95.0 (OCH2O), 84.7 (C-5),
81.0 (C-3), 77.2 (C-6), 75.1 (CH2Ar), 73.8 (C-4), 72.9 (C-1), 72.0
(C-2), 71.9 (CH2Ph), 69.7 (CH2Ph), 69.2 (CH2Ph), 55.3 (Ar OCH3),
21.0 (COCH3), 18.2 (CH3 TIPS) and 13.5 (CH TIPS); HRMS m/z
(ES+) [Found: (M+Na)+ 851.4155. C48H64O10SiNa requires M+,
851.4166]; m/z (ES+) 851 ([M+Na]+, 100%); Anal. Calcd for
C48H64O10Si: C, 69.53; H, 7.78. Found: C, 69.41; H, 7.91.
4.12. (+)-D-3-O-Benzyl-2,6-O-bisbenzyloxymethyl-4-O-triisopro-
pylsilyl-myo-inositol 12
To a solution of (ꢀ)-D-3-O-benzyl-2,6-O-bisbenzyloxymethyl-5-
O-(4-methoxybenzyl)-4-O-triisopropylsilyl-myo-inositol 11 (1.23 g,
1.56 mmol) in CH2Cl2 (60 mL) and water (3.3 mL) was added
2,3-dichloro-5,6-dicyanobenzoquinone (780 mg, 3.44 mmol) in a
single portion, turning the mixture dark green. After 10 min stir-
ring at rt, the colour changed to orange/yellow and TLC analysis
indicated consumption of the starting material. The solution was
diluted with aqueous saturated sodium hydrogen carbonate solu-
tion (40 mL) and CH2Cl2 (80 mL), forming a dark biphasic mixture.
The biphasic mixture was filtered through a pad of Celite and the
layers were subsequently separated. The aqueous phase was re-ex-
tracted with CH2Cl2 (3 ꢂ 80 mL). The combined organic phases
were washed with aqueous saturated sodium hydrogen carbonate
solution (100 mL) and brine (100 mL), dried over magnesium sul-
fate, filtered and concentrated in vacuo affording a pale brown
oil. Purification of the oil by silica gel chromatography, eluting with
4.11. (ꢀ)-
D-3-O-Benzyl-2,6-O-bisbenzyloxymethyl-5-O-(4-meth-
oxybenzyl)-4-O-triisopropylsilyl-myo-inositol 11
hexane and diethyl ether (60:40), furnished (+)-D-3-O-benzyl-2,6-
O-bisbenzyloxymethyl-4-O-triisopropylsilyl-myo-inositol 12 (993
mg, 95%) as a colourless oil: Rf 0.21 (hexane/diethyl ether 50:50);
In an open flask, (ꢀ)-
D-1-O-acetyl-3-O-benzyl-2,6-O-bisbenzyl-
oxymethyl-5-O-(4-methoxybenzyl)-4-O-triisopropylsilyl-myo-ino-
sitol 10 (3.48 g, 4.20 mmol) was dissolved in a mixture of methanol
(90 mL) and tetrahydrofuran (30 mL). To this solution was added
lithium hydroxide (422 mg, 17.63 mmol) in a single portion. The
mixture was stirred vigorously at rt for 15 min, after which time
TLC analysis indicated consumption of the starting material. The
reaction was quenched by the addition of saturated aqueous
ammonium chloride solution (20 mL) and the volatile components
were removed in vacuo. The resulting residue was partitioned be-
tween water (150 mL) and CH2Cl2 (150 mL). The layers were sepa-
rated and the aqueous layer was re-extracted with CH2Cl2 (3 ꢂ 100
mL). The combined organic phases were washed with brine
(100 mL), dried over magnesium sulfate, filtered and concentrated
in vacuo, furnishing a colourless oil. Purification of the oil by silica
gel chromatography, eluting with hexane and diethyl ether
½
a 2D0
ꢁ
¼ þ3:6 (c 2.45, CHCl3); mmax (thin film)/cmꢀ1 3439 (s), 3065
(m), 3032 (m), 2493 (s), 2866 (s), 1719 (m), 1497 (m), 1455 (s),
1368 (s), 1273 (m), 1025 (s), 884 (s), 826 (s), 737 (s) and 698 (s);
1H NMR (400 MHz; CDCl3) d 7.30–7.17 (15H, m, Ar H), 4.89 (1H,
d, J 10.4, OCHAHBO), 4.87 (1H, d, J 10.4, OCHAHBO), 4.85 (1H, d, J
6.6, OCHAHBO0), 4.68 (1H, d, J 6.6, OCHAHBO0), 4.65 (1H, d, J 11.9,
OCHAHBPh0), 4.62 (2H, s, OCH2Ph), 4.59 (1H, d, J 11.6, CHAHBPh00),
4.51 (1H, d, J 11.9, OCHAHBPh0), 4.49 (1H, d, J 11.6, CHAHBPh00),
4.10 (1H, dd, J 2.3, 2.3, H-2), 4.06 (1H, dd, J 9.3, 8.9, H-4), 3.60
(1H, dd, J 9.2, 9.0, H-6), 3.47 (1H, d, J 5.6, OH-1), 3.44–3.38 (1H,
m, H-1), 3.32 (1H, ddd, J 9.0, 8.9, 2.0, H-5), 3.19 (1H, dd, J 9.3, 2.3,
H-3), 3.01 (1H, d, J 2.0, OH-5) and 1.11–0.95 (21H, m, TIPS); 13C
NMR (100 MHz; CDCl3) d 138.0, 137.6, 137.0, 128.5, 128.4, 128.2,
128.1, 128.0, 127.9, 127.7, 127.6, 127.6, 96.5 (OCH2O), 95.9
(OCH2O’), 84.1 (C-6), 80.3 (C-3), 76.3 (C-2), 75.5 (C-5), 74.3 (C-4),
72.2 (CH2Ph00), 70.9 (C-1), 70.4 (CH2Ph), 69.8 (CH2Ph0), 18.3 (CH3
TIPS) and 13.0 (CH TIPS); HRMS m/z (ES+) [Found: (M+Na)+
689.3475. C38H54O8SiNa requires M+, 689.3486]; m/z (ES+) 689
([M+Na]+, 100%); Anal. Calcd for C38H54O8Si: C, 68.44; H, 8.16.
Found: C, 68.02; H, 8.30.
(70:30), afforded (ꢀ)-
D-3-O-benzyl-2,6-O-bisbenzyloxymethyl-5-O-
(4-methoxybenzyl)-4-O-triisopropylsilyl-myo-inositol 11 (3.19 g,
97%) as a colourless oil, which solidified on standing to give a waxy
solid: Rf 0.37 (hexane/diethyl ether 50:50); ½a D20
¼ ꢀ17:9 (c 1.47,
ꢁ
CHCl3); mmax (thin film)/cmꢀ1 3449 (m), 2942 (s), 2863 (s), 1617
(w), 1514 (s), 1464 (s), 1382 (w), 1245 (s), 1164 (s), 1133 (s),
1069 (s), 1030 (s), 885 (w), 824 (m), 749 (m) and 699 (m); 1H
NMR (500 MHz; CDCl3) d 7.32–7.25 (15H, m, Ar CH), 7.24–7.22
(2H, m, H-2,6 ArOCH3), 6.86–6.83 (2H, m, H-3,5 ArOCH3), 4.92
(1H, d, J 6.8, OCHAHBO), 4.83 (1H, d, J 6.6, OCHAHBO0), 4.79 (1H, d,
J 6.6, OCHAHBO0), 4.78 (1H, d, J 10.9, OCHAHBPhOCH3), 4.77 (1H,
d, J 6.8, OCHAHBO), 4.74 (1H, d, J 10.9, OCHAHBPhOCH3), 4.73 (1H,
d, J 11.9, CHAHBPh), 4.72 (1H, d, J 12.0, CHAHBPh0), 4.70 (1H, d, J
11.5, CHAHBPh00), 4.58 (1H, d, J 12.0, CHAHBPh0), 4.53 (1H, d, J
11.9, CHAHBPh), 4.49 (1H, d, J 11.5, CHAHBPh00), 4.27 (1H, dd, J 8.9,
8.6, H-4), 4.36 (1H, dd, J 2.5, 2.3, H-2), 3.88 (1H, d, J 4.8, OH-1),
3.86 (1H, dd, J 9.1, 8.9, H-6), 3.80 (3H, s, OCH3), 3.53 (1H, ddd, J
9.1, 4.8, 2.5, H-1), 3.33 (1H, dd, J 8.9, 8.6, H-5), 3.31 (1H, dd, J 8.9,
2.3, H-3) and 1.09–0.99 (21H, m, TIPS); 13C NMR (100 MHz; CDCl3)
d 158.8 (CHOCH3), 137.9, 137.8, 137.3, 131.4, 128.6, 128.5 (C-2,6
ArOCH3 ring), 128.3, 128.1, 128.0, 127.8, 127.6, 127.6, 127.5,
113.5 (C-3,5 ArOCH3 ring), 96.5 (OCH2O’), 95.5 (OCH2O), 83.8 (C-
6), 83.7 (C-5), 80.9 (C-3), 74.8 (OCH2Ar), 74.7 (C-2), 73.7 (C-4),
71.9 (CH2Ph00), 71.2 (C-1), 70.1 (CH2Ph), 69.5 (CH2Ph’), 55.3
4.13. (+)-D-3-O-Benzyl-2,6-O-bisbenzyloxymethyl-myo-inositol
13
To a solution of (+)-D-3-O-benzyl-2,6-O-bisbenzyloxymethyl-4-
O-triisopropylsilyl-myo-inositol 12 (947 mg, 1.42 mmol) in CH2Cl2
(30 mL) was added a solution of tetrabutylammonium fluoride
(1.0 M, 14.2 mL) in tetrahydrofuran. The solution was stirred at rt
for 24 h, after which time TLC analysis indicated consumption of
the starting material. The reaction was quenched by the addition
of saturated aqueous sodium hydrogen carbonate solution
(50 mL) and the layers were separated. The aqueous phase was
re-extracted with CH2Cl2 (4 ꢂ 50 mL). The combined organic
phases were washed with brine (50 mL), dried over magnesium
sulfate, filtered and concentrated in vacuo, providing a brown oil.
Purification by silica gel chromatography, eluting with hexane
and ethyl acetate (40:60), provided (+)-D-3-O-benzyl-2,6-O-bisb-
enzyloxymethyl-myo-inositol 13 (655 mg, 90%) as a colourless so-
lid: Rf 0.26 (hexane/ethyl acetate 65:35); ½a D20
¼ þ27:3 (c 0.11,
ꢁ

N. S. Keddie et al. / Tetrahedron: Asymmetry 20 (2009) 857–866
865
CHCl3) {lit.28
½
a 2D4
ꢁ
¼ þ26:8 (c 0.45, CHCl3)}; mp 113–114 °C (from
nitrogen gas and then with hydrogen gas. The suspension was then
vigorously stirred under an atmosphere of hydrogen for 16 h. The
heterogeneous mixture was filtered through a pad of Celite and
the residue was washed with diethyl ether (2 ꢂ 5 mL); the organic
filtrates were discarded. The palladium residue was washed with
water (6 ꢂ 10 mL), and the combined aqueous filtrates were lyph-
ethyl acetate) [lit.28 112 °C]; mmax (thin film)/cmꢀ1 3438 (s), 2899
(m), 1498 (w), 1454 (m), 1378 (m), 1171 (s), 1129 (s), 1105 (s),
1065 (s), 1020 (s), 942 (m), 737 (s) and 698 (s); 1H NMR
(500 MHz; CDCl3) d 7.37–7.28 (15H, m, Ar H), 5.00 (1H, d, J 6.8,
OCHAHBO), 4.98 (1H, d, J 7.0, OCHAHBO0), 4.92 (1H, d, J 7.0,
OCHAHBO0), 4.82 (1H, d, J 6.8, OCHAHBO), 4.74 (1H, d, J 11.7,
CHAHBPh), 4.73 (1H, d, J 12.0, CHAHBPh’), 4.72 (1H, d, J 11.9,
CHAHBPh00), 4.69 (1H, d, J 11.7, CHAHBPh), 4.64 (1H, s, J 12.0,
CHAHBPh0), 4.62 (1H, d, J 11.9, CHAHBPh00), 4.25 (1H, dd, J 2.4, 2.3,
H-2), 3.99 (1H, dd, J 9.7, 9.3, H-4), 3.65 (1H, dd, J 9.2, 9.1, H-6),
3.66–3.62 (1H, m, OH-5), 3.54 (1H, d, J 5.6, OH-1), 3.51–3.48 (1H,
m, H-1), 3.43 (1H, dd, J 9.3, 9.1, H-5), 3.32 (1H, dd, J 9.7, 2.4, H-3)
and 2.71 (1H, br s, OH-4); 13C NMR (100 MHz; CDCl3) d 137.6,
137.5, 136.8, 128.6, 128.6, 128.5, 128.1, 128.1, 128.0, 127.9, 127.9,
127.8, 96.6 (OCH2O0), 96.0 (OCH2O), 84.6 (C-6), 78.9 (C-3), 76.0 (C-
2), 74.0 (C-5), 72.4 (C-4), 72.2 (CH2Ph), 71.0 (C-1), 70.5 (CH2Ph)
and 70.0 (CH2Ph); HRMS m/z (ES+) [Found: (M+Na)+ 533.2159.
C29H34O8SiNa requires M+, 533.2151]; m/z (ES+) 533 ([M+Na]+,
100%). The data are in good agreement with the literature values.28
olised, providing (ꢀ)-
D-myo-inositol 1,4,5-trisphosphate hexakis
sodium salt 15 (40.5 mg, 95%) as a colourless solid:); ½a D25
¼
ꢁ
ꢀ19:2 (c 0.25, H2O, pH ꢃ9) {lit.51
½
a 2D4
ꢁ
¼ ꢀ20 (c 0.05, H2O, pH
9)}; 1H NMR (300 MHz; D2O) d 4.21–4.15 (1H, m, H-2), 4.16–4.06
(1H, m, inositol ring), 3.92–3.75 (3H, m, inositol ring) and 3.59
(1H, dd, J 9.8, 2.7, H-1); 31P NMR (121 MHz; D2O) d 3.82, 3.03
and 2.33; HRMS m/z (ES+) [Found: (M-Na+2H)+ 530.8791.
C6H11O15P3Na5 requires M+, 530.8794]; m/z (ES-) 485 ([M-
3Na+2H]-, 12%), 463 (40), 441 (63), 419 (52) and 339 (73). The data
are in agreement with the literature values.28 It should be noted
that there is a large variation in the [a] values quoted for InsP3
D
(ꢀ3.2 to ꢀ30) in the literature.
4.16. (ꢀ)-
D-3-O-Benzyl-2,6-O-bisbenzyloxymethyl-5-O-(4-meth-
oxybenzyl)-myo-inositol 16
4.14. (ꢀ)-
D-3-O-Benzyl-2,6-O-bisbenzyloxymethyl-myo-inositol
1,4,5-tris(dibenzyl phosphate) 14
To a solution of (ꢀ)-D-1-O-acetyl-3-O-benzyl-2,6-O-bisbenzyl-
oxymethyl-5-O-(4-methoxybenzyl)-4-O-triisopropylsilyl-myo-ino-
sitol 11 (234 mg, 0.30 mmol) in THF (5 mL) was added a solution of
tetrabutylammonium fluoride (1.0 M, 5.95 mL) in tetrahydrofuran.
The solution was stirred at rt overnight, after which time TLC anal-
ysis showed consumption of the starting material. The reaction
solution was diluted with water (20 mL), then extracted with
diethyl ether (30 mL). The aqueous phase was re-extracted with
CH2Cl2 (2 ꢂ 30 mL). The combined organic phases were washed
with brine (20 mL), dried over magnesium sulfate, filtered and con-
centrated in vacuo, providing a colourless solid. Purification by sil-
ica gel chromatography, eluting with hexane and ethyl acetate
To a solution of bis(benzyloxy)-N,N-bisisopropylamine phos-
phine (2.30 g, 6.65 mmol) in CH2Cl2 (100 mL) was added a solution
of 1H-tetrazole (0.43 M in acetonitrile, 15.5 mL, 6.65 mmol). The
mixture was stirred at rt for 20 min, before a solution of (+)-D-3-
O-benzyl-2,6-O-bisbenzyloxymethyl-myo-inositol 13 (566 mg,
0.04 mmol) in CH2Cl2 (50 mL) was added dropwise via a pressure
equalised dropping funnel over a period of 1 h. The resulting
solution was stirred overnight at rt, after which time TLC analysis
indicated the consumption of the starting material. The solution
was cooled to ꢀ78 °C and 3-chloroperoxybenzoic acid (1.49 g,
6.65 mmol) was added in a single portion. The mixture was al-
lowed to warm to rt and stirred for a further 3 h, after which TLC
indicated consumption of the presumed intermediate (PIII species).
The reaction was quenched by the addition of aqueous sodium sul-
fite solution (10% w/v, 100 mL). The layers were separated and the
aqueous phase was re-extracted with CH2Cl2 (3 ꢂ 100 mL). The
combined organic layers were washed with brine (100 mL), dried
over magnesium sulfate, filtered then concentrated in vacuo, pro-
viding a light brown oil. Purification of the oil by silica gel chroma-
tography, eluting with hexane and ethyl acetate (50:50) provided
(70:30, 50:50), provided (ꢀ)-
D-3-O-benzyl-2,6-O-bisbenzyloxymeth-
yl-5-O-(4-methoxybenzyl)-myo-inositol 16 (167 mg, 89%) as a col-
ourless solid: Rf 0.13 (hexane/ethyl acetate 70:30); ½a D20
¼ ꢀ16:9
ꢁ
(c 0.54, CHCl3); mp 105–106 °C (from hexane/ethyl acetate); mmax
(thin film)/cmꢀ1 3465 (s), 2892 (w), 1611 (m), 1514 (s), 1498 (m),
1454 (m), 1370 (m), 1251 (s), 1175 (s), 1065 (s), 1029 (s), 818
(w), 735 (s), 700 (m) and 586 (w); 1H NMR (400 MHz; CDCl3) d
7.38–7.26 (17H, m, Ar H), 6.91–6.87 (2H, m, H-3,5 ArOCH3 ring),
5.00–4.97 (2H, m, overlapping 2 ꢂ CHAHB), 4.91 (1H, d, J 6.6,
CHAHBPh), 4.85–4.58 (9H, m, multiple CHAHB), 4.28 (1H, dd, J 2.5,
2.5, H-2), 4.10 (1H, dd, J 9.7, 9.3, H-4), 3.85 (1H, dd, J 9.6, 9.3, H-
6), 3.81 (3H, s, OCH3), 3.78 (1H, d, J 4.7, OH-1), 3.29 (1H, ddd, J
9.6, 4.7, 2.4, H-1), 3.37 (1H, dd, J 9.3, 9.3, H-5), 3.31 (1H, dd, J 9.8,
2.5, H-3) and 2.55 (1H, br s, OH-4); 13C NMR (100 MHz; CDCl3) d
159.2, 137.7, 137.6, 137.3, 130.9, 129.5, 128.6, 128.5, 128.4, 128.0,
128.0, 127.9, 127.7, 113.9 (C-3,5 ArOCH3 ring), 96.7 (OCH2O), 95.7
(OCH2O’), 82.9 (C-6), 82.4 (C-5), 79.2 (C-3), 75.2 (C-2), 75.0 (CH2),
73.0 (C-4), 72.1 (CH2), 71.5 (C-1), 70.2 (CH2), 69.7 (CH2) and 55.3
(OCH3); HRMS m/z (ES+) [Found: (M+Na)+ 653.2723. C37H42O9Na
requires M+, 653.2727]; m/z (ES+) 653 ([M+Na]+, 100%); Anal. Calcd
for C37H42O9: C, 70.46; H, 6.71. Found: C, 70.58; H, 6.62.
(ꢀ)-
D-3-O-benzyl-2,6-O-bisbenzyloxymethyl-myo-inositol 1,4,5-
tris(dibenzyl phosphate) 14 (1.32 g, 93%) as a colourless oil: Rf
0.13 (hexane/ethyl acetate 1:1); ½a D20
¼ ꢀ18:1 (c 1.00, CHCl3)
ꢁ
{lit.28
½
a 2D4
ꢁ
¼ ꢀ17:6 (c 0.85, CHCl3)}; 1H NMR (300 MHz; CDCl3) d
7.32–7.14 (45H, m, Ar H), 5.16–4.78 (17H, m, multiple CH2), 4.71
(1H, d, J 12.1, CHAHBPh), 4.65 (2H, s, CH2Ph), 4.56–4.48 (4H, m,
2 ꢂ ring H and CH2), 4.43 (1H, dd, J 18.8, 9.4, ring H), 4.30 (1H,
dd, J 9.5, 9.5, ring H), 4.17–4.10 (1H, m, H-5) and 3.37 (1H, dd, J
9.9, 2.1, H-3); 31P NMR (121 MHz; CDCl3) d ꢀ1.12, ꢀ1.23 and
ꢀ1.66; m/z (ES+) 1313 ([M+Na]+, 100%). The data are in good agree-
ment with the literature values.28
4.15. (ꢀ)-
D
-myo-Inositol 1,4,5-trisphosphate hexakis sodium
4.17. 45Ca2+-flux assay in permeabilised Lvec and L15 fibroblasts
salt 15
These experiments were performed as described in Kasri et al.,
2004.52 Briefly, Lvec cells were seeded in 12-well clusters (Greiner)
at a density of 40,000 cells per well, whereas L15 cells were seeded
at a density of 60,000 cells per well. Experiments were carried out
on confluent cell monolayers between the 6th and 8th day after
seeding. Cells were incubated for 10 min with a solution containing
120 mM KCl, 30 mM imidazole–HCl (pH 6.8), 2 mM MgCl2, 1 mM
To a solution of (ꢀ)-
D-3-O-benzyl-2,6-O-bisbenzyloxymethyl-
myo-inositol 1,4,5-tris(dibenzyl phosphate) 14 (120.0 mg,
0.093 mmol) in a mixture of t-butanol (6 mL) and water (1 mL)
were added palladium black (198 mg, 1.859 mmol) and sodium
hydrogen carbonate (46.8 mg, 0.558 mmol). The mixture was
placed under vacuum and the atmosphere was replaced first with

866
N. S. Keddie et al. / Tetrahedron: Asymmetry 20 (2009) 857–866
ATP, 1 mM EGTA and 40 lg/mL saponin at 30 °C. The non-mito-
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Acknowledgements
The authors are grateful to the BBSRC for funding and the
EPSRC mass spectrometry service at Swansea for the provision
of some mass spectrometry data. We thank to the NMR service
at the University of St Andrews for assistance. The authors are
grateful to Marina Crabbe, Anja Florizoone and Wendy Janssens
for technical assistance. They thank Professor Colin Taylor and
Dr. Stephen Tovey for their help in establishing the Flexstation
Ca2+ assays. Work in the laboratory of GB was supported by
GOA 2004/07 and Grant P6/28 of the Belgian State (Belgian
Science Policy).
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