A Definitive Synthesis of D-myo-Inositol 1,4,5,6-Tetrakisphosphate and Its Enantiomer D-myo-Inositol 3,4,5,6-Tetrakisphosphate from a Novel Butane-2,3-diacetal-Protected Inositol

Article abstract of DOI:10.1002/chem.200305207

New and rapid syntheses of the enantiomeric intracellular signalling molecules D-myo-inositol 1,4,5,6-tetrakisphosphate (1a) and D-myo-inositol 3,4,5,6-tetrakisphosphate (1b) are described. The synthetic strategy employs the novel butane-2,3-diacetal-prot

Full text of DOI:10.1002/chem.200305207

FULL PAPER
A Definitive Synthesis of d-myo-Inositol 1,4,5,6-Tetrakisphosphate and Its
Enantiomer d-myo-Inositol 3,4,5,6-Tetrakisphosphate from a Novel Butane-
2,3-diacetal-Protected Inositol
Stephen J. Mills,[a] Andrew M. Riley,[a] Changsheng Liu,[a] Mary F. Mahon,[b] and
Barry V. L. Potter*[a]
Abstract: New and rapid syntheses of
the enantiomeric intracellular signal-
ling molecules d-myo-inositol 1,4,5,6-
tetrakisphosphate (1a) and d-myo-ino-
sitol 3,4,5,6-tetrakisphosphate (1b) are
described. The synthetic strategy em-
ploys the novel butane-2,3-diacetal-
protected (BDA-protected) myo-inosi-
tol (Æ)-3ab, directly accessible from
myo-inositol on a large scale, and an
optical resolution with diastereoisomer-
ic (R)-(À)-acetylmandelate esters. The
X-ray crystal structure of (Æ)-4, an un-
usual side product of acid-catalysed re-
action of myo-inositol with butane-
dione is also presented, and the abso-
lute configurations of 1a and 1b are
definitively assigned by conversion of
key precursors into (+)-bornesitol and
l-iditol hexaacetate, respectively. Bio-
logical activity of synthetic 1b was con-
firmed in comparison with the natural
polyphosphate.
Keywords: butane-2,3-diacetal
chiral resolution cyclitols
inositol ¥ signal transduction
¥
¥
¥
Introduction
Since the discovery in 1983 that d-myo-inositol 1,4,5-tris-
phosphate, Ins(1,4,5)P3, acts as a Ca2+-mobilising intracellu-
lar second messenger, many other inositol phosphates have
been discovered, although it is only in recent years that
their physiological functions are beginning to be under-
stood.[1]
d-myo-Inositol
1,3,4,5-tetrakisphosphate,
Ins(1,3,4,5)P4, for example, may act in a coordinated way to
assist Ins(1,4,5)P3-initiated Ca2+ mobilisation, and has re-
cently been shown to have a physiological role, inhibiting
Ins(1,4,5)P3 5-phosphatase.[2]
Two other inositol tetrakisphosphates, d-myo-inositol
1,4,5,6-tetrakisphosphate, Ins(1,4,5,6)P4, 1a, and its enan-
dramatically increased in response to Salmonella invasion,
leading to inhibition of the phosphatidylinositol 3,4,5-tris-
phosphate, PtdIns(3,4,5)P3, signalling pathway,[3] and a role
for Ins(1,4,5,6)P4 in the regulation of transcription has also
been proposed.[4] Recently, it was reported that the tumour
suppressor protein PTEN, which is known to have
PtdIns(3,4,5)P3 3-phosphatase activity, can also hydrolyse
the 3-phosphate group of Ins(1,3,4,5,6)P5 to generate
Ins(1,4,5,6)P4 (1a),[5] thus raising the possibility that pertur-
bations in the Ins(1,3,4,5,6)P5/Ins(1,4,5,6)P4 cycle may con-
tribute to the neoplastic consequences of PTEN gene muta-
tions.[5]
Ins(3,4,5,6)P4 (1b) is now thought to behave as an intra-
cellular signal that inhibits the conductance of Ca2+-activat-
ed ClÀ channels in the plasma membrane,[6] thereby contri-
buting to the control of salt and fluid secretion from epithe-
lial cells. Recently, it has been shown that signalling by
Ins(3,4,5,6)P4 is regulated in vivo by inositol 1,3,4-trisphos-
phate, Ins(1,3,4)P3,[7] which acts by activating an
Ins(1,3,4,5,6)P5 1-phosphatase.[8] In cystic fibrosis (CF),
tiomer,
d-myo-inositol
3,4,5,6-tetrakisphosphate,
Ins(3,4,5,6)P4, 1b, are also attracting growing attention.
Ins(1,4,5,6)P4 levels in human colonic epithelial cells are
[a] Prof. B. V. L. Potter, S. J. Mills, A. M. Riley, C. Liu
Wolfson Laboratory of Medicinal Chemistry
Department of Pharmacy and Pharmacology
University of Bath
Claverton Down, Bath, BA2 7AY (UK)
Fax : (+44)1225-386-114
E-mail: B.V.L.Potter@bath.ac.uk
[b] M. F. Mahon
Department of Chemistry
University of Bath
Claverton Down, Bath, BA2 7AY (UK)
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DOI: 10.1002/chem.200305207
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FULL PAPER
cyclic AMP-activated ClÀ channels are defective, leaving the
Ca2+-activated channels unimpaired. Thus, drugs that could
antagonise the synthesis or actions of Ins(3,4,5,6)P4 might
have therapeutic value in CF by stimulating ClÀ secretion
through Ca2+-activated channels.[9]
The fact that Ins(1,4,5,6)P4 (1a) and Ins(3,4,5,6)P4 (1b)
are enantiomeric, and yet appear to have distinct physiologi-
cal roles, requires that homochiral materials of high purity
and certain absolute configurations are available in suffi-
cient quantities for biological studies. This demand cannot
be met with material obtained from biological sources be-
cause the two enantiomers cannot be separated by standard
HPLC techniques, nor can the absolute configurations of
the tiny quantities involved be determined without difficulty.
Thus, a simple synthetic route to both 1a and 1b is required,
and the absolute configurations of the products must be es-
tablished beyond doubt. Various syntheses of Ins(1,4,5,6)P4
(1a) and Ins(3,4,5,6)P4 (1b) have been reported,[10] although
there are contradictions in the chemical literature regarding
the optical rotation of Ins(1,4,5,6)P4 (1a) and Ins(3,4,5,6)P4
(1b), and also of key synthetic intermediates used in their
synthesis.[10g] Thus, specific rotations opposite in sign but
similar in magnitude have been reported for the same com-
pounds. Ins(1,4,5,6)P4 itself, for example, has been designat-
ed as either the levorotatory[10b,c,g] or the dextrorotatory[10e,f]
enantiomer.
Scheme 1. i) Butanedione, (MeO)3CH, CSA, MeOH, reflux.
polyols by using cheap and commercially available butane-
dione instead of TMB.[15] We have recently applied the bu-
tanedione method to both myo- and chiro-inositol on a
large scale in syntheses of the rare neo isomer of inositol[13a]
and of novel chiro-inositol-based analogues[13b] of 1a and
1b.
Here we report that, in addition to the symmetrical diol
2, a second major product also results from the acid-cata-
lysed reaction of butanedione with myo-inositol: namely the
asymmetrical diol dl-1,6:4,5-bis-O-(2,3-dimethoxybutane-
2,3-diyl)-myo-inositol (Æ)-3ab (Scheme 1). When the cooled
reaction mixture is filtered, compound 2 is obtained as a
solid precipitate, while (Æ)-3ab, which is much more soluble
in methanol, remains in the mother liquor and can be isolat-
ed by flash chromatography followed by recrystallisation.
With reaction times of around 40 h, the asymmetrical diol
[(Æ)-3ab] and the symmetrical diol 2 were both obtained
from 25 g of inositol, in 20% and 26% yield, respectively.
The pattern of protection in (Æ)-3ab clearly makes it an
ideal precursor for the synthesis of the enantiomers
Ins(1,4,5,6)P4 (1a) and Ins(3,4,5,6)P4 (1b). Furthermore, the
reaction gives rapid access to (Æ)-3ab on a multigram scale
in a single step.
Here we present new syntheses of Ins(1,4,5,6)P4 (1a)
and Ins(3,4,5,6)P4 (1b) from a novel butane-2,3-diacetal-pro-
tected inositol. The absolute configurations of the two tetra-
kisphosphates were definitively assigned by reference to two
independent standards. In the course of this study, we also
identified an unusual side product of the inositol protection
reaction and established its structure by a single-crystal X-
ray study.
Results and Discussion
The major challenge in the synthesis of inositol polyphos-
phates is to obtain a chiral intermediate suitable for phos-
phorylation. This usually requires multiple regiospecific pro-
tection of the hydroxy groups in inositol with various perma-
nent and temporary protecting groups, and an optical reso-
lution of enantiomeric intermediates.[11] Rapid and simpli-
fied routes are therefore of great advantage. Recent years
have seen an increasing use of 1,2-diacetals in organic syn-
thesis,[12] and their application to the selective protection of
diequatorial 1,2-diols gives them great potential in the syn-
thesis of many inositol phosphates and analogues. The cur-
rent route arises out of our recent studies[13] on the protec-
tion of inositols with the butane-2,3-diacetal (BDA) protect-
ing group.
The application of the BDA group to the protection of
myo-inositol was first demonstrated by Montchamp et al.,[14]
who showed that the acid-catalysed reaction of 2,2,3,3-tetra-
methoxybutane (TMB) with myo-inositol on a small scale
gave the symmetrical diol 1,6:3,4-bis-O-(2,3-dimethoxybu-
tane-2,3-diyl)-myo-inositol (2, Scheme 1). It was later shown
that the BDA group could be introduced into a variety of
In our initial report[13a] we noted that prolonged reaction
times gave a higher yield of the symmetrical diol 2, but also
that a less polar product steadily accumulated in the equili-
brium mixture. By use of extended reaction times (up to 28
days on this scale) it was later possible to isolate this third
product [(Æ)-4], in low yield, as a highly crystalline materi-
1
al. The H NMR spectrum of (Æ)-4 (see Experimental Sec-
tion) was unusual for a myo-inositol derivative. It was im-
mediately obvious that the compound was asymmetrical,
and abnormal coupling constants between vicinal protons in
the myo-inositol ring implied that the inositol ring did not
take on the usual chair conformation in (Æ)-4. It was also
clear that only three O-methyl groups were present in the
molecule, and derivatisation to give a monoacetate con-
firmed that (Æ)-4 contained only one hydroxy group. Final-
ly, a significant downfield shift for a quaternary carbon atom
in the 13C NMR spectrum of (Æ)-4 [d=107.62 ppm, com-
pared to d=99.15, 99.33, 99.88 and 100.39 ppm for the
acetal carbon atoms in (Æ)-3ab] suggested the presence of a
five-membered dioxolane ring.[16,17] A single-crystal X-ray
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Synthesis of Intracellular Signalling Molecules
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Figure 1. X-ray crystal structure of (Æ)-4 (ellipsoids are represented at
the 30% level).
study established the structure of (Æ)-4 to be as shown in
Figure 1. It is hard to define a unique mechanism to account
for the origin of (Æ)-4, but a feasible route involves the
elimination of a molecule of methanol from an unstable
bis(butane-2,3-diacetal) intermediate with the 1,2:4,5 protec-
tion pattern, presumably formed as part of competing acid-
catalysed equilibria. It seems likely that the formation of
(Æ)-4, in which the inositol ring is held in a twist-boat con-
formation by the formation of a rigid cage involving O-1, O-
2 and O-3, is favoured by stabilizing anomeric effects, to-
gether with the avoidance of steric clashes.[16] Thus, both di-
oxane rings in (Æ)-4 adopt chair conformations with axial
O-methyl groups. While this work was nearing completion,
a similar side product of acid-catalysed reaction of myo-ino-
sitol with butanedione was reported,[18] although that struc-
ture, inferred from the X-ray crystal structure of its benzyl
ether, apparently has the opposite relative stereochemistry
at C-2’’ to that found in (Æ)-4 in the present work. To the
best of our knowledge, no crystal structure of the benzyl
ether derivative[18] has yet been published to allow formal
direct comparison with ours.
Because the asymmetrical diol (Æ)-3ab is racemic at this
stage, a resolution step is required (Scheme 2). It was found
that DCC-promoted regioselective esterification of (Æ)-3ab
with (R)-(À)-acetylmandelic acid (1.06 equivalents) gave di-
astereoisomeric acetylmandelate esters 5 and 6, which were
conveniently separated by flash chromatography. The indi-
vidual diastereoisomers were then saponified in refluxing
methanolic sodium hydroxide to give the individual homo-
chiral diols (+)-3a and (À)-3b in good yield. Their specific
rotations (+270 and À270 respectively) were unusually
large for myo-inositol derivatives.
Scheme 2. i) (R)-(À)-acetylmandelic acid, DCC, DMAP, CH2Cl2, 36.5%
(5), 36.5% (6); ii) NaOH in MeOH, reflux, 30 min, 91% for 3a and 3b;
iii) BnBr, NaH, DMF, RT 2 h, 84% for 7a and 86% for 7b; iv) 95%
aqueous trifluoroacetic acid in CH2Cl2 (1:1), 82% for 8a, 75% for 8b;
v) bis(benzyloxy)diisopropylaminophosphine, 1H-tetrazole, CH2Cl2, then
8a or 8b, then mCPBA, À788C, 88% for 9a, 54% for 9b; vi) 20%
Pd(OH)2 on carbon, H2 (50 psi), MeOH/H2O (5:1), 20 h. DCC=N,N’-di-
cyclohexylcarbodiimide, DMAP=4-dimethylaminopyridine, mCPBA=3-
chloroperoxybenzoic acid.
As noted above, there are contradictions in the literature
regarding the specific rotations of synthetic Ins(1,4,5,6)P4
(1a) and Ins(3,4,5,6)P4 (1b). While it is likely that the spe-
cific rotations of these tetrakisphosphates will be dependent
on salt form and pH, the specific rotations reported for key
intermediates used in some syntheses of Ins(1,4,5,6)P4 (1a)
and Ins(3,4,5,6)P4 (1b) have also been reported with oppo-
site signs for the same material. It was therefore vital, in the
present case, to establish the absolute configurations of in-
termediates, and thereby those of our synthetic
Ins(1,4,5,6)P4 (1a) and Ins(3,4,5,6)P4 (1b) by reliable means.
Accordingly, (+)-3a was subjected to regioselective stann-
ylene-mediated methylation of the equatorial hydroxy
group, followed by hydrolysis of BDA acetals with TFA to
1
give pentaol 10 (Scheme 3). The H and 13C NMR spectra of
10 were identical to those reported for 1-O-methyl-myo-ino-
sitol [(À)-bornesitol],[19] although the specific rotation of 10
was opposite in sign, identifying it as (+)-bornesitol (3-O-
methyl-myo-inositol).[20] This identified (+)-3a as the
1,6:4,5-protected compound, and enabled the absolute con-
figurations of all chiral intermediates and products to be as-
signed.
For additional confirmation, the enantiomeric diol (À)-
3b was subjected to oxidative glycol cleavage with silica-
supported sodium metaperiodate[21] followed by reduction of
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B. V. L. Potter et al.
FULL PAPER
ly. Phosphitylation of the individual tetraols was achieved by
treatment of 8a and 8b with the tetrazolide formed by treat-
ment of bis(benzyloxy)diisopropylaminophosphine with 1H-
tetrazole. At this stage, the 31P NMR spectrum (Figure 2) of
each resulting tetrakisphosphite (not isolated) showed two
5
doublets (corresponding to P-1 and P-4, JP, P ꢀ6 Hz) and two
5
apparent triplets (for P-5 and P-6, JP, P ꢀ6 Hz); the same
pattern of signals was obtained for the enantiomer. This fur-
5
ther exemplifies our original observation of JP, P couplings in
such species.[23] The spectrum obtained in Figure 2 can only
arise from the presence of four adjacent equatorial phos-
phites in an asymmetrical structure, thus confirming the re-
quired substitution pattern of the myo-inositol ring.
Oxidation of the tetrakisphosphite intermediates with 3-
chloroperoxybenzoic acid gave the protected tetrakisphos-
phates 9a and 9b, respectively. Deprotection of 9a and of
9b was accomplished by hydrogenolysis over 20% Pd(OH)2/
C, at 50 psi, to provide Ins(1,4,5,6)P4 (1a) and Ins(3,4,5,6)P4
(1b), respectively. For biological evaluation, 1a and 1b were
further purified by ion-exchange chromatography on Q-Se-
pharose Fast Flow resin, with elution with a gradient of tri-
ethylammonium hydrogen carbonate buffer, to give the
pure triethylammonium salts of Ins(1,4,5,6)P4 (1a) or
Ins(3,4,5,6)P4 (1b). Measurement of the specific rotations of
1a and 1b showed that Ins(1,4,5,6)P4 (1a) was the levorota-
tory enantiomer. Accurate quantification of each tetrakis-
phosphate was carried out by a modification of the Briggs
phosphate assay.[24]
Synthetic Ins(1,4,5,6)P4 (1a) and Ins(3,4,5,6)P4 (1b) were
evaluated biologically in a preliminary fashion by using a
single CFPAC-1 cell in whole-cell mode, with 500 nm Ca2+
as the stimulus for activation. While 1b was able to inhibit
the conductance of the Ca2+-activated ClÀ channels to the
same degree as seen with Ins(3,4,5,6)P4 from other sources,
1a was, as expected, without effect.
Scheme 3. i) a) Bu2SnO, CH3CN, CH3I, tetrabutylammonium bromide;
b) 95% aqueous trifluoroacetic acid in CH2Cl2 (1:1), 81%; ii) a) NaIO4/
SiO2,[21] CH2Cl2; b) NaBH4, EtOH, 87%; iii) a) 50% aqueous trifluoro-
acetic acid; b) Ac2O, pyridine, 46%.
the resulting dialdehyde with sodium borohydride to give
the highly crystalline C2-symmetric iditol derivative 11
(Scheme 3). Treatment of 11 with 50% aqueous trifluoro-
acetic acid for 2 h, followed by peracetylation of the prod-
uct, gave 12 in modest (46%) yield. NMR spectra, melting
point and specific rotation of 12 identified it as l-iditol hexa-
acetate,[22] thus identifying (À)-3b as the 3,4:5,6-protected
diacetal, and confirming the configurational assignments
made above.
The diols 3a and 3b were then benzylated (Scheme 2)
with benzyl bromide and sodium hydride in DMF to give
the fully blocked dibenzyl derivatives 7a and 7b in good
yield. Treatment of 7a and of 7b with aqueous trifluoroace-
tic acid in dichloromethane at room temperature removed
the BDA protecting groups to give the previously unknown
crystalline 1,4,5,6- and 3,4,5,6-tetraols 8a and 8b respective-
Conclusions
In summary, we have described short and practical syntheses
of Ins(1,4,5,6)P4 (1a) and Ins(3,4,5,6)P4 (1b) starting from a
novel BDA-protected myo-inositol. To resolve any uncer-
tainty concerning the absolute configurations of the tetrakis-
phosphates produced by some earlier reported syntheses,
the absolute configurations of the products were established
unambiguously by correlation with (+)-bornesitol and l-
iditol. Also 1b was biologically active. These tetrakisphos-
phates, accessible in large quantities by this route, should be
useful in further evaluation of the disparate biological roles
of these two enantiomers in cellular signalling pathways.
Experimental Section
General: Chemicals were purchased from Aldrich and Fluka. Dichloro-
methane, pyridine and dimethylformamide (DMF) were purchased in an-
hydrous form. TLC was performed on precoated plates (Merck TLC alu-
minium sheets silica 60F254. Art. No. 5554). Products were viewed under
UV light at 254 nm or with phosphomolybdic acid in methanol followed
by heating. Flash chromatography was carried out with Sorbsil C60 silica
Figure 2. 31P NMR spectrum (162 MHz in CDCl3) of d-2,3-di-O-benzyl-
1,4,5,6-myo-inositol-tetrakis[di(benzyloxy)phosphite] (intermediate not
isolated).
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Synthesis of Intracellular Signalling Molecules
6207 6214
gel. NMR spectra were recorded on either JEOL EX-270 or Varian Mer-
cury EX-400 NMR spectrometers. 1H chemical shifts were measured in
ppm relative to tetramethylsilane (TMS). 31P chemical shifts were meas-
ured in ppm relative to external 85% H3PO4 and are positive when
downfield from this reference. Melting points (uncorrected) were deter-
mined with a Reichert Jung Thermo Galen Kofler Block. Microanalysis
was carried out by the University of Bath microanalysis service. Optical
rotations were measured with an Optical Activity Ltd. AA-10 polarime-
ter, and [a]D values are given in 10À1 degcm2 gÀ1. Fast atom bombardment
(FAB) mass spectra were recorded at the Mass Spectrometry Service of
the University of Bath, with 3-nitrobenzyl alcohol (NBA) as matrix. Ion-
exchange chromatography was performed on a LKB-Pharmacia medium-
pressure ion-exchange chromatograph on Q Sepharose Fast Flow with
gradients of triethylammonium hydrogen carbonate (TEAB) as eluent.
Column fractions containing inositol polyphosphates were assayed for
total phosphate by a modification of the Briggs test.[24]
Isolation of dl-(2’S*,3’S*,2’’R*,3’’S*)-4,5-O-(2’,3’-dimethoxybutane-2’,3’-
diyl)-1,2,3-O-(2’’-methoxybutane-2’’-yl-3’’-ylidene)-myo-inositol [(Æ)-4]:
Trimethyl orthoformate (200 mL), butanedione (50 mL, 570 mmol) and
(Æ)-camphorsulphonic acid (1 g) were added to a stirred suspension of
myo-inositol (50.0 g, 277 mmol) in MeOH (500 mL). The mixture was
heated at reflux for 28 days and then allowed to cool. The suspension
was filtered, and the filtrate was washed with MeOH (200 mL) and al-
lowed to dry. The resulting white solid (50 g) was suspended in propan-2-
ol (600 mL) and the mixture was heated at reflux for 2 h with vigorous
stirring. The hot suspension was filtered, and as the filtrate cooled to
room temperature, a white solid (5 g) precipitated. This was found to
consist of a 4:1 mixture of (Æ)-4 and 2, as judged by 1H NMR spectros-
copy. Crystallisation of this solid from boiling acetonitrile (250 mL) gave
alcohol (Æ)-4 (3.42 g, 3.4%) as colourless needles: Rf 0.60 (CH2Cl2/ace-
tone 2:1); the needles undergo a phase change above 1658C to plates,
which then sublime, with softening, above 2708C.
1H 1H COSY (400 MHz, [D5]pyridine, TMS): d = 1.37, 1.45, 1.47, 1.57
(4îs, 12H; 4îCH3), 3.20, 3.33, 3.40 (3îs, 9H; 3îOCH3), 4.06 (dd, J =
12.0, 7.0 Hz, 1H; H-5), 4.26 (m, D2O exch. gives dd, J = 7.0, 1.5 Hz, 1H;
H-6), 4.43 (brs, 1H; H-1), 4.60 (ddd, J = 5.5, 4.7, 0.8 Hz, 1H; H-3), 4.78
(dd, J = 12.1, 4.7 Hz, 1H; H-4), 5.02 (brs, D2O exch; 1 H; OH-6), 5.08
(dd, J = 5.5, 1.6 Hz, 1H; H-2) ppm; 13C NMR (100 MHz, [D5]pyridine):
d = 18.07, 18.14, 18.91, 18.94 (4îCH3), 47.64, 47.77, 48.30 (3îOCH3),
71.08 (CH), 72.36 (2îCH), 74.24, 74.69, 78.56 (3îCH), 99.40, 99.45,
99.79 (3îCq), 107.62 (C-3’’); MS (+ve ion FAB) 345 (70%)
[MÀOCH3]+, 255 (40%), 173 (50%), 101 (100%); elemental analysis
calcd (%) for C17H28O9 (376.40): C 54.25, H 7.50; found: C 54.3, H 7.48.
X-ray crystallography of (Æ)-4: A crystal of approximate dimensions
0.03î0.01î0.075 mm was used for data collection. Crystal data:
C34H56O18, Mr = 752.79, monoclinic, a = 11.840(2), b = 12.176(2), c =
13.606(2) ä, b = 113.63(2)8, U = 1797.0(5) ä3, space group P21/c, Z =
2, 1calcd = 1.391 gcmÀ3, m(MoKa) = 0.113 mmÀ1, F(000) = 808. Crystallo-
graphic measurements were made at 293(2) K on a Nonius Kappa CCD
diffractometer in the range 1.88<q<27.49o. Data (23153 reflections)
were corrected for Lorentz and polarisation and also for absorption.
[SORTAV program].
In the final least squares cycles all atoms were allowed to vibrate aniso-
tropically. Hydrogen atoms were included at calculated positions where
relevant. Analysis of the gross structure revealed that molecules interact
through hydrogen bonding between the alcoholic proton H6 of one mole-
cule and the proximate O3 of a lattice neighbour, to generate one-dimen-
sional polymers.
The solution of the structure (SHELXS-86)[25] and the refinement
(SHELXL-97)[26] converged to a conventional [that is, based on 2580 F2
data with Fo >4s(Fo)] R1 = 0.0469 and wR2 = 0.1059. Goodness of fit
= 0.938. The maximum and minimum residual densities were 0.281 and
À0.308 eäÀ3, respectively. The asymmetric unit (shown in Figure 1) along
with the labelling scheme used was produced by using ORTEX.[27]
1d-3-O-[(R)-(À)-Acetylmandelyl]-1,6:4,5-bis-O-(2,3-dimethoxybutane-
2,3-diyl)-myo-inositol (5) and 1d-1-O-[(R)-(À)-acetylmandelyl]-3,4:5,6-
bis-O-(2,3-dimethoxybutane-2,3-diyl)-myo-inositol (6):
A solution of
DCC (5.16 g, 25.0 mmol) in dry CH2Cl2 (50 mL) was added dropwise at
À788C over 3 h with stirring to a mixture of (Æ)-3ab (9.62 g, 23.5 mmol),
DMAP (50 mg, 0.41 mmol) and (R)-(À)-acetylmandelic acid (4.85 g,
25.0 mmol) in dry CH2Cl2 (200 mL). The resulting mixture was stirred
overnight at room temperature and then filtered through a bed of Celite,
which was then washed thoroughly with CH2Cl2 (2î50 mL). The com-
bined filtrate and washings were evaporated under reduced pressure to
give a foam. The individual diastereoisomers were separated by flash
chromatography on silica (CHCl3/acetone 15:1 and then EtOAc/toluene
2:3) to give the less polar diastereoisomer 5 (5.04 g, 36.5%) and then the
more polar diastereoisomer 6 (5.03 g, 36.5%) as waxy solids. Both dia-
stereoisomers were recrystallised from hexane. Compound 5 (m.p. 101
1038C), 6 (m.p. 119 1228C).
CCDC-199689 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge via www.ccdc.cam.
ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
(+44)1223-336033; or deposit@ccdc.cam.uk).
dl-1,6:4,5-Bis-O-(2,3-dimethoxybutane-2,3-diyl)-myo-inositol [(Æ)-3ab]:
A mixture of methanol (400 mL), camphorsulfonic acid (1 g) and tri-
methyl orthoformate (100 mL) was stirred vigorously at room tempera-
ture. myo-Inositol (25 g, 138.5 mmol) was added to the stirred solution,
followed by butanedione (25 mL, 285 mmol), and the mixture was heated
under reflux for 41 h. The cherry red suspension was allowed to cool and
was then filtered through a large sinter funnel. The precipitate was
washed with methanol and then with diethyl ether to leave the symmetri-
cal bis(butane-2,3-diacetal) 2 [(14.75 g, 26%) Rf = 0.37, (CH2Cl2/acetone
3:1)] as a white solid. The red mother liquor and combined washings
were concentrated and then purified by flash chromatography (CH2Cl2/
acetone 3:1) to give a second product (Rf = 0.34) as a foam. Addition of
ether to the foam gave a white solid, which was recrystallised from ace-
tone/hexane to give pure dl-1,6:4,5-bis-O-(2,3-dimethoxybutane-2,3-
diyl)-myo-inositol [(Æ)-3ab] (11.15 g, 20%) from acetone/hexane; m.p.
215 2188C.
1H 1H COSY (400 MHz, CDCl3): d = 1.29, 1.30, 1.32, 1.33 (4îs, 12H;
4îCH3), 2.70 (br, D2O exch, 1H; OH-3), 2.94 (br, D2O exch, 1H; OH-
2), 3.24 (s, 3H; OCH3), 3.27 (s, 6H; 2îOCH3), 3.29 (s, 3H; OCH3), 3.51
(dd, J = 2.6, 9.9 Hz, 1H; H-3), 3.61 (dd, J = 9.9, 9.9 Hz, 1H; H-5), 3.64
(br, D2O exch. gives dd, J = 9.9, 2.7 Hz, 1H; H-3), 3.92 (dd, J = 9.9,
9.9 Hz, 1H; H-4 or H-6), 4.06 (dd, J = 9.9, 9.9 Hz, 1H; H-4 or H-6), 4.11
(br, exch. gives dd, J = 2.7, 2.7 Hz, 1H; H-2) ppm; 13C NMR (100 MHz,
CDCl3): d = 18.07, 18.12, 18.17 (4îCH3), 48.20, 48.27, 48.41 (4îOCH3),
65.93, 68.21, 69.35, 70.13, 70.28, 70.64 (6îCH), 99.15, 99.33, 99.88, 100.39
(4îCq of BDA) ppm; elemental analysis calcd (%) for C18H32O10
(408.45): C 52.93, H 7.90; found: C 52.5, H 7.95.
Compound 5: Rf = 0.22 (CHCl3/acetone 15:1); [a]2d0 = +174 (c = 0.5 in
CHCl3); 1H NMR (400 MHz, CDCl3): d = 1.27 (s, 3H; CH3), 1.29 (s,
3H; CH3), 1.30 (s, 6H; 2îCH3), 2.17 (s, 3H; COCH3), 3.22, 3.25, 3.26,
3.27 (4îs, 12H; 4îOCH3), 3.51 (dd, J = 2.5, 9.9 Hz, 1H; H-1), 3.67 (dd,
J = 9.8, 9.8 Hz, 1H; H-5), 4.06 (dd, J = 10.1, 10.1 Hz; 1 H; H-4 or H-6),
4.12 4.20 [m, 2H; H-2 and (H-4 or H-6)], 4.84 (dd, J = 2.7, 9.7 Hz, 1H;
H-3), 5.89 (s, 1H; acetylmandelyl CH), 7.35 7.40 (m, 3H; ArH), 7.47
7.52 (m, 2H; ArH) ppm; 13C NMR (100 MHz, CDCl3): d = 18.01, 18.07,
18.12 (4îCH3), 21.10 (CH3CO), 48.00, 48.42, 48.47 (4îOCH3), 65.46,
67.16, 68.25, 68.42, 68.60, 72.87, 75.47 (6îmyo-inositol ring carbons and
1îCH acetylmandelate), 99.09, 99.33, 99.89, 100.37 (4îCq), 127.88,
129.00, 129.63 (3îCH, Ar), 133.60 (Cq, Ar), 167.81, 171.24 (2îCq carbon-
yl) ppm; elemental analysis calcd (%) for C28H40O13 (584.62): C 57.53, H
6.90; found: C 57.9, H 7.13.
Compound 6: Rf = 0.12 (CHCl3/acetone 15:1); [a]2d0 = À210 (c = 0.5 in
CHCl3); 1H NMR (400 MHz, CDCl3): d = 1.01, 1.22, 1.26, 1.30 (4îs,
12H; 4îCH3), 2.17 (s, 3H; COCH3), 2.72, 3.22, 3.23, 3.25 (4îs, 12H; 4î
OCH3), 3.53 (dd, J = 2.4, 10.1 Hz, 1H; H-3), 3.61 (dd, J = 9.8, 9.8 Hz,
1H; H-5), 4.05 (dd, J = 9.8, 9.8 Hz, 1H; H-4 or H-6), 4.06 (dd, J = 9.8,
9.8 Hz, 1H; H-4 or H-6), 4.23 (dd, J = 2.4, 2.4 Hz, 1H; H-2), 4.91 (dd, J
= 3.0, 10.7 Hz, 1H; H-1), 5.97 (s, 1H; acetylmandelyl CH), 7.34 7.39 (m,
3H; ArH), 7.46 7.51 (m, 2H; ArH) ppm; 13C NMR (100 MHz, CDCl3):
d = 17.87, 17.96, 18.05, 18.12 (4îCH3), 21.10 (CH3CO), 47.79, 48.40,
48.42, 48.48 (4îOCH3), 65.45, 66.96, 68.34, 68.94, 68.98, 72.44, 74.91 (6î
myo-inositol ring carbons and 1îCH acetylmandelate), 99.13, 99.25,
99.59, 100.40 (4îCq), 128.43, 128.83, 129.47 (CH, Ar), 133.62 (Cq, Ar),
168.42, 170.56 (Cq, carbonyl) ppm; elemental analysis calcd (%) for
C28H40O13 (584.62): C 57.53, H 6.90; found: C 57.9, H 7.05.
6211
Chem. Eur. J. 2003, 9, 6207 6214
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

B. V. L. Potter et al.
FULL PAPER
d-1,6:4,5-Bis-O-(2,3-dimethoxybutane-2,3-diyl)-myo-inositol (3a): A mix-
= 4.7, 9.0, 9.4, D2O exch. gives dd, J = 9.4, 9.4 Hz, 1H; H-4 or H-6),
3.94 (dd, J = 2.3, 2.3 Hz; 1 H; H-2), 4.62 (s, 2H; CH2Ph), 4.69 4.72
(brm, D2O exch. gives AB, J = 11.7 Hz, 3H; CH2Ph and OH), 4.75 (d, J
= 4.7 Hz, D2O exch., 1H; OH), 4.81 (d, J = 4.7 Hz, D2O exch., 2H; 2î
OH), 7.21 7.41 (m, 10H; CH2Ph) ppm; 13C NMR (100 MHz, (CD3)2SO):
d = 71.26, 73.87 (2îCH2Ph), 71.93, 72.38, 72.73, 75.28, 78.54, 80.19 (6î
myo-inositol ring carbons), 126.77, 126.86, 126.94, 127.16, 127.72, 127.86
(CH, Ar), 138.94, 139.48 (Cq, Ar) ppm; elemental analysis calcd (%) for
C20H24O6 (360.41): C 66.65, H 6.71; found: C 66.3, H 6.77.
ture of compound
5 (3.10 g, 5.3 mmol), sodium hydroxide (1.20 g
30 mmol) and methanol (150 mL) was heated at reflux for 30 min. The
mixture was cooled and then neutralised with carbon dioxide. The re-
maining solid was dissolved in water (50 mL), and evaporated to dryness
in vacuo. The crude product was extracted with CH2Cl2 (4î100 mL), and
purification by flash chromatography (CH2Cl2/acetone 2:1) gave 3a as a
solid (1.96 g, 91%). Rf
= 0.32 (CH2Cl2/acetone 2:1); m.p. 235 2378C
(from acetone/hexane); [a]2d0 = +270 (c = 0.5 in CHCl3); (FAB)+ acc.
mass found 377.1812 [MÀOMe]+ ; C17H29O9 calcd 377.1811; elemental
analysis calcd (%) for C18H32O10 (408.45): C 52.93, H 7.90; found: C 52.5,
H 7.94.
d-1,2-Di-O-benzyl-myo-inositol (8b): A mixture of compound 7b (1.64 g,
2.78 mmol) and 95% aqueous trifluoroacetic acid (10 mL) was stirred in
CH2Cl2 (10 mL) at room temperature for 30 minutes. Workup and purifi-
cation as for compound 8a gave the title compound (0.747 g, 75%). Rf =
The NMR data were the same as for the racemic compound.
0.24 (CHCl3/MeOH 6:1); m.p. 197 1988C; [a]d20
DMF); elemental analysis calcd (%) for C20H24O6 (360.41): C 66.65, H
6.71; found: C 67.0, H 6.71.
=
À47 (c
= 0.5 in
d-3,4:5,6-Bis-O-(2,3-dimethoxybutane-2,3-diyl)-myo-inositol (3b): A mix-
ture of compound
6 (3.00 g, 5.13 mmol), sodium hydroxide (1.20 g
30 mmol) and methanol (150 mL) was heated at reflux for 30 min.
Workup and purification as for compound 3a gave 3b (1.91 g, 91%). Rf
The NMR data were the same as for compound 8a.
=
0.32 (CH2Cl2/acetone 2:1); m.p. 236 2388C (from acetone/hexane);
d-2,3-Di-O-benzyl-1,4,5,6-tetrakis[di(benzyloxy)phospho]-myo-inositol
(9a): A mixture of bis(benzyloxy)diisopropylaminophosphine (1.035 g,
3.00 mmol) and 1H-tetrazole (0.35 g, 5 mmol) in CH2Cl2 (10 mL) was stir-
red at room temperature for 30 minutes in order to give the tetrazolide
intermediate (dP 127 ppm). The tetraol 8a (216 mg, 0.6 mmol) was then
added, and the solution was stirred for a further 30 minutes. The reaction
mixture was cooled to À788C, and MCPBA (1.60 g, 4.6 mmol) was
added. The cooling bath was removed and stirring was continued at room
temperature for a further 30 minutes. The solvent was evaporated to give
a white solid, which was dissolved in EtOAc (50 mL) and washed succes-
sively with 10% aqueous sodium metabisulfite and a saturated aqueous
solution of sodium hydrogen carbonate (50 mL of each). The organic
layer was separated and dried (MgSO4), and the organic solvent was
evaporated to give a syrup. The product was purified by flash chromatog-
raphy, first with CHCl3/acetone (5:1) and then with EtOAc/hexane (2:1),
[a]2d0 = À270 (c = 0.5 in CHCl3); (FAB)+ acc. mass found 377.1816
[MÀOMe]+ ; C17H29O9 calcd 377.1811; elemental analysis calcd (%) for
C18H32O10 (408.45): C 52.93, H 7.90; found: C 52.4, H 7.86.
The NMR data were the same as for the racemic compound.
d-2,3-Di-O-benzyl-1,6:4,5-bis-O-(2,3-dimethoxybutane-2,3-diyl)-myo-inosi-
tol (7a): A mixture of compound 3a (1.73 g, 4.25 mmol), DMF (40 mL),
benzyl bromide (2.0 mL, 20 mmol) and sodium hydride (0.8 g, 20 mmol)
was stirred at room temperature for 2 h. TLC (ether/hexane 1:1) showed
a new product (Rf = 0.36), and no starting material. The excess sodium
hydride was destroyed with methanol (5 mL) and the solvents were
evaporated in vacuo. The remaining syrup was partitioned between water
and ether (100 mL of each) and washed with 0.1m aqueous hydrochloric
acid, a saturated solution of sodium hydrogen carbonate and water
(100 mL of each). The organic layer was dried (MgSO4) and the remain-
ing syrup was purified by flash chromatography (ether/hexane 1:1) to
to provide the title compound as a syrup (740 mg, 88%). Rf
= 0.12
1
(CHCl3/acetone 5:1); [a]d20 = 0 (c = 0.5 in CHCl3); H NMR (400 MHz,
CDCl3): d = 3.51 (dd, J = 9.8 Hz, 1H; H-1), 4.30 (ddd, J = 2.7, 7.4,
9.8 Hz, 1H; H-3), 4.44 (brs, 1H; H-2), 4.43, 4.52 (AB, J = 11.3 Hz, 2H;
CH2Ph), 4.59 (ddd, J = 9.4, 9.4, 9.8 Hz, 1H; H-5), 4.64, 4.79 (AB, J =
11.3 Hz, 2H; CH2Ph), 4.82 5.10 (m, 18H; 8îCH2Ph, H-4 and H-6), 7.06
7.33 (m, 50H; Ar) ppm; 13C NMR (100 MHz, CDCl3): d = 69.14, 69.38,
69.51, 69.62, 69.77 (CH2Ph of benzyl phosphate), 72.25, 75.25 (CH2Ph),
74.54, 75.75, 76.80, 77.30, 77.59 (6îmyo-inositol ring carbons), 127.35,
127.43, 127.56, 127.58, 127.62, 127.72, 127.81, 127.85, 127.90, 128.00,
128.04, 128.09, 128.12, 128.14, 128.16, 128.38 (CH, Ar), 132.61, 134.16,
135.35, 135.45, 135.75, 135.82, 137.04, 137.86 (Cq, Ar) ppm; (FAB)+ acc.
mass found 1401.4119; C76H77O18P4 calcd 1401.4060; elemental analysis
calcd (%) for C76H77O18P4 (1401.32): C 65.14, H 5.47; found: C 64.8, H
5.45.
give a white foam (2.11 g, 84%). Rf = 0.36 (Et2O/hexane 1:1); [a]d20
=
+182 (c = 0.5 in CHCl3); 1H NMR (400 MHz, CDCl3): d = 1.27, 1.29,
1.31, 1.33 (4îs, 12H; 4îCH3), 3.21, 3.27, 3.28 (4îs, 12H; 4îOCH3),
3.45 (dd, J = 2.3, 10.1 Hz, 1H; H-1 or H-3), 3.46 (dd, J = 2.7, 10.1 Hz,
1H; H-1 or H-3), 3.65 (dd, J = 9.8, 10.1 Hz, 1H; H-5), 3.91 (dd, J = 2.3,
2.3 Hz, 1H; H-2), 4.18 (dd, J = 10.15, 10.15 Hz, 1H; H-4 or H-6), 4.19
(dd, J = 9.8, 10.15 Hz, 1H; H-4 or H-6), 4.63, 4.82 (AB, J = 11.7 Hz,
2H; CH2Ph), 4.85, 4.76 (AB, J = 11.7 Hz, 2H; CH2Ph), 7.22 7.52 (m,
10H; CH2Ph) ppm; 13C NMR (100 MHz, CDCl3): d
= 17.77, 17.86,
17.89, 18.01 (4îCH3), 47.81, 47.88, 48.03, 48.08 (4îOCH3), 72.78, 74.20
(2îCH2Ph), 65.78, 68.77, 69.95, 70.32, 76.33, 77.66 (6îmyo-inositol ring
carbons), 98.52, 98.97, 99.42, 99.55 (4îCq of BDA), 126.92, 127.10,
127.73, 127.87, 128.05 (CH, Ar), 138.96, 139.10 (Cq, Ar) ppm; elemental
analysis calcd (%) for C32H44O10 (588.70): C 65.29, H 7.53; found: C 65.4,
H 7.41.
d-1,2-Di-O-benzyl-3,4,5,6-tetrakis[di(benzyloxy)phospho]-myo-inositol
(9b): A mixture of bis(benzyloxy)diisopropylaminophosphine (1.035 g,
3.00 mmol) and 1H-tetrazole (0.35 g, 5.00 mmol) in CH2Cl2 (10 mL) was
stirred at room temperature for 30 minutes in order to give the tetrazo-
lide intermediate (dP 127 ppm). The tetraol 8b (216 mg, 0.60 mmol) was
added, and the solution was stirred for a further 30 minutes. Workup and
purification as for the enantiomer 9a gave the title compound as a syrup
(457 mg, 54%). Rf = 0.12 (CHCl3/acetone 5:1); [a]2d0 = 0 (c = 0.5 in
CHCl3); (FAB)+ acc. mass found 1401.4123; C76H77O18P4 calcd
1401.4060; elemental analysis calcd (%) for C76H77O18P4 (1401.32): C
65.14, H 5.47; found: C 65.0, H 5.49.
d-1,2-Di-O-benzyl-3,4:5,6-bis-O-(2,3-dimethoxybutane-2,3-diyl)-myo-inosi-
tol (7b): A mixture of compound 3b (1.43 g, 3.51 mmol), DMF (40 mL),
benzyl bromide (2.0 mL, 20 mmol) and sodium hydride (0.8 g, 20 mmol)
was stirred at room temperature for 2 h. TLC (ether/hexane 1:1) showed
a new product (Rf = 0.36), and no starting material. Workup and purifi-
cation as for the enantiomer 7a gave the title compound (1.78 g, 86%),
as a white foam.Rf = 0.36 (Et2O/hexane 1:1); [a]2d0 = À182 (c = 0.5 in
CHCl3); elemental analysis calcd (%) for C32H44O10 (588.70): C 65.29, H
7.53; found: C 65.3, H 7.54.
The NMR data were the same as for compound 7a.
The NMR data were identical to those for compound 9a.
d-2,3-Di-O-benzyl-myo-inositol (8a): A mixture of compound 7a (1.94 g,
3.30 mmol) and 95% aqueous trifluoroacetic acid (10 mL) was stirred in
CH2Cl2 (10 mL) at room temperature for 30 minutes. The solvents were
evaporated under reduced pressure and co-evaporated with chloroform
(2î25 mL). The solid was suspended in cold MeOH (50 mL) and filtered
off to give a white powder, which was recrystallised from acetonitrile
1d-myo-Inositol 1,4,5,6-tetrakisphosphate (1a): Compound 9a (473 mg,
338 mmol) was hydrogenolysed in a mixed solvent containing methanol
and water (60 mL, 5:1) in the presence of palladium hydroxide (1.0 g,
20% on carbon) at 50 psi for 20 h. The reaction mixture was filtered
through a PTFE syringe filter to remove the catalyst, and the solvents
were evaporated in vacuo to give a glassy residue. The compound was
then subjected to ion-exchange chromatography on Q-Sepharose Fast
Flow with a triethylammonium hydrogen carbonate buffer gradient of
0.40m to 1.0m over 80 tubes and 1.0m to 2.0m buffer over 15 tubes. The
compound eluted between 0.8m to 1.0m buffer, and was detected by the
Briggs test[24] for the detection of inorganic phosphate. The required frac-
(0.975 g, 82%). Rf = 0.24 (CHCl3/MeOH 6:1); m.p. 197 1988C; [a]d20
=
+47 (c = 0.5 in DMF); 1H NMR (400 MHz, (CD3)2SO): d = 2.97 (ddd,
J = 4.7, 9.0, 9.4 Hz, D2O exch. gives dd, J = 9.0, 9.4 Hz, 1H; H-5), 3.21
(dd, J = 2.3, 10.15 Hz, 1H; H-3), 3.26 (ddd, J = 2.3, 4.7, 9.8 Hz, H-1,
D2O exch. gives dd, J = 2.0, 9.8 Hz, 1H; H-1), 3.44 (ddd, J = 4.7, 9.4,
9.4, D2O exch. gives dd, J = 9.4, 9.4 Hz, 1H; H-4 or H-6), 3.59 (ddd, J
6212
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 6207 6214

Synthesis of Intracellular Signalling Molecules
6207 6214
tions were pooled and further quantified by the Briggs test[24] to give the
pure title compound (178 mmol, 53%). [a]2d0 = À7.9 (c = 1.4 in MeOH)
[lit.[10a] À6.2 (c = 2.15 in H2O, pH 9.5); lit.[10b] À6.9 (c = 0.65 in H2O,
8 K+ salt); lit.[10c] À10.2 (c = 2.46 in H2O, pH 10.7); lit.[10e] +8.0 (c =
1.05 in H2O, pH 7); lit.[10f] +8.0 (c = 1.1 in H2O, pH 7); lit.[10g] À4.8 (c =
46%) as a colourless, crystalline solid. Rf = 0.16 (ethyl acetate/hexane
1:1); m.p. 122 1248C (from ethanol) (lit.[22] 119 1208C); [a]d20 = À23 (c
=
0.95 in CHCl3) [lit.[22] À22.7 (c
=
1.27 in CHCl3)]; 1H NMR
(400 MHz, CDCl3): d = 2.07 (6H; 2îCH3), 2.10 (6H; 2îCH3), 2.11
(6H; 2îCH3), 4.04 (J = 12.1, 5.9Hz, 2H; H-1a and H-6a), 4.31 (J =
12.1, 4.7Hz, 2H; H-1band H-6b), 5.24 5.31 (m, 2H; 2îCH), 5.34 5.39
(m, 2H; 2îCH) ppm; 13C NMR (100 MHz, CDCl3): d = 20.65 (2îCH3),
20.72 (2îCH3), 20.76 (2îCH3), 61.67 (C-1 and C-6), 68.65 (2îCH),
69.10 (2îCH), 169.47 (2îC=O), 169.68 (2îC=O), 170.07 (2îC=
O) ppm.
1
2.7 in H2O, free acid)]; H NMR (400 MHz, D2O): d = 3.56 (dd, J = 2.7,
9.7 Hz, 1H; H-3), 4.08 (ddd, J = 2.7, 9.7, 9.7 Hz, 1H; H-1), 4.16 (br, 1H;
H-2), 4.17 (ddd, J = 9.5, 9.5, 9.5 Hz, 1H; CH), 4.41 (ddd, J = 9.4, 9.4,
9.4 Hz, 1H; CH), 4.60 (ddd, J = 9.8, 9.8, 9.8 Hz, 1H; CH) ppm; 31P
1
NMR (160 MHz, D2O H-decoupled): d = 3.26 (s, 1P), 2.39 (s, 1P), 2.14
(s, 1P), 2.03 (s, 1P) ppm; m/z (Àve ion FAB): 498.9 (100%) [MÀH]À;
(FAB)À acc. mass found: 498.9209; C6H15O18P4 calcd 498.9209.
1d-myo-Inositol 3,4,5,6-tetrakisphosphate (1b): Deprotection of com-
pound 9b as described for 9a, and purification as described for enantiom-
Acknowledgement
er 1a gave tetrakisphosphate 1b (72 mmol, 37%); [a]d20
= +8.1 (c =
1.72 in MeOH) [lit.[10a] +6.2 (c = 2.15 in H2O, pH 9.5); lit.[10b] +7.2 (c =
2.3 in H2O, 8 K + salt); lit.[10c] +9.8 (c = 1.43 in H2O, pH 11.1); lit.[10d]
À3.0 (c = 1 in H2O, free acid); lit.[10f] À2.9 (c = 0.3, H2O, pH 1.6); lit.[10f]
À5.6 (c = 0.2 in H2O, pH 7); lit.[10g] + 4.1 (c = 2.7 in H2O, free acid)].
We thank the Wellcome Trust for Programme Grant support (060554)
and the EPSRC crystallographic service, University of Southampton, for
the collection of a crystallographic data set for (Æ)-4. We also thank Dr.
S. B. Shears, National Institute of Environmental Health Sciences, North
Carolina, U.S.A for preliminary biological evaluation of synthetic
Ins(3,4,5,6)P4 and Ins(1,4,5,6)P4.
NMR spectra and mass spectra were as for 1a.
1d-3-O-methyl-myo-inositol [(+)-bornesitol, 10]: Methyl iodide (2.5 mL,
40 mmol) was added to a mixture of diol (+)-3a (408 mg, 1.00 mmol), di-
butyl tin oxide (274 mg, 1.1 mmol) and tetrabutylammonium bromide
(355 mg, 1.1 mmol) in acetonitrile (40 mL). The mixture was heated at
reflux for 17 h under a Soxhlet apparatus containing 3 ä sieves, allowed
to cool, and then concentrated by evaporation under reduced pressure.
The residue was purified by flash chromatography (EtOAc/hexane 2:1)
to give a white solid (411 mg). Aqueous TFA (90%, 4 mL) was added to
this solid and the solution was stirred at room temperature for 1.5 h,
after which time TLC showed a single product (Rf = 0.22, acetone/water
5:1). The solution was concentrated by evaporation under reduced pres-
sure, and then under high vacuum to remove traces of TFA and butane-
dione, giving pentaol 10 (157 mg, 81%) as a crystalline solid. Rf = 0.22
(acetone/water 5:1); m.p. 202 2058C (from MeOH/EtOH) (lit.[20]
[1] a) R. F. Irvine, M. J. Schell, Nat. Rev. Mol. Cell Biol. 2001, 2, 327
338 and references therein; b) S. B. Shears, Biochim. Biophys. Acta
1998, 1436, 49 67, and references therein.
[2] M. C. Hermosura, H. Takeuchi, A. Fleig, A. M. Riley, B. V. L.
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205 2078C); [a]2d0
=
+34 (c = 0.6 in H2O) [lit.[20] +31.9 (c = 1 in
H2O)].
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l-2,3:4,5-Bis-O-(2,3-dimethoxybutane-2,3-diyl)-iditol (11): A solution of
the diol (À)-3b (320 mg, 0.783 mmol) in CH2Cl2 (5 mL) was added to a
[21]
suspension of SiO2/NaIO4
(2.0 g) in CH2Cl2 (5 mL). The suspension
was stirred vigorously at room temperature for 16 h and was then filtered
through a pad of Celite. The Celite was washed well with CHCl3 (4î
20 mL) and the combined washings were concentrated by evaporation
under reduced pressure to leave a colourless oil. The oil was taken up in
ethanol (10 mL), and NaBH4 (78 mg, 2.0 mmol) was added. The mixture
was stirred at room temperature for 30 min, water (2 mL) was added,
and after a further 30 min the mixture was concentrated by evaporation
under reduced pressure. The residue was taken up in water (20 mL) and
extracted with CH2Cl2 (5î20 mL). The combined organic extracts were
dried (MgSO4) and then concentrated by evaporation under reduced
pressure to give the diol 11 (281 mg, 87%) as a white crystalline solid. Rf
= 0.24 (ethyl acetate); m.p. 205 2078C, with sublimation (from EtOAc/
hexane); [a]2d0 = À190 (c = 1 in CHCl3); 1H NMR (270 MHz, CDCl3): d
= 1.27 (s, 6H; 2îCH3), 1.29 (s, 6H; 2îCH3), 3.25 (s, 12H; 4îOCH3),
3.49 (dd, J = 10.4, 4.8 Hz, D2O exch., 2H; 1-OH and 6-OH), 3.62 (ddd,
J = 10.4, 10.4, 3.0 Hz, 2H; H-1a and H-6a), 3.75 3.85 (m, 4H; H-1b, H-
6band 2îCH), 3.95 4.05 (m, 2H; 2îCH) ppm; 13C NMR (100 MHz,
CDCl3): d = 17.60 (2îCH3), 17.70 (2îCH3), 48.36 (2îOCH3), 48.43
(2îOCH3), 63.85 (C-1 and C-6), 70.03 (2îCH), 72.43 (2îCH), 99.03
(2îCq of BDA), 99.44 (2îCq of BDA) ppm; m/z (+ve ion FAB): 347.3
(10), 315.2 (50), 116.2 (50), 101.1 (100), 73.1 (75); elemental analysis
calcd (%) for C18H34O10 (410.46): C 52.67, H 8.35; found: C 52.6, H 8.3.
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Angew. Chem. Int. Ed. Engl. 1995, 34, 1933 1972.
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(2 mL of each) was added to compound 11 (100 mg, 0.244 mmol). The
solution was stirred at room temperature for 2h and was then concentrat-
ed by evaporation under reduced pressure. Pyridine (2 mL) and acetic
anhydride (1 mL) were added to the residue and the solution was stirred
at room temperature for 16 h. The solution was concentrated by evapora-
tion under reduced pressure, and the residue was purified by flash chro-
matography (hexane/EtOAc 3:2 to 1:1) to give 12 (49 mg, 0.113 mmol,
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6213
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

B. V. L. Potter et al.
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Received: June 5, 2003
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Revised: August 15, 2003 [F5207]
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Chem. Eur. J. 2003, 9, 6207 6214