Synthesis of the enantiomers of myo-inositol 1,2,4,5-tetrakisphosphate, a regioisomer of myo-inositol 1,3,4,5-tetrakisphosphate

Article abstract of DOI:10.1039/a608337d

Routes for the synthesis of racemic myo-inositol 1,2,4,5-tetrakisphosphate DL-Ins(1,2,4,5)P₄ 5ab and the chiral antipodes D- and L-myo-inositol 1,2,4,5-tetrakisphosphate 5a and 5b, respectively, are described. For the synthesis of racemate 5ab, 3,6-di-O-benzoyl-1,2:4,5-di-O-isopropylidene-myo-inositol 7ab is prepared in two steps from myo-inositol. The ketals are hydrolysed under acidic conditions to give DL-1,4-di-O-benzoyl-myo-inositol 8ab. Phosphitylation of compounds 8ab using chloro(diethoxy)-phosphine in the presence of base, followed by oxidation and a three-step deprotection strategy, gives DL-Ins(1,2,4,5)P₄ 5ab. The chiral tetrakisphosphates 5a and 5b are synthesized using a different route. The 4,5-isopropylidene group of DL-3,6-di-O-benzyl-1,2:4,5-di-O-isopropylidene-myo-inositol 13ab are selectively removed under mild acidic conditions to give diol 14ab. p-Methoxybenzylation at the 4,5-positions followed by acid hydrolysis of the cis-isopropylidene ketal affords cis-diol 16ab. Selective coupling of (S)-(+)-O-acetylmandelic acid with diol 16ab at the equatorial hydroxy group provides two diastereoisomers 18 and 19, which are separated by chromatography. Basic hydrolysis of the individual diastereoisomers provides the enantiomers 16a and 16b. Acidic hydrolysis gives D- and L-3,6-di-O-benzyl-myo-inositol 20a and 20b, respectively. Phosphitylation and oxidation of tetraols 20a and 20b gives the fully blocked derivatives, which are deprotected to give tetrakisphosphates 5a and 5b, respectively. The absolute configuration of compound 20a is established by a chemical method. DL-1,2:4,5-Di-O-isopropylidene-myo-inositol 12ab is coupled to (S)-(+)-O-acetylmandelic acid to give a mixture of bis-esters 26 and 27 and crystallisation of the mixture of diastereoisomers affords pure isomer 27. Basic hydrolysis gives the pure enantiomer 12a (for which the absolute configuration is known) and benzylation followed by acid hydrolysis gives tetraol 20a with the same physical properties as compound 20a prepared by a different route described previously. D-Ins(1,2,4,5)P₄ 5a is a potent mobiliser of intracellular Ca²⁺ ions in permeabilised platelets, while L-Ins(1,2,4,5)P₄ 5b is inactive.

Full text of DOI:10.1039/a608337d

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Synthesis of the enantiomers of myo-inositol 1,2,4,5-tetrakis-
phosphate, a regioisomer of myo-inositol 1,3,4,5-tetrakisphosphate
Stephen J. Mills and Barry V. L. Potter *
Department of Medicinal Chemistry, School of Pharmacy and Pharmacology,
University of Bath, Claverton Down, Bath, BA2 7AY, UK
Routes for the synthesis of racemic myo-inositol 1,2,4,5-tetrakisphosphate D L-Ins(1,2,4,5)P 4 5ab and the
chiral antipodes D - and L-myo-inositol 1,2,4,5-tetrakisphosphate 5a and 5b, respectively, are described.
For the synthesis of racemate 5ab, 3,6-di-O-benzoyl-1,2:4,5-di-O-isopropylidene-myo-inositol 7ab is
prepared in two steps from myo-inositol. The ketals are hydrolysed under acidic conditions to give
D L-1,4-di-O-benzoyl-myo-inositol 8ab. Phosphitylation of compounds 8ab using chloro(diethoxy)-
phosphine in the presence of base, followed by oxidation and a three-step deprotection strategy, gives
D L-Ins(1,2,4,5)P 4 5ab.
The chiral tetrakisphosphates 5a and 5b are synthesized using a different route. The 4,5-isopropylidene
group of D L-3,6-di-O-benzyl-1,2:4,5-di-O-isopropylidene-myo-inositol 13ab are selectively removed under
mild acidic conditions to give diol 14ab. p-M ethoxybenzylation at the 4,5-positions followed by acid
hydrolysis of the cis-isopropylidene ketal affords cis-diol 16ab. Selective coupling of (S)-(؉)-O-acetyl-
mandelic acid with diol 16ab at the equatorial hydroxy group provides two diastereoisomers 18 and 19,
which are separated by chromatography. Basic hydrolysis of the individual diastereoisomers provides the
enantiomers 16a and 16b. Acidic hydrolysis gives D - and L-3,6-di-O-benzyl-myo-inositol 20a and 20b,
respectively. Phosphitylation and oxidation of tetraols 20a and 20b gives the fully blocked derivatives,
which are deprotected to give tetrakisphosphates 5a and 5b, respectively. The absolute configuration of
compound 20a is established by a chemical method. D L-1,2:4,5-D i-O-isopropylidene-myo-inositol 12ab is
coupled to (S)-(؉)-O-acetylmandelic acid to give a mixture of bis-esters 26 and 27 and crystallisation of
the mixture of diastereoisomers affords pure isomer 27. Basic hydrolysis gives the pure enantiomer 12a (for
which the absolute configuration is known) and benzylation followed by acid hydrolysis gives tetraol 20a
with the same physical properties as compound 20a prepared by a different route described previously.
D -Ins(1,2,4,5)P 4 5a is a potent mobiliser of intracellular Ca2؉ ions in permeabilised platelets, while
L-Ins(1,2,4,5)P 4 5b is inactive.
GTPase activating protein-1 (GAP-1) family member. The
Introduction
GTPase activating protein-1 site has been designated
GAP1IP4BP. When Ins(1,3,4,5)P4 binds at this site it may pos-
sibly have a second messenger function in its own right. The
synthesis of regioisomeric inositol tetrakisphosphates is there-
fore of clear current interest.
The involvement of myo-inositol polyphosphates in signal
transduction via the polyphosphoinositide pathway has stimu-
lated the need for the synthesis of molecules that will somehow
interfere with, or modulate, the processes of cellular signalling,1
whether it be at phospholipase C, the intracellular D-myo-
inositol 1,4,5-trisphosphate receptor, or even further down-
stream, where the second messenger D-myo-inositol 1,4,5-
trisphosphate [Ins(1,4,5)P3, 1] is deactivated and the signal is
terminated. The process of signal transduction via Ins(1,4,5)P3
starts when a cell-surface receptor activates the enzyme phos-
pholipase C-β via a G-protein. This enzyme hydrolyses the
minor membrane phospholipid, phosphatidylinositol 4,5-
bisphosphate, to provide the hydrophobic diacylglycerol and
hydrophilic Ins(1,4,5)P3 as signalling molecules. Ins(1,4,5)P3
interacts specifically at an N-terminal binding site of a tetra-
meric Ins(1,4,5)P3 receptor-operated Ca2ϩ channel, in order to
release Ca2ϩ from non-mitochondrial stores.1 After the Ca2ϩ
-
release event, the signal must be deactivated by one or more
metabolic pathways. First, an Ins(1,4,5)P3 5-phosphatase
removes a 5-phosphate moiety from Ins(1,4,5)P3 to give D-myo-
inositol 1,4-bisphosphate Ins(1,4)P2 2 which is inactive for Ca2ϩ
release, but has been reported to be an allosteric activator of the
enzyme 6-phosphofructo-1-kinase,2 and also activates the
enzyme DNA polymerase α.3 Second, Ins(1,4,5)P3 can also
be phosphorylated to give D-myo-inositol 1,3,4,5-tetrakisphos-
phate [Ins(1,3,4,5)P4, 3], and the production of Ins(1,4)P2 and
Ins(1,3,4,5)P4 is considered as an off signal. The function of
Ins(1,3,4,5)P4 has not been unambiguously resolved; however,
it may gate a plasma membrane Ca2ϩ channel.4 An Ins(1,3,4,5)P4
binding protein has been purified from platelets5 and is a
DL-myo-Inositol 2,4,5-trisphosphate [Ins(2,4,5)P3, 4ab] is an
unnatural trisphosphate, which has a potency some 30-fold
6,7
lower than Ins(1,4,5)P3 but has been used as a metabolic
J. Chem. Soc., Perkin Trans. 1, 1997
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resistant analogue6 of Ins(1,4,5)P3, since it is a weak substrate
for Ins(1,4,5)P3 5-phosphatase and a poor substrate for
Ins(1,4,5)P3 3-kinase.6,8 Recently, Bird and Putney, Jr,9 micro-
injected Ins(2,4,5)P3 into mouse lacrimal acinar cells and it
stimulated intracellular Ca2ϩ mobilisation and Ca2ϩ entry.
However, microinjection of purified D-Ins(1,3,4,5)P4 into these
cells was ineffective at Ca2ϩ mobilisation or activation of Ca2ϩ
entry. Thus, the introduction of high concentrations (final cellu-
lar concentration 100–200 µM) of Ins(1,3,4,5)P4 somehow
blocked the Ins(2,4,5)P3 Ca2ϩ entry phase. These results in-
dicated that physiological concentrations of Ins(1,3,4,5)P4 in
this cell type do not cause Ca2ϩ mobilisation 10–12 nor do they
potentiate Ins(1,4,5)P3 induced Ca2ϩ entry.
Since it was unclear whether substitution at the 2-hydroxy
group or the lack of a phosphate at the 1-hydroxy moiety was
responsible for the properties described above, we synthesized
the unnatural tetrakisphosphate myo-inositol 1,2,4,5-tetrakis-
phosphate, DL-Ins(1,2,4,5)P4 5ab, first in racemic form and then
as the individual enantioners D-Ins(1,2,4,5)P4 5a and L-Ins-
(1,2,4,5)P4 5b. D-Ins(1,2,4,5)P4 5a could be considered as a
relative of Ins(1,4,5)P3 but with a charged phosphate at the
2-position (several articles have focused upon substitution at
the 2-position with neutral bulky groups).1,7,13 This analogue
can also be related to Ins(1,3,4,5)P4, but with a 3-phosphate
being transposed onto the adjacent 2-hydroxy moiety. These
compounds were synthesized in order to evaluate structure–
activity profiles with respect to the Ins(1,4,5)P3 and Ins-
(1,3,4,5)P4 binding proteins and the enzymes Ins(1,4,5)P3 3-
kinase and Ins(1,4,5)P3 5-phosphatase, the latter of which also
hydrolyses Ins(1,3,4,5)P4.
Scheme 1 Reagents and conditions: i, 2,2-dimethoxypropane, PTSA
Ins(1,2,4,5)P4 has previously been synthesized in racemic14
and chiral15 form and a preliminary report of the present work
concerning the racemic modification has appeared.14a However,
only D-Ins(1,2,4,5)P4 5a has been reported in chiral form. We
now report here a useful route to the synthesis of both enan-
tiomers of Ins(1,2,4,5)P4 (5a and 5b) by resolution of partially
blocked myo-inositol derivatives, using the chiral auxiliary (S)-
(ϩ)-O-acetylmandelic acid 17.
(cat), DMF, 100–120 ЊC, 2 h; then pyridine, benzoyl chloride, 2 h (30%);
ii, 80% (aq.) AcOH, reflux, 30 min (93%); iii, (EtO)2PCl, DIPE, DMF, 1
h; then 70% tert-butyl hydroperoxide (83%); iv, TMSBr, CH2Cl2, room
temp. overnight then water, and final purification by Q-Sepharose Fast
Flow ion-exchange chromatography (81%); v, 1 mol dmϪ3 NaOH,
60 ЊC, 1 h; then purification by Q-Sepharose Fast Flow ion exchange
chromatography (80%). All compounds are racemic.
The enantiomers of Ins(1,2,4,5)P4 were synthesized by a dif-
ferent route from that for the racemic mixture. Previously,14a we
demonstrated that DL-Ins(1,2,4,5)P4 5ab competitively inhibited
the dephosphorylation of [3H]Ins(1,4,5)P3 by human erythro-
cyte membrane Ins(1,4,5)P3 5-phosphatase with a Ki-value of
15.9 µM. However, either isomer could potentially inhibit the
enzyme. The reasons for synthesizing the title compounds were,
first, to establish which isomer was responsible for the inhib-
ition of the 5-phosphatase, and second, to discover the true
EC50-value for Ca2ϩ release of D-Ins(1,2,4,5)P4 5a in com-
parison with those of Ins(1,4,5)P3 and scyllo-Ins(1,2,4,5)P4. It
is also known that L-Ins(2,4,5)P3 can release Ca2ϩ from intra-
cellular stores, albeit with a low potency (EC50-value of 110 µM)
and thus it would be interesting to discover if L-Ins(1,2,4,5)P4
5b made some contribution to the Ca2ϩ-releasing properties
observed for DL-Ins(1,2,4,5)P4.
strengths may not show the following effect due to chemical-
shift anisotropy), showed eight peaks resulting from two
5JPP AB coupling systems18 centred around δP = 141.8 and
141.3 (for the 4,5-positions) and δP = 140.4 and 139.8 (for the
1,2-positions), for each doublet of the AB coupling pattern
demonstrating phosphitylation of a pair of vicinal diols at
the 1,2-positions (5JPP 1.8 Hz) and for the 4,5-positions (5JPP
3.7 Hz). Oxidation of phosphites 9ab provided crystalline
DL-3,6-di-O-benzoyl-1,2,4,5-tetrakis-O-(diethoxyphosphoryl)-
myo-inositol 10ab. The eight ethyl groups of compound 10ab
were replaced by transesterification quantitatively (checked by
31P NMR spectroscopy) using bromotrimethylsilane in methyl-
ene dichloride. Hydrolysis with water gave DL-3,6-di-O-benzoyl-
Ins(1,2,4,5)P4 11ab quantitatively. A small sample was purified
by ion-exchange chromatography using a gradient of triethyl-
ammonium hydrogen carbonate (TEAB) on Q-Sepharose Fast
Flow to give pure tetraol 11ab. DL-Ins(1,2,4,5)P4 5ab was
prepared by basic hydrolysis of the two benzoate esters. Pure
DL-Ins(1,2,4,5)P4 5ab was obtained as the triethylammonium
salt after ion exchange chromatography and eluted at ca. 550
mmol dmϪ3 TEAB buffer and was quantified by phosphate
analysis.
Racemic 1,4-di-O-benzyl-5,6-bis-O-(p-methoxybenzyl)-myo-
inositol 16ab was prepared according to Scheme 2 and has also
been synthesized by another group.19 Basic methanolysis of the
two benzoyl esters of compound 7ab gave DL-1,2:4,5-di-O-
isopropylidene-myo-inositol 12ab. Benzylation of diol 12ab
with benzyl bromide provided fully blocked DL-3,6-di-O-
benzyl-1,2:4,5-di-O-isopropylidene-myo-inositol 13ab. The less
stable trans-acetal was removed selectively using a catalytic
quantity of PTSA and ethane-1,2-diol to provide DL-1,4-di-O-
benzyl-2,3-O-isopropylidene-myo-inositol 14ab in 80% yield.
Results and discussion
DL-3,6-Di-O-benzoyl-1,2:4,5-di-O-isopropylidene-myo-inositol
7ab (Scheme 1) was prepared using the method developed by
Gigg et al.16 A mixture of myo-inositol 6, 2,2-dimethoxy-
propane and toluene-p-sulfonic acid (PTSA) was stirred at
100 ЊC in N,N-dimethylformamide (DMF). After benzoylation
the highly insoluble product 7ab was suspended in aq. acetic
acid and the mixture was heated under reflux to give DL-1,4-di-
O-benzoyl-myo-inositol 8ab. This tetraol has also been syn-
thesized by Meek et al.17
Commerically available chloro(diethoxy)phosphine (δP 167)
was used to phosphitylate tetraol 8ab. The 31P NMR spectrum
of the intermediate 1,2,4,5-tetrakisphosphite 9ab, operating at
36.2 MHz with a sweep width of 2500 KHz (higher field
1280
J. Chem. Soc., Perkin Trans. 1, 1997

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This compound has also been prepared from 13ab by Gigg et
al.20 under different acidic conditions in only 55% yield. Diol
14ab was alkylated with p-methoxybenzyl chloride to provide
fully blocked product 15ab. The cis isopropylidene group was
removed by careful acid treatment to give diol 16ab. Caution
must be taken at this stage, to avoid hydrolysis of the p-
methoxybenzyl group. It was envisaged that the introduction of
a chiral auxiliary at the equatorial position of the cis diol 16ab
would result in the formation of two separable diastereo-
isomers.
blocked myo-inositol derivatives for the synthesis of D- and
L-Ins(1,4,6)P4.23
Coupling of DL-1,4-di-O-benzyl-5,6-bis-O-(p-methoxybenz-
yl)-myo-inositol 16ab with (S)-(ϩ)-O-acetylmandelic acid 17
at low temperature afforded the two diastereoisomers 18 and
19 (structure established retrospectively, after the determination
of the chirality of derived tetraols 20a and 20b respectively, vide
infra). By keeping the temperature at Ϫ20 ЊC, selectivity was
achieved and there was no acylation at the 2-hydroxy position
1
(by H NMR analysis); isomers 18 and 19 were separated by
flash chromatography and were obtained as crystalline solids.
The proton 1-H (shifted downfield due to esterification of
1-OH) could not be identified in either diastereoisomer (18 and
19) because the methylene AB coupling pattern of the benzyl
group overlapped with the expected dd for 1-H. However, 2-H
was identified as a broad doublet at δ 4.15 (J 1.8 Hz) for com-
pound 18 and as a broad doublet (J 1.8 Hz) at δ 4.40 for com-
pound 19. The 2-OH signal was also significant because it was
seen at δ 2.16 for compound 18 and at δ 2.69 for compound 19,
which indicated that the proton and hydroxy groups attached to
C-2 were more deshielded than for the less polar diastereo-
isomer 18. The unique singlet at δ 5.94 for isomer 18, and at δ
5.98 for isomer 19, of CH3CO2CH(Ph)CO2Ins is indicative of
the high purity of each diastereoisomer and no other impurities
were detected in either the 1H or the 13C NMR spectra.
Deacylation of isomers 18 and 19 under basic conditions gave
the pure enantiomers 16a and 16b and the optical rotations of
products 16a and 16b were equal and opposite. The two chiral
1,2,4,5-tetraols were prepared by acid hydrolysis of the p-
methoxybenzyl ethers in aq. HCl at reflux temperature. The
resulting solid was filtered off, and recrystallised from ethanol
to give the pure enantiomers D- 20a and L-3,6-di-O-benzyl-myo-
inositol 20b which had specific rotations of ϩ16 and Ϫ16 ×
10Ϫ1 deg cm2 gϪ1, respectively. The mp of the racemic mixture
(lit.,16 205–207 ЊC) was considerably higher than for the chiral
antipodes (172–173 ЊC).
The absolute configuration of antipodes 20a and 20b was
determined by a chemical method. A simple way to establish
the absolute configuration of D-3,6-di-O-benzyl-myo-inositol
20a would be to resolve DL-1,2:4,5-di-O-isopropylidene-myo-
inositol 12ab, followed by benzylation and acidic hydrolysis of
the isopropylidene groups to give the individual enantiomers
20a and 20b. The resolution of DL-1,2:4,5-di-O-isopropyl-
idene-myo-inositol has been previously accomplished using the
chiral auxiliary (S)-(Ϫ)-ω-camphanoyl chloride.24 In this reso-
lution, the 3-position was blocked by using tert-butyldiphenyl-
silyl chloride, the 6-position was acylated with the chiral auxil-
iary, and separation of the diastereoisomers was achieved by
tedious HPLC. Therefore, a simple resolution to provide the
chiral diol would be appropriate, because single-crystal X-ray
analysis of the 6-O-camphanate has been determined, and
derived from this the specific rotation, [α]D ϩ22, and mp (159–
161 ЊC) for L-1,2:4,5-di-O-isopropylidene-myo-inositol has
been established.24 Moreover, in a recent article, D-1,2:4,5-di-O-
isopropylidene-myo-inositol was synthesized from D-mannitol,
however, the mp was found to be 176–177 ЊC with a specific
rotation of [α]D Ϫ21.7.25 Thus, the specific rotation is in
agreement for both enantiomers, but there appears to be some
discrepancy over the mp
DL-1,2:4,5-Di-O-isopropylidene-myo-inositol 12ab was acyl-
ated with (S)-(ϩ)-O-acetylmandelic acid 17 in the presence of
a coupling reagent to afford a mixture of diastereoisomers
(26 and 27) which could not be separated by chromatography
(see later, Scheme 4). The mixture of diastereoisomers was
recrystallised from hot methanol and one compound, 27, was in
abundance by a factor of 2.5 by 1H NMR spectroscopy; further
recrystallisation from the same solvent gave the pure diastereo-
isomer 27 in 18% yield (unoptimised). Basic methanolysis of
the two acyl groups followed by chromatography and recrystal-
lisation of the diol from ethyl acetate gave D-1,2:4,5-di-O-
Scheme 2 Reagents and conditions: i, NaOH, MeOH, reflux, 30 min
(82%); ii, BnBr, NaH, DMF, 2 h; room temp. (91%); iii, ethane-1,2-diol,
methylene dichloride, PTSA (cat), rt (80%); iv, p-methoxybenzyl chlor-
ide, NaH, DMF, rt, 2 h (80%); v, MeOH–1 mol dmϪ3 HCl (aq), (9:1),
50 ЊC, 30 min (90%); vi, DMAP, DCC, methylene dichloride, Ϫ20 ЊC,
(36% for 18; 37% for 19); vii, NaOH, MeOH, reflux, 30 min (99% for
16a, 91% for 16b); viii, EtOH–1 mol dmϪ3 HCl (aq), (2:1), reflux, 4 h
(86% for 20a, 83% for 20b)
(S)-(ϩ)-O-Acetylmandelic acid 17 was chosen for resolution
of the cis-diol because it is relatively inexpensive and is 99%
pure by GLC, and unlike the enantiomers of the commonly
used camphanic acid chloride, both R and S isomers are
cheaply available. (S)-(ϩ)-O-Acetylmandelic acid has not
been widely used for the resolution of myo-inositol derivatives,
so we took the opportunity to investigate its potential as a
resolving reagent. Previously, it was used successfully to resolve
several blocked myo-inositol derivatives. Two diastereoisomers
were derived, by coupling (S)-(ϩ)-O-acetylmandelic acid to
DL-1,4,5,6-tetra-O-benzyl-myo-inositol21 at the equatorial 1-
hydroxy position and were easily separated by flash chrom-
atography and used to synthesize previously inaccessible hex-
oses21 and the β-glucosidase and α-mannosidase inhibitors (ϩ)-
and (Ϫ)-norjirimycin.22 We have recently employed (S)-(ϩ)-O-
acetylmandelic acid as a chiral auxiliary to resolve partially
J. Chem. Soc., Perkin Trans. 1, 1997
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isopropylidene-myo-inositol 12a, [α]D Ϫ22, with a mp of 174–
176 ЊC. These physical properties agreed with the data pub-
lished by Chiara and Martín-Lomas.25 Gigg and co-workers26
have synthesized L-1,2:4,5-di-O-isopropylidene-myo-inositol
by a different route, [α]D ϩ23.3, (mp 175–177 ЊC). The dispute
over the mp of the chiral diol has now been resolved because
the value (159–161 ЊC) stated by Young and co-workers24
appeared to be a little low. Compound 12a was then benzylated
to give D-3,6-di-O-benzyl-1,2:4,5-di-O-isopropylidene-myo-
inositol 13a, ([α]D Ϫ44, mp 157–159 ЊC). Recently, Gigg and
co-workers26 synthesized L-3,6-di-O-benzyl-1,2:4,5-di-O-iso-
propylidene-myo-inositol 13b, [α]D ϩ85, which had a mp of
159–161 ЊC. The acetal protecting groups at the 1,2- and 4,5-
positions were removed by acid hydrolysis and the solvents were
evaporated off in vacuo. The resulting solid was recrystallised
from ethanol to give D-3,6-di-O-benzyl-myo-inositol 20a,
which was identical (1H NMR, mp, specific rotation) with the
previously described compound.
yields. Use of m-chloroperbenzoic acid (MCPBA) is preferable
to tert-butyl hydroperoxide since the latter gives lower yields
resulting from the formation of polar by-products. All the
benzyl protective groups were removed from the fully blocked
compound in one step by using sodium in liquid ammonia.28
Purification of crude D-Ins(1,2,4,5)P4 5a and L-Ins(1,2,4,5)P4
5b was carried out by ion-exchange chromatography on Q-
Sepharose Fast Flow and both compounds were eluted at ~700
mmol dmϪ3 TEAB buffer and were isolated as their triethyl-
ammonium salts and quantified by phosphate analysis.
Scheme 4 Reagents and conditions: i, DCC, DMAP, methylene di-
chloride, 0 ЊC (18% for 27); ii, NaOH, MeOH, reflux, 30 min (86%); iii,
NaH, BnBr, DMF, 2 h (91%); iv, 1 mol dmϪ3 HCl–MeOH (1:9), reflux,
30 min (95%)
Full experimental data for DL-Ins(1,2,4,5)P4 5ab have been
published for Ca2ϩ release29 and its interaction with the enzymes
Ins(1,4,5)P3 3-kinase and 5-phosphatase.30 The full data for
antipodes 5a and 5b will be published elsewhere. However,
notably L-Ins(1,2,4,5)P4 5b was not found to release intra-
cellular Ca2ϩ and D-Ins(1,2,4,5)P4 5a was only ~2-fold less
potent than was Ins(1,4,5)P3 at Ca2ϩ release in rabbit platelets.
Scheme 3 Reagents and conditions: i, 1H-tetrazole 22, methylene
dichloride, reagent 21, room temp., 15 min, add tetraols 20a and 20b in
separate experiments, 10 min; ii, MCPBA, 0 ЊC, 30 min (94% for 25a,
88% for 25b); iii, Na/liquid NH3, then purification by ion-exchange
chromatography
Experimental
TLC was performed on pre-coated plates (Merck TLC alu-
minium sheets silica 60 F254, Art. no. 5554): the product was
visualised by spraying with methanolic phosphomolybdic acid,
followed by heating. Flash chromatography refers to the pro-
cedure developed by Still et al.31 and was carried out on Sorbsil
C60 silica gel.
A PIII approach was adopted to introduce the phosphate sub-
stituents, as shown in Scheme 3. Thus, phosphitylating agent 27
21 (2 mole equivalents per hydroxy group) and 1H-tetrazole 22
(4 mole equivalents per hydroxy group) reacted to form the
tetrazolide intermediate 23 (δP ϩ126.73 ppm; cf. δP ϩ147.86
ppm for compound 21). Any moisture present in the solvent is
indicated by the formation of H-phosphonate (δP ϩ7.54 ppm).
The procedure was carried out for both enantiomers in the
same way. Thus, in separate experiments the enantiomers of
3,6-di-O-benzyl-myo-inositol 20a and 20b were allowed to react
with intermediate 23. The 31P NMR spectrum again showed
eight peaks and the distinctive five-bond 31P–31P spin–spin
NMR spectra for the nuclei 31P, 1H and 13C were recorded on
JEOL FX-90Q, GX270 and GX400 spectrometers. Chemical
shifts were measured in parts per million (ppm) relative to
tetramethylsilane (TMS), deuterium oxide (D2O) or [2H6]-
dimethyl sulfoxide ([2H6]DMSO). Samples recorded in D2O
were approximately pH 4–5. The 31P NMR shifts were meas-
ured in ppm relative to external 85% phosphoric acid. M.p.s
(uncorrected) were determined using a Reichert-Jung Thermo
Galen Kofler block. Microanalysis was carried out by the Uni-
versity of Bath microanalysis service. Low-resolution mass
spectra were recorded by the University of Bath Mass Spec-
trometry Service using ϩve and Ϫve Fast Atom Bombardment
5
coupling systems18 for isomers 24a and 24b (5J1,2 1.8 Hz; J4,5
3.7 Hz). Oxidation of the tetrakisphosphite intermediates
afforded the fully protected D 25a and L-3,6-di-O-benzyl-1,2,4,5-
tetrakis-O-(dibenzyloxyphosphoryl)-myo-inositol 25b in high
1282
J. Chem. Soc., Perkin Trans. 1, 1997

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(FAB) with 3-nitrobenzyl alcohol (NBA) as the matrix. High-
resolution accurate mass spectrometry was carried out by
the EPSERC Mass Spectrometry Service in Swansea. Optical
rotations were measured using an Optical Activity Ltd. AA-10
polarimeter; [α]D -values are given in 10Ϫ1 deg cm2 gϪ1 and all
rotations were measured at ambient temperature.
Ion-exchange chromatography was performed on an LKB-
Pharmacia Medium-Pressure Ion Exchange Chromatograph
using Q-Sepharose and gradients of TEAB as eluent. Fractions
containing phosphate were assayed by a modification of the
Briggs phosphate test.32
myo-inositol 10ab (0.932 g, 0.1 mmol) in dry methylene
dichloride (5 cm3) was stirred at room temperature under nitro-
gen. Bromotrimethylsilane (0.264 cm3, 2 mmol) was added to
the dry solution, and the mixture was stirred overnight. The
solvents were evaporated off and the residue was stirred with
water (2 cm3) for 1 h. Final purification of one-third of the
compound was carried out by ion-exchange chromatography,
on Q-Sepharose Fast Flow, using a buffer gradient of TEAB
(200–1000 mmol dmϪ3) and flow rate 5 cm3 minϪ1. The fractions
which gave a positive Briggs test and eluted at ~500 mmol dmϪ3
buffer were pooled to give pure title compound 11ab (27 µmol,
81%), δH (270 MHz; D2O) 4.54–4.62 (2 H, m, 1- and 5-H), 4.80 (1
H, 4-H, obscured by HDO peak), 5.07 (1 H, d, J 10.25, 3-H),
5.23 (1 H, d, J 10.1, 2-H), 5.61 (1 H, t, J 9.9, 6-H), 7.47–7.66 [6
H, m, Ins-OC(O)Ph] and 8.09–8.16 [4 H, m, Ins-OC(O)Ph];
δC(68 MHz; D2O) 71.51, 72.39, 73.88, 74.95, 128.04, 128.56,
128.79, 128.98, 129.54, 129.70, 133.22, 167.52 and 167.74;
δP(162 MHz; D2O) Ϫ0.22 (d, J 9.8, CHOPO32Ϫ), Ϫ0.39 (d, J
10.7, CHOPO32Ϫ), Ϫ0.49 (d, J 11.9, CHOPO32Ϫ) and Ϫ0.79 (d,
J, 8.8, CHOPO32Ϫ); m/z (FABϪ) 707 [M Ϫ H (100%)], 460 (12),
387 (30), 232 (95), 177 (20), 159 (44) and 97 (30) [Found: m/z
706.9730. C20H24O20P4 requires (M Ϫ H)Ϫ, 706.9732].
Light petroleum refers to the fraction with distillation range
60–80 ЊC.
DL-1,4-Di-O-benzoyl-myo-inositol 8ab
DL-3,6-Di-O-benzoyl-1,2:4,5-di-O-isopropylidene-myo-inositol
7ab (9.36 g, 20 mmol) was suspended in 80% aq. acetic acid (200
cm3). The mixture was heated under reflux for 30 min, cooled,
and poured into an ice–water mixture (1000 cm3). The precipi-
tated solid was filtered off, washed thoroughly with diethyl
ether and recrystallised from DMF–water to give the title com-
pound 8ab (7.25 g, 93%), mp 253–254 ЊC (from DMF–water)
(Found: C, 61.9; H, 5.11. C20H20O8 requires C, 61.85; H,
5.15%); δH (270 MHz; [2H6]DMSO) 3.48 (1 H, dt, J 4.65 and 9.3,
5-H), 3.73 (1 H, ddd, J 2.25, 6.9 and 9.5, 3-H), 3.93 (1 H, dt, J
5.5 and 9.5, 6-H), 4.07 (1 H, br t, J 2.0, 2-H), 4.83 (1 H, dd, J 2.0
and 10.0, 1-H), 4.97 (1 H, d, J 6.6, D2O ex, OH), 5.25–5.33 (2 H,
m, D2O ex, OH), 5.35 (1 H, t, J 9.9, 4-H), 5.38 (1 H, d, J 4.2,
D2O ex, OH), 7.50–7.69 [6 H, m, OC(O)Ph] and 8.00–8.09 [4
H, m, OC(O)Ph]; δC(68 MHz; [2H6]DMSO) 69.83, 70.77, 70.93,
73.07, 75.70, 76.28, 129.34, 130.12, 130.25, 130.64, 131.13,
133.85, 134.14 and 166.58; m/z (FABϩ) 389 [M ϩ H (100%)],
306 (62), 274 (28), 243 (20), 199 (40) and 105 (88).
DL-myo-Inositol 1,2,4,5-tetrakisphosphate 5ab
Crude DL-3,6-di-O-benzoyl-myo-inositol 1,2,4,5-tetrakisphos-
phate 11ab (0.1 mmol) was heated with 1 mol dmϪ3 sodium
hydroxide (3 cm3) at 60 ЊC for 1 h. Dowex (Hϩ-form) was added
with water (30 cm3) until the pH was ~6. The Dowex was fil-
tered off, washed with water (2 × 20 cm3), and the benzoic acid
was removed by washing with methylene dichloride (2 × 30
cm3). The aqueous layer was then concentrated and the residue
was purified by ion-exchange chromatography, using a gradient
of TEAB (0–1000 mmol dmϪ3). The pure title compound 5ab
eluted at ~550 mmol dmϪ3 TEAB (80 µmol, 80%), δH (400 MHz;
D2O) 3.70 (1 H, d, J 10.1, 3-H), 3.90 (1 H, t, J 9.5, 6-H), 3.97–
4.04 (2 H, m, 1- and 5-H), 4.29 (1 H, q, J 9.2, 4-H) and 4.80 (1
H, 2-H, obscured by HDO peak); δC(68 MHz; D2O) 69.86,
70.86, 73.91, 74.88, 76.41 and 77.71; δP(162 MHz; D2O) ϩ1.31
(d, J 8.0, CHOPO32Ϫ), ϩ1.15 (d, J 7.9, CHOPO32Ϫ), ϩ1.04 (d, J
9.9, CHOPO32Ϫ) and Ϫ0.04 (d, J 6.0, CHOPO32Ϫ); m/z (FABϪ)
499 [M Ϫ H (100%)], 481 (5), 401 (5), 154 (10) and 97 (7)
[Found: m/z, 498.9210. C6H16O18P4 requires (M Ϫ H)Ϫ,
498.9210].
DL-3,6-Di-O-benzoyl-1,2,4,5-tetrakis-O-(diethoxyphosphoryl)-
myo-inositol 10ab
A mixture of DL-1,4-di-O-benzoyl-myo-inositol 8ab (0.776 g, 2
mmol), dry DMF (10 cm3) and dry N,N-diisopropylethylamine
(2.8 cm3, 16 mmol) was stirred under nitrogen at room temper-
ature. The solution was cooled in an ice-bath and chloro(di-
ethoxy)phosphine (2.32 cm3, 16 mmol) was added dropwise over
a period of 5 min and then the mixture was warmed to room
temperature. After being stirred for 1 h, 70% tert-butyl hydro-
peroxide, (3 cm3, 21.8 mmol) was added to the reaction mixture
at Ϫ78 ЊC to give the crude product, Rf (ethyl acetate) 0.20. The
solvents were evaporated off in vacuo and the remaining solid
was partitioned between water and methylene dichloride (50
cm3 of each). The organic layer was washed successively with
10% aq. sodium metabisulfite, brine (20 cm3 of each) and finally
water (2 × 20 cm3). The organic layer was dried over (MgSO4)
and evaporated off to give a solid. The crude product was puri-
fied over silica gel (ethyl acetate) and the solvent was evapor-
ated off to give the pure title compound 10ab (1.55 g, 83%), mp
122–123 ЊC (from ethyl acetate–hexane) (Found: C, 46.1; H,
6.03. C36H56O20P4 requires C, 46.35; H, 6.00%); δH (400 MHz;
CDCl3) 0.82–0.88 [9 H, m, OP(O)OCH2CH3], 1.20–1.33 [15 H,
m, OP(O)OCH2CH3], 3.52–3.85 [6 H, m, OP(O)OCH2CH3],
4.01–4.21 [10 H, m, OP(O)OCH2CH3], 4.79 (2 H, q, J 9.5, 1- and
5-H), 5.15 (1 H, q, J 9.2 and 9.5, 4-H), 5.25 (1 H, td, J 2.4 and
9.2, 2- or 3-H), 5.29 (1 H, td, J 2.1 and 10.1, 3- or 2-H), 5.90 (1
H, t, J 10.1, 6-H), 7.43–7.59 [6 H, m, OC(O)Ph] and 8.17–8.24
[4 H, m, OC(O)Ph]; δC(68 MHz; CDCl3) 14.34, 15.24, 15.79,
15.89, 63.66, 63.79, 64.22, 70.02, 73.27, 75.05, 75.37, 76.35,
129.02, 129.70, 128.11, 128.21, 130.12, 130.38, 133.14, 133.37
and 165.32; δP(162 MHz; CDCl3) Ϫ1.53, Ϫ2.11, Ϫ2.18 and
Ϫ2.49 (1H–31P decoupled); m/z (FABϩ) 933 [M ϩ H (18%)], 779
(5) and 105 (100).
DL-1,4-Di-O-benzyl-2,3-O-isopropylidene-myo-inositol 14ab
DL-3,6-Di-O-benzyl-1,2:4,5-di-O-isopropylidene-myo-inositol16
13ab (5.28 g, 12 mmol) was dissolved in methylene dichloride
(100 cm3), followed by the addition of a catalytic amount of
PTSA (20 mg, 0.1 mmol) and one mole equivalent of ethane-
1,2-diol (0.57 cm3, 12 mmol). The mixture was stirred at room
temperature until the solvent became slightly turbid. TLC
(Et2O) showed a major product Rf = 0.30, a trace product
Rf = 0.06, and a trace of starting material Rf = 0.80. Triethyl-
amine (2 cm3) was added to the reaction mixture and the sol-
vent was evaporated off. Purification by flash chromatography
(methylene dichloride–ethyl acetate, 1:1) gave the title com-
pound 14ab (3.84 g, 80%), mp 160–161 ЊC (from ethyl acetate)
(lit.,20 161–163 ЊC), δH (CDCl3; 270 MHz) 1.33, 1.48 (6 H, 2 s,
CMe2), 2.96 (1 H, br s, D2O ex, OH), 3.01 (1 H, br s, D2O ex,
OH), 3.35 (1 H, t, J 9.3, 5-H), 3.51 (1 H, d, J 10.25, 3-H), 3.52 (1
H, t, J 9.9, 6-H), 3.92 (1 H, t, J 9.5, 4-H), 4.06 (1 H, dd, J 5.3
and 6.8, 1-H), 4.27 (1 H, dd, J 4.2 and 5.1, 2-H), 4.68 and 4.91
(2 H, AB, J 11.5, OCH2Ph), 4.77 (2 H, apparent s, OCH2Ph)
and 7.24–7.41 (10 H, m, OCH2Ph); δC(68 MHz; CDCl3) 25.88,
27.99, 71.51, 72.55, 72.97, 73.27, 73.98, 76.93, 79.17, 81.89,
109.85, 127.66, 127.96, 128.02, 128.31, 128.44, 137.78 and
138.07.
DL-3,6-Di-O-benzoyl-myo-inositol 1,2,4,5-tetrakisphosphate
11ab
DL-3,6-Di-O-benzyl-1,2-O-isopropylidene-4,5-bis-O-(p-methoxy-
benzyl)-myo-inositol 15ab
DL-3,6-Di-O-benzoyl-1,2,4,5-tetrakis-O-(diethoxyphosphoryl)-
A mixture of DL-1,4-di-O-benzyl-2,3-O-isopropylidene-myo-
J. Chem. Soc., Perkin Trans. 1, 1997
1283

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inositol 14ab (2.8 g, 7 mmol) and sodium hydride (0.72 g, 30
mmol) was dissolved in dry DMF (50 cm3). p-Methoxybenzyl
chloride (2.9 cm3, 20 mmol) was added dropwise at room tem-
perature and the mixture was stirred for 2 h. TLC (diethyl
ether–light petroleum, 2:1) showed a new product, Rf = 0.40.
The excess of sodium hydride was destroyed with methanol (10
cm3) and the solvents were evaporated off in vacuo. The remain-
ing syrup was partitioned between water (100 cm3) and diethyl
ether (100 cm3), and washed successively with aq. 0.1 mol
dmϪ3 HCl (100 cm3), saturated aq. sodium hydrogen carbonate
(100 cm3), and water (100 cm3). The organic layer was dried
(MgSO4), the remaining syrup was purified by flash chrom-
atography (diethyl ether–light petroleum, 2:1) and the product
15ab was isolated as a syrup (3.60 g, 80%) (Found: C, 73.0; H,
6.64. C39H44O8 requires C, 73.09; H, 6.93%); δH (270 MHz;
CDCl3) 1.35 and 1.51 (6 H, 2 s, CMe2), 3.39 (1 H, t, J 8.8, 5-H),
3.67 (1 H, dd, J 3.6 and 8.8, 3- or 1-H), 3.74–3.80 (1 H,
obscured, 1- or 3-H), 3.77 (3 H, s, OCH2C6H4OMe), 3.79 (3 H,
s, OCH2C6H4OMe), 3.92 (1 H, t, J 8.6, 4- or 6-H), 4.09 (1 H, t, J
6.6, 6- or 4-H), 4.25 (1 H, dd, J 4.0 and 5.3, 2-H), 4.71–4.88 (8
H, m, OCH2Ph), 6.84 (4 H, 2 d, J 9.1, OCH2C6H4OMe) and
7.21–7.41 (14 H, m, OCH2Ph and OCH2C6H4OMe); δC(68
MHz; CDCl3) 25.53, 27.59, 55.04, 73.10, 73.65, 74.37, 74.73,
77.00, 78.91, 80.47, 81.70, 82.35, 109.56, 113.52, 113.58, 114.10,
127.30, 127.63, 127.72, 127.82, 128.05, 128.21, 129.44, 130.61,
131.78, 138.04, 138.40 and 158.96; m/z (FABϪ) 549 (M Ϫ
benzyl, 8%), 519 (M Ϫ p-methoxybenzyl, 40)], 335 (10), 258
(30), 137 (OCH2C6H4OMe, 100) and 107 (OCH2Ph, 70).
thoroughly with methylene dichloride (100 cm3). The solvent
was evaporated off to give a solid, and the individual dia-
stereoisomers were separated by flash chromatography (chloro-
form–acetone, 30:1) to give isomers 18, Rf 0.44 (36% yield);
mp 120–121 ЊC (from EtOH); [α]D ϩ12 (c 1, CH2Cl2) and 19,
Rf 0.34 (37% yield); mp 147–148 ЊC (from EtOH); [α]D ϩ42 (c
1, CH2Cl2); for isomer 18 (Found: C, 71.1; H, 6.27. C46H48O11
requires C, 71.10; H, 6.23%) and for isomer 19 (Found: C, 70.8;
H, 6.22%); isomer 18 δH (400 MHz; CDCl3) 2.16 (1 H, s, D2O
ex, OH), 2.19 [3 H, s, O2CCH(OAc)Ph], 3.44 (1 H, dd, J 2.45
and 9.5, 3-H), 3.49 (1 H, t, J 9.5, 5-H), 3.77 (3 H, s,
OCH2C6H4OMe), 3.78 (3 H, s, OCH2C6H4OMe), 3.91 (1 H, t,
J 9.5, 4-H), 4.05 (1 H, t, J 10.1, 6-H), 4.15 (1 H, br d, J 1.8),
4.61–4.81 (9 H, m, OCH2Ph, OCH2OMe, and 1-H), 5.94 [1 H,
s, O2CCH(OAc)Ph], 6.82 (2 H, d, J 8.5, OCH2C6H4OMe), 6.83
(2 H, d, J 8.85, OCH2C6H4OMe) and 7.16–7.44 [19 H, m,
OCH2Ph, OCH2C6H4OMe and O2CCH(OAc)Ph]; δC(100 MHz;
CDCl3) 20.70, 55.25, 67.32, 72.80, 74.78, 75.25, 75.46, 75.58,
78.42, 79.61, 80.72, 82.73, 113.77, 127.34, 127.52, 127.76,
127.91, 128.29, 128.47, 128.82, 129.26, 129.42, 129.55, 130.76,
130.81, 133.37, 137.71, 138.31, 159.14, 159.18, 168.27 and
170.75; m/z (FABϪ) 929 (M ϩ NBA, 30%), 775 (M Ϫ H, 58),
599 (50), 193 (55) and 149 (100).
For isomer 19 δH (400 MHz; CDCl3) 2.17 [3 H, s, O2CCH-
(OAc)Ph], 2.69 (1 H, s, D2O ex, OH), 3.43 (1 H, t, J 9.5, 5-H),
3.49 (1 H, dd, J 2.75 and 9.8, 3-H), 3.75 (3 H, s, OCH2-
C6H4OMe), 3.77 (3 H, s, OCH2C6H4OMe), 3.93 (1 H, t, J 9.8, 4-
H), 4.00 (1 H, t, J 9.8, 6-H), 4.14 and 4.46 (2 H, AB, J 11.0,
OCH2Ph or OCH2C6H4OMe), 4.40 (1 H, br d, J 1.8, 2-H),
4.61–4.84 (7 H, m, OCH2Ph, OCH2C6H4OMe, and 1-H), 5.98 [1
H, s, O2CCH(OAc)Ph], 6.75 (2 H, d, J 8.5, OCH2C6H4OMe),
6.82 (2 H, d, J 8.85, OCH2C6H4OMe) and 6.83–7.46 [19 H, m,
OCH2Ph, OCH2C6H4OMe and O2CCH(OAc)Ph]; δC(100 MHz;
CDCl3) 20.65, 55.23, 55.27, 67.43, 72.80, 74.94, 74.98, 75.40,
75.58, 78.40, 79.79, 80.74, 82.70, 113.68, 113.75, 127.19, 127.25,
127.89, 128.02, 128.49, 128.84, 129.41, 129.50, 130.65, 130.83,
132.92, 137.63, 138.20, 159.08, 159.16, 168.58 and 170.66; m/z
(FABϪ) 929 (M ϩ NBA, 15%), 775 (M Ϫ H, 28), 599 (25), 193
(55) and 149 (100).
DL-1,4-Di-O-benzyl-5,6-bis-O-(p-methoxybenzyl)-myo-inositol
16ab
DL-3,6-Di-O-benzyl-1,2-O-isopropylidene-4,5-bis-O-(p-meth-
oxybenzyl)-myo-inositol 15ab (2.25 g, 3.5 mmol) was dissolved
in a mixture of methanol–1 mol dmϪ3 aq. HCl (9:1; 30 cm3),
which solution was kept at 50 ЊC for 30 min. TLC (Et2O)
showed a new product, Rf 0.40. Sodium hydrogen carbonate (2
g) was added and the solvents were evaporated off under
reduced pressure. The product was extracted with methylene
dichloride (3 × 100 cm3), and the organic solvent was evapor-
ated off to give a solid. The crude product was purified by flash
chromatography (diethyl ether–chloroform, 3:1) to give the
title compound 16ab (1.9 g, 90%), mp 130–132 ЊC (from ethyl
acetate–hexane) (lit.,19 130.4–130.6 ЊC); δH (270 MHz; CDCl3)
2.53 (1 H, d, J 4.4, D2O ex, OH), 2.62 (1 H, s, D2O ex, OH), 3.43
(2 H, overlapping, 3- and 1-H), 3.44 (1 H, t, J 9.3, 5-H), 3.78 (3
H, s, OCH2C6H4OMe), 3.79 (3 H, s, OCH2C6H4OMe), 3.81 (1
H, t, J 9.3, 4- or 6-H), 3.94 (1 H, t, J 9.5, 6- or 4-H), 4.25 (1 H,
br s, 2-H), 4.70–4.96 (8 H, m, OCH2Ph and OCH2C6H4OMe),
6.83 (2 H, d, J 8.8, OCH2C6H4OMe), 6.84 (2 H, d, J 8.8,
OCH2C6H4OMe) and 7.21–7.36 (14 H, m, OCH2Ph and
OCH2C6H4OMe); δC(68 MHz; CDCl3) 55.04, 69.15, 71.71,
72.68, 75.34, 75.50, 80.01, 81.28, 81.37, 82.93, 113.74, 127.82,
127.89, 128.54, 129.41, 129.54, 130.68, 130.84, 138.49, 138.81
and 159.12;m/z(FABϪ)753(M ϩ NBA,40%),599(M Ϫ H,100),
509 (M Ϫ benzyl, 10), 479 (M Ϫ p-methoxybenzyl, 20), 335
(15), 137 (OCH2C6H4OMe, 30) and 107 (OCH2Ph, 30).
D-3,6-Di-O-benzyl-4,5-bis-O-(p-methoxybenzyl)-myo-inositol
16a
A
mixture of D-1-O-[(S)-(ϩ)-O-acetylmandelyl]-3,6-di-O-
benzyl-4,5-bis-O-(p-methoxybenzyl)-myo-inositol 18 (0.956 g,
1.23 mmol), sodium hydroxide (0.40 g, 10 mmol) and methanol
(100 cm3) was heated at reflux temperature for 30 min. The
mixture was cooled, and neutralised with carbon dioxide. The
resulting solid was diluted with water (50 cm3) and evaporated
to dryness in vacuo. The crude product was extracted with
methylene dichloride (4 × 100 cm3) which was then evaporated
off to give a solid, compound 16a, Rf (Et2O) 0.40 (0.729 g, 99%);
mp 133–134 ЊC (from ethyl acetate–hexane); [α]D Ϫ25 (c 1,
CH2Cl2) (Found: C, 72.1; H, 6.77. C36H40O8 requires C, 71.98;
H, 6.71%). The mass spectrum and NMR data were identical
with those of racemate 16ab.
L-3,6-Di-O-benzyl-4,5-bis-O-(p-methoxybenzyl)-myo-inositol
D- 18 and L-1-O-[(S)-(ϩ)-O-Acetylmandelyl]-3,6-di-O-benzyl-
4,5-bis-O-(p-methoxybenzyl)-myo-inositol 19
16b
A
mixture of L-1-O-[(S)-(ϩ)-O-acetylmandelyl]-3,6-di-O-
A mixture of DL-1,4-di-O-benzyl-5,6-bis-O-(p-methoxybenzyl)-
myo-inositol 16ab (2.5 g, 4.17 mmol), (S)-(ϩ)-O-acetylmandelic
acid 17 (0.835 g, 4.3 mmol) and 4-(dimethylamino)pyridine
(DMAP) (0.03 g, 0.25 mmol) was stirred in methylene dichlor-
ide (15 cm3) at Ϫ20 ЊC (solid CO2 alone). A solution of
dicyclohexylcarbodiimide (DCC) (0.877 g, 4.33 mmol) in
methylene dichloride (5 cm3) was added dropwise over a period
of 90 min with stirring of the reaction mixture, which was then
stirred at room temperature overnight after which TLC
(chloroform–acetone, 30:1) showed two products, Rf 0.44
and 0.34. The mixture was filtered through Celite and washed
benzyl-4,5-bis-O-(p-methoxybenzyl)-myo-inositol 19 (0.929 g,
1.19 mmol), sodium hydroxide (0.40 g, 10 mmol) and methanol
(100 cm3) was heated at reflux temperature for 30 min. Work-up
as for the D-enantiomer gave the title compound 16b Rf (Et2O)
0.40 (0.655 g, 91%); mp 133–134 ЊC (from ethyl acetate–
hexane); [α]D ϩ25 (c 1, CH2Cl2) (Found: C, 72.0; H, 6.86%).
The mass spectrum and NMR data were identical with those of
racemate 16ab.
D-3,6-Di-O-benzyl-myo-inositol 20a
D-3,6-Di-O-benzyl-4,5-bis-O-(p-methoxybenzyl)-myo-inositol
1284
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
16a (0.624 g, 1.04 mmol) was suspended in 1 mol dmϪ3 aq.
HCl–ethanol (60 cm3; 1:2). The mixture was heated at reflux
temperature for 4 h, cooled and the solvents were evaporated in
vacuo. The resulting solid was filtered off and washed with
water (10 cm3) and ether (2 × 10 cm3). The solid was then
recrystallised from ethanol to give the pure title compound 20a,
Rf (chloroform–methanol, 6:1) 0.60 (0.323 g, 86%); mp 172–
173 ЊC (from ethanol); [α]D ϩ16 (c 1, MeOH) (Found: C, 66.6;
H, 6.73. C20H24O6 requires C, 66.65; H, 6.71%); δH (400 MHz;
[2H6]DMSO) 3.11 (1 H, dd, J 2.4 and 9.8, 3-H), 3.15 (1 H, dt, J
4.9 and 8.85, D2O ex, t, J 9.15, 5-H), 3.31 (1 H, ddd, J 2.4, 6.7
and 9.5, D2O ex, dd, J 2.4 and 9.8, 1-H), 3.44 (1 H, t, J 9.5,
6-H), 3.60 (1 H, dt, J 2.4 and 5.8, D2O ex, t, J 2.4, 2-H), 4.51
and 4.60 (2 H, AB, J 12.2, OCH2Ph), 4.67 (1 H, d, J 6.7, D2O
ex, OH), 4.74–4.81 (4 H, m, OH and OCH2Ph), 4.84 (1 H, d, J
4.9, D2O ex, OH) and 7.11–7.44 (10 H, m, OCH2Ph); δC(100
MHz; [2H6]DMSO) 69.73, 70.72, 71.43, 72.25, 73.59, 75.03,
79.79, 81.82, 126.92, 127.08, 127.48, 127.52, 127.63, 127.85,
127.99, 139.32 and 139.94; m/z (FABϪ) 513 (M ϩ NBA, 100%),
359 (M Ϫ H, 75), 291 (50) and 228 (30).
methylene dichloride (5 cm3) was stirred at room temperature
for 15 min in order to form the tetrazolide intermediate 23.
L-3,6-Di-O-benzyl-myo-inositol 20b (0.108 g, 0.30 mmol) was
added to the solution, which was stirred for a further 10 min.
The reaction mixture was cooled to 0 ЊC, MCPBA (0.8 g, 2.3
mmol) (50–60%) was added and the mixture was stirred for a
further 30 min. The product 25b was extracted, and purified by
chromatography in the same way as its antipode 25a (0.37 g,
88%), [α]D ϩ3.3 (c 1.26, CH2Cl2) (Found: C, 65.0; H, 5.72%.
The mass spectrum and NMR data were identical with those of
isomer 25a.
D-myo-Inositol 1,2,4,5-tetrakisphosphate 5a
Ammonia (80 cm3) was distilled into a three-neck flask (cooled
with solid CO2) and small pieces of freshly cut sodium metal
(0.80 g, 34.8 mmol) were added until the solution remained
blue. The solid-CO2 condenser was moved to the reaction flask
and ammonia (40 cm3) was gently transferred to the flask by
heating. Small slivers of sodium (0.40 g, 17.4 mmol) were
added to the ammonia until the colour remained blue once
again.
D-3,6-Di-O-benzyl-1,2,4,5-tetrakis-o-[di(benzyloxy)-
L-3,6-Di-O-benzyl-myo-inositol 20b
phosphoryl]-myo-inositol 25a (0.178 g, 126 µmol) was dissolved
in dry 1,4-dioxane (1 cm3), and the solution was then added to
the mixture of sodium in liquid ammonia. The reaction was left
for 2 min and was then quenched with methanol (20 cm3). The
ammonia was evaporated off under a stream of nitrogen and
MilliQ water was then added to the residue, which was evapor-
ated to dryness in vacuo. The deprotected phosphate was puri-
fied by ion-exchange chromatography on Q-Sepharose, using a
gradient of TEAB buffer (0–1000 mmol dmϪ3) and eluted at
~800 mmol dmϪ3, to give title compound 5a (50.02 µmol, 40%),
[α]D Ϫ27.2 (c 0.50, TEAB, pH 8.6); δH (400 MHz; D2O) 3.59 (1
H, d, J 9.8, 3-H), 3.76 (1 H, t, J 9.5, 6-H), 3.90 (2 H, q, J 9.15,
1- and 5-H), 4.16 (1 H, q, J 9.5, 4-H) and 4.59 (1 H, d, J 9.8,
2-H); δP(162 MHz; D2O) ϩ0.65 (d, J 9.3, CHOPO32Ϫ), ϩ0.27
(d, J 9.0, CHOPO32Ϫ), Ϫ0.01 (d, J 9.0, CHOPO32Ϫ) and Ϫ0.33
(d, J 8.1, CHOPO32Ϫ); m/z (FABϪ) 499 [M Ϫ H (100%)], 419
(5), 159 (10) and 97 (9) [Found: m/z, 498.9226 (M Ϫ H)Ϫ
requires m/z, 498.9208].
L-3,6-Di-O-benzyl-4,5-bis-O-(p-methoxybenzyl)-myo-inositol
16b (0.590 g, 0.98 mmol) was suspended in 1 mol dmϪ3 aq.
HCl–ethanol (60 cm3; 1:2). The mixture was heated at reflux
temperature for 4 h, cooled, and evaporated in vacuo. The
resulting solid was filtered off, and washed successively with
water (10 cm3) and diethyl ether (2 × 10 cm3). The solid was
then recrystallised from ethanol to give the pure title compound
20b, Rf (chloroform–methanol, 6:1) 0.60 (0.293 g, 83%); mp
172–173 ЊC (from EtOH); [α]D Ϫ16 (c 1, MeOH) (Found: C,
66.4; H, 6.73%). The mass spectrum and NMR data were iden-
tical with those of compound 20a.
D-3,6-Di-O-benzyl-1,2,4,5-tetrakis-O-[di(benzyloxy)phosphoryl]-
myo-inositol 25a
A mixture of bis(benzyloxy)diisopropylaminophosphine 21
(0.69 g, 2 mmol) and 1H-tetrazole 22 (0.28 g, 4 mmol) in dry
methylene dichloride (5 cm3) was stirred at room temperature
for 15 min in order to form the tetrazolide intermediate 23.
D-3,6-Di-O-benzyl-myo-inositol 20a (0.108 g, 0.30 mmol) was
added to compound 23 and the mixture was stirred for a further
10 min before being cooled to 0 ЊC; MCPBA (0.8 g, 2.3 mmol)
(50–60%) was added and the mixture was stirred for a further
30 min, diluted with ethyl acetate (50 cm3), and washed succes-
sively with 10% aq. sodium metabisulfite (50 cm3), 1 mol dmϪ3
HCl, saturated aq. sodium hydrogen carbonate, brine and water
(50 cm3 of each). The organic layer was separated, then dried
(MgSO4), and evaporated to give a syrup. The product was
purified by flash chromatography, Rf (chloroform–acetone,
5:1) 0.20, then (ethyl acetate–pentane, 2:1), in order to obtain
the pure title compound 25a as a syrup (0.395 g, 94%); [α]D Ϫ3.5
(c 2, CH2Cl2) (Found: C, 65.2; H, 5.54. C76H76O18P4 requires C,
65.14; H, 5.47%); δH (400 MHz; CDCl3) 3.55 (1 H, d, J 9.8, 3-H),
3.98 (1 H, t, J 9.5, 6-H), 4.41 (1 H, t, J 9.5, 5-H), 4.48–5.11 [22
H, m, O(O)POCH2Ph, OCH2Ph, 1- and 4-H], 5.43 (1 H, d, J
8.9, 2-H) and 6.94–7.41 [50 H, m, O(O)POCH2Ph and
OCH2Ph]; δC(100 MHz; CDCl3) 69.18, 69.23, 69.29, 69.24,
69.42, 69.47, 69.53, 69.60, 69.65, 72.24, 74.37, 74.37, 74.63,
75.43, 77.38, 78.66, 127.19, 127.39, 127.57, 127.74, 127.85,
127.96, 128.09, 128,16, 128.29, 128.45, 128.51, 128.65, 135.48,
135.55, 135.60, 135.73, 135.78, 135.84, 135.91, 136.01, 136.46
and 137.94; δP(162 MHz; CDCl3) Ϫ1.16, Ϫ1.66, Ϫ1.71 and
Ϫ2.07 (31P–1H-decoupled); m/z (FABϩ) 1401 (M ϩ H, 7%), 181
(5), 107 (2) and 91 (100).
L-myo-Inositol 1,2,4,5-tetrakisphosphate 5b
L-3,6-Di-O-benzyl-1,2,4,5-tetrakis-O-[di(benzyloxy)phos-
phoryl]-myo-inositol 25b (0.10 g, 71 µmol) was deprotected
as for its antipode 25a to give pure L-myo-inositol 1,2,4,5-
tetrakisphosphate 5b after ion-exchange chromatography (15.56
µmol, 22%). The NMR spectra were slightly different due to the
different pH of the solution sample; [α]D ϩ25.8 (c 0.31, TEAB,
pH 8.6); δH (270 MHz; D2O) 3.72 (1 H, d, J 9.7, 3-H), 3.90 (1 H,
t, J 9.5, 6-H), 4.03 (2 H, q, J 9.3, 1- and 5-H), 4.30 (1 H, q, J 9.5,
4-H) and 4.71 (1 H, br d, J 9.7, 2-H); δP(109 MHz; D2O) ϩ1.78
(d, J 10.1, CHOPO32Ϫ), ϩ1.44 (d, J 6.7, CHOPO32Ϫ), ϩ1.20 (d,
J 6.7, CHOPO32Ϫ) and ϩ0.67 (d, J 6.7, CHOPO32Ϫ); m/z
(FABϪ) 499 [M Ϫ H (100%)], 419 (10), 159 (10) and 97 (10)
[Found: m/z, 498.9187].
D-3,6-Bis-O-[(S)-(ϩ)-O-acetylmandelyl]-1,2:4,5-di-O-
isopropylidene-myo-inositol 27
A
mixture of DL-1,2:4,5-di-O-isopropylidene-myo-inositol
12ab (2.08 g, 8 mmol), DCC (4.13 g, 20 mmol) and DMAP
(0.05 g, 0.4 mmol) in dry methylene dichloride (50 cm3) was
stirred at 0 ЊC under nitrogen. A solution of (S)-(ϩ)-O-
acetylmandelic acid 17 (3.88 g, 20 mmol) in dry methylene
dichloride (30 cm3) was added dropwise over a period of 15
min and the mixture was stirred overnight. The precipitated
dicyclohexylurea was filtered off over Celite and the filtrate
was evaporated to give a solid. The mixture of diastereoisomers
was purified by flash chromatography [Rf (chloroform–acetone,
16:1) 0.30] but they could not be separated. The mixture was
recrystallised four times from methanol to give the single pure
diastereoisomer 27 (0.89 g, 18%) in an unoptimised yield, mp
L-3,6-Di-O-benzyl-1,2,4,5-tetrakis-O-[di(benzyloxy)phosphoryl]-
myo-inositol 25b
A mixture of bis(benzyloxy)diisopropylaminophosphine 21
(0.69 g, 2 mmol) and 1H-tetrazole 22 (0.28 g, 4 mmol) in dry
J. Chem. Soc., Perkin Trans. 1, 1997
1285

View Article Online
212–214 ЊC; [α]D ϩ64 (c 1, CH2Cl2) (Found: C, 62.7; H, 5.88.
C32H36O12 requires C, 62.74; H, 5.92%); δH (270 MHz; CDCl3)
1.21, 1.29, 1.32 and 1.57 (12 H, 4 s, CMe2), 2.17 and 2.18 [6 H, 2
s, O2CCH(OAc)Ph], 3.23 (1 H, dd, J 9.3 and 11.0, 5-H), 4.04 (1
H, t, J 10.45, 4-H), 4.15 (1 H, dd, J 4.8 and 6.6, 1-H), 4.54 (1 H,
t, J 4.6, 2-H), 5.08 (1 H, dd, J 4.2 and 10.6, 3-H), 5.22 (1 H, dd,
J 6.6 and 11.0, 6-H), 6.01 [1 H, s, O2CCH(OAc)Ph], 6.11 [1 H,
s, O2CCH(OAc)Ph] and 7.33–7.52 [10 H, m, O2CCH(OAc)Ph];
δC(CDCl3; 68 MHz) 20.59, 20.72, 25.72, 26.56, 26.69, 27.73,
71.34, 74.05, 74.34, 74.48, 75.04, 75.67, 76.37, 78.65, 110.72,
112.61, 127.92, 128.17, 128.54, 128.59, 129.07, 129.20, 167.74,
168.40, 169.85 and 170.54; m/z (FABϩ) 613 (M ϩ H, 50%), 555
(30), 149 (90) and 107 (100).
Council for financial support. B. V. L. P. is a Lister Institute
Research Professor. We also thank Dr Christine Murphy and
Professor John Westwick for the preliminary biological data.
References
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D-1,2:4,5-Di-O-isopropylidene-myo-inositol 12a
A mixture of D-3,6-di-O-[(S)-(ϩ)-O-acetylmandelyl]-1,2:4,5-
di-O-isopropylidene-myo-inositol 27 (0.74 g, 1.21 mmol),
sodium hydroxide (0.40 g, 10 mmol) and methanol (100 cm3)
was heated at reflux temperature for 30 min. The mixture was
cooled, and neutralised with carbon dioxide. The solid was then
diluted with water (50 cm3) and evaporated to dryness in vacuo.
The crude product was extracted with methylene dichloride
(4 × 100 cm3) and the solvent was evaporated off to give a solid.
The title compound 12a was purified by flash chromatography
(ethyl acetate–methylene dichloride, 1:1), Rf (Et2O) 0.20, and
dried (MgSO4), and the solvent was evaporated off. The remain-
ing solid was recrystallised from ethyl acetate to give compound
12a (0.27 g, 86%), mp 174–176 ЊC (from ethyl acetate) (lit.,25
176–177 ЊC); [α]D Ϫ22 (c 1, MeCN) (lit.,24 Ϫ21.7, c 0.46 MeCN)
(Found: C, 55.6; H, 7.88. C12H20O6 requires: C, 55.37; H,
7.74%). The NMR data were identical with those for racemate
12ab.33
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and B. V. L. Potter, Eur. J. Biochem., 1994, 223, 115.
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D-3,6-Di-O-benzyl-1,2:4,5-di-O-isopropylidene-myo-inositol
13a
A mixture of D-1,2:4,5-di-O-isopropylidene-myo-inositol 12a
(0.209 g, 0.80 mmol), DMF (10 cm3) and sodium hydride (0.096
g, 4 mmol) was stirred at room temperature. Benzyl bromide
(0.2 cm3, 2 mmol) was added and the mixture was stirred for a
further 2 h. TLC (diethyl ether–light petroleum, 1:1) then
showed a new product, Rf 0.60. Methanol (2 cm3) was added to
destroy the excess of sodium hydride and the solvents were
evaporated off in vacuo. The residue was partitioned between
water and diethyl ether (30 cm3 each) and the organic layer was
evaporated off to give a solid. The title compound 13a was
purified by flash chromatography (diethyl ether–pentane, 1:2)
and recrystallised from hexane (0.32 g, 91%); mp 157–159 ЊC
(from hexane) (lit.,26 159–161 ЊC, for L-enantiomer); [α]D Ϫ44 (c
1, CH2Cl2) (lit.,26 ϩ85, c 1, CHCl3, for L-enantiomer) (Found:
C, 71.1; H, 7.35. C26H32O6 requires C, 70.89; H, 7.32%). The
NMR data were identical with those of racemate 13ab.33
D-3,6-Di-O-benzyl-myo-inositol 20a
A mixture of D-3,6-di-O-benzyl-1,2:4,5-di-O-isopropylidene-
myo-inositol 13a (0.27 g, 0.62 mmol) and methanol–1 mol dmϪ3
HCl (50 cm3; 9:1) was heated at reflux temperature for 30 min.
The solution was cooled and the solvents were evaporated off
to give a solid, which was recrystallised from ethanol [Rf
(chloroform–methanol; 6:1) 0.60] (0.21 g, 95%), mp 172–
173 ЊC (from EtOH); [α]D ϩ16 (c 1, MeOH). The mass spec-
trum and NMR data were identical with those of compound
20a as described previously.
33 S.-K. Chung and Y. Ryu, Carbohydr. Res., 1994, 258, 145.
Paper 6/08337D
Received 11th December 1996
Accepted 21st January 1997
Acknowledgements
We thank the Biotechnology and Biological Sciences Research
1286
J. Chem. Soc., Perkin Trans. 1, 1997