Regioselective deprotection of orthobenzoates for the synthesis of inositol phosphates

Article abstract of DOI:10.1039/b901890e

Synthetic myo-inositol 1,4,5-triphosphate, Ins(1,4,5)P₃, and myo-inositol 1,3,4,5-tetraphosphate, Ins(1,3,4,5)P₄, continue to be valuable in biological studies. Inositol orthoesters have proved an important class of intermediate to a

Full text of DOI:10.1039/b901890e

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Regioselective deprotection of orthobenzoates for the synthesis
of inositol phosphates†
Joanna M. Swarbrick,a Samuel Cooper,a Geert Bultynckb and Piers R. J. Gaffney*a
Received 29th January 2009, Accepted 2nd February 2009
First published as an Advance Article on the web 6th March 2009
DOI: 10.1039/b901890e
Synthetic myo-inositol 1,4,5-triphosphate, Ins(1,4,5)P3, and myo-inositol 1,3,4,5-tetraphosphate,
Ins(1,3,4,5)P4, continue to be valuable in biological studies. Inositol orthoesters have proved an
important class of intermediate to access these compounds. We investigated the ability of steric bulk
from a 4-O protecting group to direct DIBAL-H reduction of inositol orthobenzoates to generate the
natural Ins(1,4,5)P3 precursor 2,3,6-O-tribenzyl myo-inositol. Introduction of an equatorial 4-C-methyl
group imparts totally selective reduction and we report the synthesis of novel 4-C-methyl-Ins(1,4,5)P3
and 4-C-methyl-Ins(1,3,4,5)P4.
principal advantages of orthoesters lie in their simultaneous
Introduction
protection of the 1-, 3- and 5-hydroxyls, restricting the myo-inositol
conformation, and subsequent easy differentiation between the
remaining three hydroxyls.3 Past synthetic focus has been on
the orthoformates.4,5 However, trans-esterification of myo-inositol
with triethyl orthobenzoate generates myo-inositol orthobenzoate
(2) which is easily purified by recrystallisation in good yield.6
Full or partial deprotection of orthoesters can be carried
out by either acidic hydrolysis or reduction. Manipulation of
this deprotection has the potential to generate further pro-
tecting groups and selectively expose hydroxyl functionalities
(Scheme 1).
The inositol phosphates, particularly inositol 1,4,5-tri- and 1,3,4,5-
tetraphosphates (Fig. 1, 1a and 1b respectively), are best known
as cytosolic secondary messengers.1 They can be phosphorylated
on all six hydroxyls, with most possible regioisomers from inositol
mono- to hexa-phosphate known in nature, and are interconverted
by a battery of kinases and phosphatases. Disentanglement of
this signalling network is difficult due to the often interdependent
mechanisms of action, and the number of different pathways in
which they are involved. Identification of the participation of these
species in signalling pathways, perturbation of which leads to
disease states such as cancer and diabetes, has further increased the
need to understand this complex system. Inositol phosphates are
neither readily synthesised, nor isolable from nature, leading to a
wealth of multi-step synthetic routes.2 Use of unnatural analogues
as biological probes should help elucidate information about how
they function.
Fig. 1 Inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetraphosphate.
Inositol phosphate synthesis requires a comprehensive pro-
tecting group strategy, often commencing from readily prepared
building blocks such as inositol acetals or orthoesters. The
aDepartment of Chemistry, Imperial College London, South Kensington
Campus, SW7 2AZ, UK. E-mail: p.gaffney@imperial.ac.uk; Fax: +44
(020) 7594 5804; Tel: +44 (020) 7594 5881
bDepartment of Molecular Cell Biology, Laboratory of Molecular and
Cellular Signaling, Campus Gasthuisberg o/n-1 bus 802, Herestraat 49, BE-
3000, Leuven, Belgium. E-mail: geert.bultynck@med.kuleuven.be; Fax: +32
(16) 330215; Tel: +32 (16) 345991
Scheme 1 Deprotection of orthoesters.
Acidic hydrolysis of an inositol orthoformate presumably
generates a formate ester initially, which is rapidly hydrolysed
to reveal all three hydroxyls.7 By contrast acidic hydrolysis of
orthobenzoates generates a 1(3)-O-benzoate ester (Bz), which can
† Electronic supplementary information (ESI) available: Preparation
of compounds 2–3d and 8–10, plus all analytical data. See DOI:
10.1039/b901890e
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be isolated. Alternatively reduction of the orthoformate with 2 eq.
DIBAL-H generates a bridging 1,3-O-methylidene acetal;8 this
is a much less acid labile protecting group than the benzylidene
acetal derived from reduction of an orthobenzoate.9 Additionally,
further reduction of the benzylidene acetal with excess DIBAL-
H produces a benzyl ether (Bn). If PG π PG¢ both the 1-
and 3-O-benzyl isomers result from reduction of the asymmetric
benzylidene acetal. Thus, if the easily prepared 2,6-O-dibenzyl
orthobenzoate 3a can be similarly reduced with DIBAL-H, the
resultant 2,3,6-O-tribenzyl ether 6a is the synthetically useful
precursor to inositol 1,4,5-triphosphate (1a). Consequently, we
sought to affect the regioselectivity of DIBAL-H reduction of 3a
to favour isomer 6a over the unwanted 1,2,6-O-tribenzyl ether 7a.
Results and discussion
IP4 (1b) may be synthesised from readily prepared dibenzyl ether
3a (Scheme 2). Key steps were direct regioselective allylation
of crude inositol orthobenzoate to give the 4-O-allyl ether in
good yield (73%).10 This directed benzylation of the 2,6-diol to
provide the established IP4 building block 4, after unblocking of
the temporary protecting groups.6,11 Phosphorylation and global
deprotection proceeded as previously reported.12a Intermediate
3a offers potential for resolution, for example with camphanate
esters,13 and was the key starting point for our investigations of
orthobenzoate hydrolysis and reduction.
Scheme 3 Reduction of myo-inositol orthobenzoates. Reagents and
conditions: a) TbdmsCl, imidazole, Et3N, DMF, 100 ◦C; b) TbdpsCl,
imidazole, Et3N, DMF, 100 ◦C; c) DIBAL-H, DCM, -78 ◦C to rt; d)
TBAF, THF; e) (BnO)2PNiPr2, tetrazole then mCPBA; f) Pd (black), H2.
6a12b and 1,2,6-O-tribenzyl-myo-inositol 7a, which are easily
separated by flash chromatography. However, the highly polar
triols were recovered in poor yield due to difficulty in handling and
there was little selectivity for the desired isomer 6a as determined
by 1H-NMR of the crude product mixture (Table 1). To complete
the synthesis of IP3 (1a), 6a was phosphorylated and globally
deprotected as previously described.12a
We had anticipated that slow addition of DIBAL-H to 3a
would favour reduction to 6a due to initial rapid reaction of
the unprotected 4-O with bulky DIBAL-H. This was thought
to occur readily because treatment of 8 with 2 eq. DIBAL-
H exclusively unblocked the acid labile 6-O-Dtpx ether to give
symmetrical mono 2-O-Dtpx ether 9, presumably by trans-annular
Scheme 2 Synthesis of IP4. Reagents and conditions: a) NaH, allylBr;
◦
t
b) NaH, BnBr, 60 C; c) BuOK; d) TsOH; e) TFA-H2O (4:1), 24 h; f)
NaOMe, MeOH, 75 ◦C; g) (BnO)2PNiPr2, tetrazole, then mCPBA; h) Pd
(black), H2.
Partial reduction of 2,4,6-O-tribenzyl ether 3b, synthesised by
exhaustive benzylation of inositol orthobenzoate (2), with limited
DIBAL-H is known to give 5b in good yield.9 However, upon
addition of 3.5 eq. DIBAL-H to 3b we observed total reduction
of the orthobenzoate to 1,2,4,6-O-tetrabenzyl inositol (6b ∫ 7b,
Scheme 3) generated by a second hydride insertion into the
1(3)-O-C bond of symmetrical 1,3-O-benzylidene acetal 5b. We
then explored the full reduction of 3a with DIBAL-H in the hope
of selectively producing the building block 6a required for IP3 (1a)
synthesis.
Table 1 Ratio of crude products in 1H-NMR of DIBAL-H reduction
of 3
Ratio of products
Starting material
R
3-O-Bn, 6
:
1-O-Bn, 7
3a
3b
3c
3d
H
Bn
Tbdms
Tbdps
4
1
4
4
:
:
:
:
3
1
5
5
Treatment of asymmetrical myo-inositol orthobenzoate 3a with
3.5 eq. DIBAL-H generates both 2,3,6-O-tribenzyl-myo-inositol
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chelation to the 6-O of the Lewis acidic 4-O-DIBAL adduct
(Scheme 4). The identity of 9 was confirmed by alkylating 2 with
DtpxCl under milder conditions than those required to give 8;
this reaction is notable for its unusual regioselectivity for the 2-O
which has previously been observed with a few large electrophiles
under mildly basic conditions.4,7,14 Unfortunately variation of the
temperature and speed of addition of DIBAL-H to 3a had little
effect on the 6a:7a ratio.
Scheme 5 Synthesis of 4-C-Me IP3. Reagents and conditions: a) Dess–
Martin periodinane, DCM; b) MeMgBr, ether, -78 ◦C to rt, 3 h; c) 4 eq.
DIBAL-H, DCM, -78 ◦C to rt, 4 h; d) (BnO)2PNiPr2, tetrazole followed
by mCPBA; e) Pd (black), H2.
with 4 eq. DIBAL-H then generated exclusively the IP3 ana-
logue precursor 11 and there was no evidence of the potential
1-O-benzyl regioisomer. Phosphitylation of the three free hydrox-
yls, including the unnatural tertiary centre, followed by oxidation
and then global deprotection of the nine benzyl groups generated
the novel IP3 analogue 12a.
Scheme 4 Selective 4-O-Dtpx unblocking with DIBAL-H. Reagents and
conditions: a) DtpxCl, MeCN, pyridine, reflux; b) DIBAL-H, DCM, 0 ◦C;
c) DtpxCl, pyridine.
The 4-C-methyl IP4 analogue, 12b, was also synthesised
(Scheme 6). Previously reported conditions for orthobenzoate
removal [TFA-water (4:1), as with 3a], were not sufficient to initiate
acid hydrolysis of 10. Treatment of 10 with conc. HCl-MeOH at
reflux generated a mixture of benzoate esters 13a and 13b. Simi-
larly to hydrolysis of orthobenzoate 3a,17 there was no preference
for the 1-O benzoate. However, as has been described for 3a,
the 1-O-benzoate may be isolated and used in the preparation
of the corresponding 1-O-phosphatidyl inositol triphosphate. The
same is assumed for the 4-C-methyl lipid head group precursor
13a. The marked increase in orthobenzoate stability compared
to 3a may be interpreted in terms of the established mechanism
for cis,cis-cyclohexane 1,3,5-triol orthoester hydrolysis18 where
the rate determining step is the chair-boat interconversion of
the inositol ring of the intermediate cation to permit irreversible
We attempted to affect the regioselectivity of the second
DIBAL-H reaction by increasing the steric congestion at the
4-O with a bulky silyl ether. From its 1H-NMR the inositol
ring of benzylidene 5b is known to occupy a boat conformation
[d5-H = 3.79 (t, J 8.6)]9 and it was anticipated that restricted
access of DIBAL-H to the 3-O in benzylidene intermediates 5c
and 5d would favour reduction of the 1-O acetal bond. Therefore
4-O-tert-butyldimethylsilyl (Tbdms) ether 3c and the bulkier
4-O-tert-butyldiphenylsilyl (Tbdps) derivative 3d were prepared
by silylation of 3a under forcing conditions. The resultant silyl
ethers were treated with excess DIBAL-H, but in neither case
was one regioisomer preferred, although the reduction products
were easier to handle and separate than 6a and 7a. The identities
of 6d and 7d were confirmed by desilylation with TBAF to give
6a and 7a respectively. This lack of selectivity may be because
the substrates for the second reduction of 3c and 3d with excess
DIBAL-H are not benzylidenes 5c and 5d but rather their 5-
O-DIBAL-adducts. The protection of trans-cyclohexane 1,2-diol
with bulky silyl ethers is known to favour the 1,2-diaxial chair
conformation15 and repulsion between the bulky 4-O-silyl and 5-
O-DIBAL groups may force the inositol ring of 5c and 5d back into
a chair-like conformation with 4,5,6-triaxial substituents reducing
the contrast between the steric environment at the 1- and 3-O.
Noting that the equatorial 2-O-Bn limits access to the 1-
and 3-O of 3, we postulated that access to the 3-O would be
further constrained by a second adjacent equatorial substituent.
4-C-Methyl inositol orthobenzoate 10 was prepared from 3a by
oxidation of the 4-OH to the inos-4-ose, followed by stereo-
selective equatorial addition of a methyl group to re-establish
the myo-inositol stereochemistry16 (Scheme 5). Treatment of 10
Scheme 6 Synthesis of 4-C-Me IP4. Reagents and conditions: a) conc.
HCl-MeOH (1:2), 70 ◦C, 3 h; b) NaOMe, MeOH, 75 ◦C, 3 h; c)
(BnO)2PNiPr2, tetrazole then mCPBA; d) Pd (black), H2.
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trapping of the dioxacarbenium ion by water. It is postulated that
the additional equatorial substituent on the inositol ring of 4-
C-methyl 10 further hinders chair-boat flipping of the bicyclic
cation. The mixed benzoyl esters 13a and 13b were treated
with sodium methoxide in methanol to give 14, the tetraacetate
derivative of which exhibited a clear NOESY cross-peak between
the 4-C-methyl singlet and the inositol ring 6-H, consistent with
the expected 4-C stereoisomer having the methyl group axial.16
Tetrol 14 was subsequently phosphorylated and deprotected (as
described for 11) to generate IP4 analogue 12b.
Both 12a and 12b occupy the chair conformation, as indicated
by the large coupling constants between the three ring protons
having an anti relationship. For example the coupling constants
between the anti-periplanar 5- and 6-H (for 12a, J5,6 9.6; for 12b,
J5,6 9.7) are very similar to that in the natural ligands (for 1a, J5,6
9.2; for 1b, J5,6 9.4); this would have been reduced in a boat-like
conformation with the 4-C-methyl occupying a pseudo-equatorial
position. The similarity of conformation between natural IP3
1a and 4-C-methyl analogue 12a was reflected by the latter’s
ability to open the IP3 receptor (IP3R) Ca2+-release channels in
permeabilised L15 cells (Fig. 2).19,20 IP3 (1a) was found to release
ca. 70% of available Ca2+ (by comparison with that released by
the Ca2+ ionophore A23187) with an EC50 of 1.1 mM, whereas
racemic 4-C-methyl IP3 (12a) released ca. 50% with a lower EC50
of 13.8 mM.
Conclusions
The utility of orthobenzoate protection of inositol is demonstrated
in the synthesis of inositol phosphates 1a and 1b, by DIBAL-
H reduction and acidic hydrolysis of a common intermediate.
Introduction of a methyl substituent on the 4-C significantly
reduces the rate of acidic hydrolysis but enforces total regiocontrol
during DIBAL-H reduction. This allows facile access to the
novel 4-C-methyl derivatives of biologically active Ins(1,4,5)P3 and
Ins(1,3,4,5)P4, 12a and 12b.
Fig. 2 (A) A typical experiment in permeabilised L15 cells showing the
fractional loss of 45Ca2+ with time. The black bar indicates the addition of
IP3 (1a) or 4-C-methyl IP3 (12a). Cells were treated with A23187 to estimate
the maximal releasable 45Ca2+. (B) Dose response for IP3R-dependent Ca2+
release provoked by IP3 (1a) or 4-C-methyl IP3 (12a) in permeabilised L15
cells. Values were normalized to the A23187-releasable Ca2+. Data points
were obtained from at least 3 independent experiments and are plotted as
mean standard error of the mean. EC50 values were obtained by sigmoidal
curve fitting (OriginꢀR 7.0).
Experimental
General experimental
sphere. Dichloromethane, acetonitrile, toluene and triethylamine
were distilled from calcium hydride; THF and diethyl ether were
distilled from sodium metal and benzophenone; methanol was
distilled from magnesium methoxide; all, except triethylamine,
1H-, 13C- and 31P-NMR spectra were recorded on Bruker AC-270,
AM-360, AV-400 and AV-500 spectrometers. Chemical shifts are
referenced with respect to residual solvent signals, dH (CHCl3)
7.25 ppm, dH (d5-DMSO) 2.50 ppm, dH (HOD) 4.60 ppm, dC
(CDCl3) 77.50 ppm, dC (d6-DMSO) 39.43 ppm, or an external
˚
were stored over 4 A molecular sieves. Flash chromatography
was carried out using flash silica and medium pressure liquid
chromatography using TLC grade silica from Merck. Thin layer
chromatography was carried out using Merck silica gel 60 F254
glass-backed plates, compounds were visualised using UV light or
KMnO4 stain.
1
reference, dP (H3PO4) 0.00 ppm. The splitting patterns for H-
NMR spectra are denoted as follows; s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet), b (broad) and combinations
thereof. Coupling constants (J) are in Hertz (Hz). 13C-NMR
1
assignments and H-NMR assignments were made with the aid
of DEPT-90 and -135, HSQC, COSY and NOESY experiments.
13C- and 31P-NMR are proton decoupled. Deuterated solvents
were purchased from Apollo Scientific Ltd (d6-DMSO) or Merck
(all others). Other reagents were purchased from Sigma-Aldrich
Ltd or Acros Organics and used as supplied except where specified.
Mass spectra were recorded on a VG AutoSpec-Q (CI) or a
Micromass LCT Premier (ESI) mass spectrometer. Reactions were
carried out under anhydrous conditions under a nitrogen atmo-
General method for the total reduction of myo-inositol
orthobenzoates with DIBAL-H
The myo-inositol 1,3,5-O-orthobenzoate (3a-d, 0.50 mmol) was
evaporated from toluene (3 ¥ 2 mL), taken up in CH2Cl2 (5 mL)
and cooled to -78 ◦C. DIBAL-H (0.7 M in hexanes, 3.5 eq.)
was added dropwise (initial vigorous effervescence observed), the
solution warmed to rt over 3 h and stirred for a further 12 h.
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The reaction was quenched with H2O (25 mL) and extracted with
CH2Cl2 (4 ¥ 100 mL). The combined organic layers were washed
with H2O (50 mL) and brine (50 mL), dried (MgSO4), filtered and
evaporated to dryness under reduced pressure.
(3 ¥ OCH2Ph), 72.18 (Ins 1-CH) ppm; MS (CI+) m/z (%) [M+H]+
540 (100).
2,3,6-O-Tribenzyl-4-O-tert-butyldimethylsilyl-myo-inositol (6c)
and
1,2,6-O-tribenzyl-4-O-tert-butyldimethylsilyl-myo-inositol
2,3,6-O-Tribenzyl-myo-inositol (6a) and 1,2,6-O-tribenzyl-myo-
inositol (7a). 2,6-O-Dibenzyl-myo-inositol 1,3,5-O-orthoben-
zoate (3b, 225 mg, 0.50 mmol) was reduced with DIBAL-H using
the general method described above. The residue was fractionated
by chromatography on flash silica. Elution with hexane-EtOAc
(9:1 → 2:8 v/v) afforded 6a (60 mg, 26%) and 7a (40 mg, 18%)
both as off-white solids; for 6a Rf (hexane-EtOAc, 3:7 v/v) 0.46;
dH (400 MHz, d6-DMSO) 7.42–7.24 (15H, m, 15 ¥ Ar CH), 4.96
(1H, d, J 5.1, ex, Ins 1-OH), 4.93 (1H, d, J 4.9, ex, Ins 4-OH), 4.91
(1H, d, J 5.2, ex, Ins 5-OH), 4.83 (1H, d, J 11.9, OCHHPh), 4.81
(1H, d, J 11.5, OCHHPh), 4.76 (1H, d, J 11.5, OCHHPh), 4.73
(1H, d, J 11.9, OCHHPh), 4.64 (2H, s, OCH2Ph), 3.96 (1H, bs,
Ins 2-H), 3.64 (1H, td, J 9.4, 4.9, ex → t, Ins 4-H), 3.51–3.44 (2H,
m, Ins 5-H + Ins 1-H), 3.25 (1H, dd, J 9.9, 2.3, Ins 3-H), 3.18 (1H,
td, J 8.8, 5.2, ex → t, Ins 6-H) ppm [the 1H-NMR in CDCl3 (not
given) is consistent with lit.,12b which is the best resolved published
data we are aware of, but is incomplete lacking two of the inositol
ring resonances]; dC (100 MHz, d6-DMSO) 140.23, 140.06, 139.59
(3 ¥ Ar C), 128.57 (2C), 128.45 (2C), 128.33 (2C), 128.03, 127.84
(2C), 127.67 (4C), 127.53, 127.43 (15 ¥ Ar CH), 82.45, 80.56,
79.42, 75.60 (4 ¥ Ins CH), 74.51, 74.10 (2 ¥ OCH2Ph), 73.17,
71.97 (2 ¥ Ins CH), 71.91 (OCH2Ph) ppm; HRMS (CI+) m/z (%)
found [M+Na]+ 473.1947 (100), C27H30O6Na requires 473.1940:
For 7a Rf (hexane-EtOAc, 3:7 v/v) 0.18; mp 136–137.5 ◦C; dH
(500 MHz, d6-DMSO) 7.31–7.21 (15H, m, 15 ¥ Ar H), 4.88 (1H,
d, J 5.2, ex, Ins 5-OH), 4.81–4.70 [6H, m, Ins 3-OH (ex) + Ins
4-OH (ex) + (2 ¥ OCH2Ph)], 4.62 (1H, d, J 11.9, OCHHPh), 4.54
(1H, d, J 11.9, OCHHPh), 3.98 (1H, t, J 2.4, Ins 2-H), 3.58 (1H,
t, J 9.5, Ins 6-H), 3.47 (1H, td, J 9.5, 4.6, ex → t, Ins 4-H), 3.45
(1H, dd, J 9.9, 2.5, Ins 1-H), 3.27 (1H, ddd, J 9.8, 4.6, 2.4, ex →
dd, Ins 3-H), 3.16 (1H, td, J 9.0, 5.2, ex → t, Ins 5-H) ppm; dC
(125 MHz, CDCl3) 138.64 (2C), 138.10 (3 ¥ Ar C), 128.48 (4C),
128.43 (2C), 128.07 (2C), 127.81 (2C), 127.78 (2C), 127.73, 127.64
(2C) (15 ¥ Ar CH), 81.11, 80.92, 77.34 (3 ¥ Ins CH), 75.48, 74.89
(2 ¥ OCH2Ph), 74.51, 73.60 (2 ¥ Ins CH), 72.85 (OCH2Ph), 72.10
(Ins CH) ppm; HRMS (CI+) m/z (%) found [M+Na]+ 473.1947
(100), C27H30O6Na requires 473.1940.
(7c). 2,6-O-Dibenzyl-4-O-tert-butyldimethylsilyl-myo-inositol
1,3,5-O-orthobenzoate (3c, 100 mg, 0.15 mmol) was reduced by
DIBAL-H using the general method described above. The residue
(86 mg) was fractionated by chromatography on flash silica.
Elution with hexane-EtOAc (9:1 → 3:7 v/v) afforded 6c (42 mg,
42%) and 7c (40 mg, 40%) both as pale yellow oils; for 6c Rf
(hexane-EtOAc, 1:1 v/v) 0.63; dH (400 MHz, CDCl3) 7.41–7.30
(15H, m, 15 ¥ Ar H), 4.94 (1H, d, J 11.6, 6-OCHHPh), 4.91
(1H, d, J 11.5, 2-OCHHPh), 4.83 (1H, d, J 11.2, 2-OCHHPh),
4.69 (1H, d, J 11.5, 6-OCHHPh), 4.66 (2H, s, 3-OCH2Ph), 4.04
(1H, t, J 9.1, Ins 4-H), 4.02 (1H, t, J 2.8, Ins 2-H), 3.68 (1H, t, J
9.3, Ins 6-H), 3.53 (1H, ddd, J 9.2, 6.4, 2.6, ex → dd, Ins 1-H),
3.45 (1H, td, J 8.9, 2.1, ex → t, Ins 5-H), 3.27 (1H, dd, J 9.5,
2.2, Ins 3-H), 2.44 (1H, d, J 2.2, Ins 5-OH), 2.33 (1H, d, J 6.6,
Ins 1-OH), 0.92 (9H, s, SiCMe3), 0.17 (3H, s, SiMe), 0.09 (3H, s,
SiMe) ppm; dC (125 MHz, CDCl3) 138.79, 138.70, 137.99 (3 ¥ Ar
C), 128.47 (2C), 128.35 (3C), 127.98 (2C), 127.73, 127.62 (7C)
(15 ¥ Ar CH), 81.77, 80.91, 77.11, 76.09 (4 ¥ Ins CH), 74.96,
74.79 (2 ¥ OCH2Ph), 74.07 (Ins CH), 72.73 (OCH2Ph), 72.32
(Ins CH), 25.99 (SiCMe3), 18.31 (SiCMe3), -4.06, -4.55 (2 ¥
SiMe) ppm; HRMS (ESI+) m/z (%) found [M+H]+ 565.2972
(100), C33H45O6Si requires 565.2985: for 7c Rf (hexane-EtOAc,
1:1 v/v) 0.72; dH (400 MHz, CDCl3) 7.41–7.31 (15H, m, 15 ¥
Ar H), 5.02 (1H, d, J 12.1, 2-OCHHPh), 4.99 (1H, d, J 11.9,
6-OCHHPh), 4.78 (1H, d, J 11.4, 6-OCHHPh), 4.75 (1H, d, J
12.1, 2-OCHHPh), 4.70 (2H, s, 1-OCH2Ph), 4.06 (1H, t, J 2.6,
Ins 2-H), 3.90 (1H, t, J 9.5, Ins 6-H), 3.80 (1H, t, J 9.1, Ins 4-H),
3.48 (1H, dd, J 9.7, 2.4, Ins 1-H), 3.37 (1H, t, J 9.0, Ins 5-H), 3.34
(1H, dd, J 9.4, 2.6, Ins 3-H), 2.43 (1H, bs, Ins OH), 2.17 (1H, bs,
Ins OH), 0.93 (9H, s, SiCMe3), 0.14 (3H, s, SiMe), 0.13 (3H, s,
SiMe) ppm; dC (100 MHz, CDCl3) 128.72 (2C), 138.7 (3 ¥ Ar C),
128.41 (2C), 128.38 (3C), 127.89 (2C), 127.70, 127.66 (2C), 127.64
(3C), 127.61 (2C) (15 ¥ Ar CH), 81.14, 80.82, 77.09, 75.47 (4 ¥ Ins
CH), 75.47 (OCH2Ph), 75.23 (Ins CH), 74.66 (OCH2Ph), 72.95
(Ins CH), 72.70 (OCH2Ph), 25.93 (SiCMe3), 18.30 (SiCMe3),
-4.28, -4.44 (2 ¥ SiMe) ppm; HRMS (ESI+) m/z (%) found
[M+H]+ 565.2992 (100), C33H45O6Si requires 565.2985.
1,2,4,6-O-Tetrabenzyl-myo-inositol (6b ∫ 7b). 2,4,6-O-Triben-
zyl-myo-inositol 1,3,5-O-orthobenzoate (3a, 100 mg, 0.19 mmol)
was reduced with DIBAL-H using the general method described
above, affording 6b (102 mg, 100%) as a clear oil; Rf (hexane-
EtOAc, 1:1 v/v) 0.48; dH (400 MHz, CDCl3) 7.41–7.29 (20H, m,
20 ¥ Ar H), 5.03 (1H, d, J 11.6, OCHHPh), 5.02 (1H, d, J 11.2,
OCHHPh), 4.93 (1H, d, J 11.3, OCHHPh), 4.82 (1H, d, J 11.4,
OCHHPh), 4.80 (1H, d, J 11.2, OCHHPh), 4.77 (1H, d, J 11.6,
OCHHPh), 4.71 (2H, s, OCH2Ph), 4.09 (1H, t, J 2.5, Ins 2-H),
3.95 (1H, t, J 9.4, Ins 4-H), 3.74 (1H, t, J 9.3, Ins 6-H), 3.58 (1H,
t, J 9.1, Ins 5-H), 3.52 (1H, bd, J 10.1, Ins 1-H), 3.48 (1H, dd,
J 9.7, 2.4, Ins 3-H), 2.57 (1H, bs, Ins 5-OH), 2.38 (1H, bs, Ins
1-OH) ppm; dC (100 MHz, CDCl3) 138.66, 138.62 (2C), 138.05
(4 ¥ Ar C), 128.50 (4C), 128.45 (2C), 128.37 (2C), 128.03 (2C),
128.00 (2C), 127.77 (4C), 127.75, 127.65, 127.60 (2C) (20 ¥ Ar
CH), 81.66 (Ins 6-CH), 81.25 (Ins 4-CH), 80.87 (Ins 3-CH), 77.05
(Ins 2-CH), 75.48 (OCH2Ph), 74.98 (Ins 5-CH), 74.98, 74.94, 72.70
2,3,6-O-Tribenzyl-4-O-tert-butyldiphenylsilyl-myo-inositol (6d)
and
1,2,6-O-tribenzyl-4-O-tert-butyldiphenylsilyl-myo-inositol
(7d). 2,6-O-Dibenzyl-4-O-tert-butyldiphenylsilyl-myo-inositol
1,3,5-O-orthobenzoate (3d, 460 mg, 1.07 mmol) was reduced
with DIBAL-H using the general method described above. The
residue (430 mg) was fractionated by chromatography on flash
silica. Elution with hexane-EtOAc (9:1 → 7:3 v/v) afforded 6d
(153 mg, 33%) and 7d (193 mg, 42%) both as clear oils; for 6d Rf
(hexane-EtOAc, 7:3 v/v) 0.34; dH (400 MHz, CDCl3) 7.76–7.68
(4H, m), 7.45–7.18 (17H, m), 7.14–7.12 (2H, m), 6.96–6.94 (2H,
m) (25 ¥ Ar H), 4.88 (1H, d, J 11.2, 6-OCHHPh), 4.71 (1H, d, J
11.2, 6-OCHHPh), 4.59 (1H, d, J 11.5, 2-OCHHPh), 4.53 (1H, d,
J 11.4, 2-OCHHPh), 4.41 (1H, d, J 11.3, 3-OCHHPh), 4.27 (1H,
t, J 8.7, Ins 4-H), 4.19 (1H, d, J 11.3, 3-OCHHPh), 4.04 (1H, t, J
2.1, Ins 2-H), 3.70 (1H, td, J 8.0, 2.7, Ins 5-H), 3.67–3.60 (2H, m,
Ins 1-H + Ins 6-H), 3.41 (1H, dd, J 9.0, 2.1, Ins 3-H), 2.41 (1H,
d, J 3.0, Ins 5-OH), 2.35 (1H, d, J 4.3, Ins 1-OH), 1.04 (9H, s,
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SiCMe3) ppm; dC (100 MHz, CDCl3) 138.69, 138.65, 137.62 (3 ¥
Ar C), 135.94 (2C), 135.87 (2C) (4 ¥ Ar CH), 133.88, 133.77 (2 ¥
Ar C), 129.48, 129.44, 128.42 (2C), 128.18 (2C), 128.02 (2C),
127.93 (2C), 127.70 (2C), 127.56 (2C), 127.45 (2C), 127.36 (5C)
(21 ¥ Ar CH), 81.60, 80.95, 76.29, 75.83, 75.03 (5 ¥ Ins CH),
74.88, 74.11 (2 ¥ OCH2Ph), 72.23 (Ins CH), 71.77 (OCH2Ph),
27.06 (SiCMe3), 19.65 (SiCMe3) ppm; HRMS (ESI+) m/z (%)
found [M+Na]+ 711.3102 (100), C43H48O6SiNa requires 711.3118:
for 7d Rf (hexane-EtOAc, 7:3 v/v) 0.62; dH (400 MHz, CDCl3)
7.75–7.72 (4H, m), 7.44–7.26 (19H, m), 7.07–7.05 (2H, m) (25 ¥
Ar H), 4.88 (2H, d, J 11.3, 2-OCHHPh + 6-OCHHPh), 4.75 (1H,
d, J 10.9, 6-OCHHPh), 4.69 (1H, d, J 11.9, 1-OCHHPh), 4.66
(1H, d, J 11.8, 1-OCHHPh), 4.49 (1H, d, J 11.8, 2-OCHHPh),
3.90 (1H, t, J 2.6, Ins 2-H), 3.89 (1H, t, J 8.9, Ins 4-H), 3.77 (1H,
t, J 9.3, Ins 6-H), 3.59 (1H, td, J 8.9, 2.3, Ins 5-H), 3.46 (1H, dd,
J 9.5, 2.6, Ins 1-H), 3.45 (1H, td, J 8.8, 2.7, Ins 3-H), 2.44 (1H,
d, J 2.4, Ins 5-OH), 1.68 (1H, d, J 8.4, Ins 3-OH), 1.21 (9H, s,
SiCMe3) ppm; dC (100 MHz, CDCl3) 138.81, 138.61, 138.13 (3 ¥
Ar C), 135.85 (2C), 135.82 (2C) (4 ¥ Ar CH), 133.85 (2C) (2 ¥ Ar
C), 129.66, 129.61, 128.40 (2C), 128.32 (2C), 128.21 (2C), 127.86
(2C), 127.71 (2C), 127.69, 127.61 (2C), 127.59 (2C), 125.53,
127.31, 127.12 (2C) (21 ¥ Ar CH), 81.12, 80.66, 77.59, 76.47,
75.53 (5 ¥ Ins CH), 75.51, 74.39 (2 ¥ OCH2Ph), 72.82 (Ins CH),
72.73 (OCH2Ph), 27.09 (SiCMe3), 19.71 (SiCMe3) ppm; HRMS
(ESI+) m/z (%) found [M+Na]+ 711.3106 (100), C43H48O6SiNa
requires 711.3118.
flash silica. Elution with hexane-EtOAc (3:1 → 0:1 v/v) afforded
the title compound (97 mg, 57%) as a pale yellow oil; Rf (hexane-
EtOAc, 7:3 v/v) 0.15; dH (400 MHz, CDCl3) 7.42–7.02 (45H, m,
45 ¥ Ar H), 5.12–4.68 [18H, m, (17 ¥ Ph-CHHO) + Ins 5-H],
4.58–4.50 (3 H, m, OCHHPh + Ins 1-H + Ins 2-H), 4.16 (1H,
d, J 2.3, Ins 3-H), 4.00 (1H, t, J 9.6, Ins 6-H), 1.02 (3H, s, Ins
4-CH3) ppm; dC (100 MHz, CDCl3) 138.38, 138.21, 137.97 (3 ¥
Ar C), 136.26 (d, J 7.8), 136.10 (d, J 7.8), 136.03 (d, J 6.0), 135.81
(d, J 7.1), 135.60 (d, J 6.5), 135.57 (d, J 6.6) (6 ¥ Ar CCH2OP),
128.56 (5C), 128.35 (5C), 128.29 (4C), 128.23 (5C), 128.13 (4C),
127.99 (3C), 127.92 (2C), 127.83 (2C), 127.76 (2C), 127.61 (2C),
127.58 (2C), 127.49, 127.44 (2C), 127.34, 127.27 (2C), 127.16 (3C)
(45 ¥ Ar CH), 89.35 (dd, Ins 4-CCH3, J 8.2, 2.6), 82.73 (bs, Ins
5-CH), 78.40 (Ins 3-CH), 77.54 (d, J 5.6, Ins 1-CH), 77.09 (d,
J 6.1, Ins 6-CH), 75.29 (2-OCH2Ph), 74.59 (6-OCH2Ph), 74.15
(Ins 2-CH), 70.84 (3-OCH2Ph), 69.63 (d, J 5.2), 69.39 (d, J 5.2),
69.22 (2C, d, J 5.2), 69.03 (2C, d, J 5.1) (6 ¥ POCH2Ph), 17.65
(d, J 2.1, Ins 4-CH3) ppm; dP (162 MHz, CDCl3) -1.88, -2.06,
-7.23 ppm; HRMS (ES+) m/z (%) found [M+H]+ 1245.4048
(100), C70H72O15P3 requires 1245.4084.
4-C-Methyl-myo-inositol 1,4,5-O-triphosphate (12a)
1,4,5-O-Tris(dibenzyloxyphosphoryl)-2,3,6-O-tribenzyl-4-C-me-
thyl-myo-inositol (64 mg, 0.05 mmol) was taken up in tBuOH-H2O
(6:1 v/v) to which was added NaHCO3 (46 mg, 0.41 mmol) and
Pd-black (145 mg, 1.03 mmol). The solution was stirred under an
atmosphere of H2 for 36 h. The catalyst was filtered off, washed
with H2O (4 ¥ 10 mL) and concentrated under reduced pressure,
before being taken up in H2O, washed with CH2Cl2 (¥ 2) and
freeze dried. The powdery solid was re-dissolved in the minimum
volume of H2O and passed through DOWEX H-100 resin. Acidic
fractions of eluent were combined, neutralised with aq. NH3 and
freeze dried to yield 12a (30 mg, 100%) as a powdery salt; dH
(400 MHz, D2O) 4.20 (1H, t, J 3.1, Ins 2-H), 4.05 (1H, dd, J 9.5,
8.9, Ins 5-H), 3.98 (1H, ddd, J 9.6, 9.1, 3.0, Ins 1-H), 3.89 (1H, d,
J 3.2, Ins 3-H), 3.75 (1H, dd, J 9.9, 9.8, Ins 6-H), 1.48 (3H, s, Ins
4-CH3) ppm; dC (125 MHz, D2O) 87.76 (d, Ins 4-CCH3, J 7.0),
83.82 (t, J 6.8), 77.65 (d, J 5.3), 75.59 (s), 72.59 (s), 72.33 (s) (5 ¥
Ins CH), 17.81 (s, Ins 4-CH3) ppm; dP (162 MHz, D2O) 2.55, 2.31,
0.30 ppm; HRMS (ES-) m/z (%) found [M-H]- 432.9716 (100),
C7H16O15P3 requires 432.9702.
2,3,6-O-Tribenzyl-4-C-methyl-myo-inositol (11). 2,6-O-Di-
benzyl-4-C-methyl-myo-inositol 1,3,5-O-orthobenzoate (10,
100 mg, 0.21 mmol) was reduced with DIBAL-H using the
general method described above. The residue was fractionated
by chromatography on flash silica. Elution with hexane-EtOAc
(9:1 → 4:6 v/v) afforded 11 (55 mg, 55%) as a clear oil which
crystallised on standing; Rf (hexane-EtOAc, 7:3 v/v) 0.09; mp
101–102 ◦C; dH (500 MHz, CD3OD) 7.44–7.21 (15H, m, 15 ¥ Ar
H), 4.84–4.81 (3H, m, 6-OCH2Ph, 2-OCHHPh), 4.73 (1H, d, J
11.7, 3-OCHHPh), 4.72 (1H, d, J 11.4, 2-OCHHPh), 4.66 (1H, d,
J 11.7, 3-OCHHPh), 3.99 (1H, t, J 2.8, Ins 2-H), 3.55–3.50 (2H,
m, Ins 1-H + Ins 6-H), 3.39 (1H, d, J 8.9, Ins 5-H), 3.37 (1H,
d, J 2.8, Ins 3-H), 1.41 (3H, s, Ins 4-CH3) ppm; dC (125 MHz,
CD3OD) 140.50, 140.40, 140.22 (3 ¥ Ar C), 129.29 (2C), 129.45
(2C), 128.14 (2C), 129.10 (2C), 128.93 (2C), 128.83 (2C), 128.53,
128.46, 128.32 (15 ¥ Ar CH), 84.04, 82.55, 79.23, 78.89 (4 ¥ Ins
CH), 78.24 (Ins C), 76.09, 75.88, 74.35 (3 ¥ OCH2Ph), 73.31
(Ins CH), 17.70 (Ins 4-CH3) ppm; HRMS (CI+) m/z (%) found
[M+NH4]+ 482.2545 (100), C28H36NO6 requires 482.2542.
1,3,4,5-O-Tetrakis(dibenzyloxyphosphoryl)-2,6-O-dibenzyl-4-
C-methyl-myo-inositol
2,6-O-Dibenzyl-4-C-methyl-myo-inositol (13, 75 mg, 0.20 mmol)
and 1-H-tetrazole (196 mg, 2.80 mmol) were evaporated from
MeCN (3 ¥ 2 mL), taken up in MeCN (5 mL) and N,N-
diisopropyldibenzyl phosphoramidite (461 mL, 1.40 mmol) added.
After 2 h the solution was cooled to -40 ◦C and mCPBA (644 mg,
2.80 mmol, 75%) added. Stirring was continued at 0 ◦C. After
2 h the solution was diluted with CH2Cl2, washed with 10% aq.
Na2S2O3, sat. NaHCO3, H2O and brine. The organic layer was
dried (MgSO4) and evaporated to dryness under reduced pressure.
The residue was fractionated by chromatography on flash silica.
Elution with hexane-EtOAc (3:1 → 0:1: v/v) afforded the title
compound (178 mg, 63%) as a clear oil; Rf (EtOAc-CH2Cl2, 1:4
v/v) 0.52; dH (400 MHz, CDCl3) 7.41–7.01 (50H, m, 50 ¥ Ar H),
1,4,5-O-Tris(dibenzyloxyphosphoryl)-2,3,6-O-tribenzyl-4-
C-methyl-myo-inositol
2,3,6-O-Tribenzyl-4-C-methyl-myo-inositol
(11,
64
mg,
0.14 mmol) and 1-H-tetrazole (116 mg, 1.66 mmol) were
evaporated from MeCN (3 ¥ 2 mL), taken up in MeCN
(5 mL) and N,N-diisopropyldibenzyl phosphoramidite (272 mL,
◦
0.83 mmol) added. After 2 h the solution was cooled to -40 C
and mCPBA (222 mg, 0.97 mmol) added. Stirring was continued
at 0 ◦C for 2 h. The solution was diluted with CH2Cl2, washed
with 10% aq. Na2S2O3, sat. NaHCO3, H2O and brine. The organic
layer was dried (MgSO4) and evaporated to dryness under reduced
pressure. The residue was fractionated by chromatography on
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5.12–4.67 [25H, m, (20 ¥ Ph-CHHO) + (5 ¥ Ins H)], 1.31 (3H, s, Ins
4-CH3) ppm; dP (162 MHz, D2O) -1.46, -1.65, -1.86, -6.30 ppm;
MS (MALDI-TOF) m/z (%) found [M+Na]+ 1438 (40).
(0:1 → 1:0 v/v) then EtOH-CH2Cl2 (0:1 → 3:97 v/v) afforded 14
(139 mg, 87%) as a clear oil; dH (400 MHz, CD3OD) 7.46–7.25
(10H, m, 10 ¥ Ar H), 4.89 (1H, d, J 11.3, OCHHPh), 4.85–4.80
(3H, m, 3 ¥ OCHHPh), 3.90 (1H, t, J 3.1, Ins 2-H), 3.67 (1H, dd
J 9.6, 3.2, Ins 1-H), 3.60–3.55 (2H, m, Ins 6-H + Ins 3-H), 3.43
(1H, d, J 9.4, Ins 5-H), 1.36 (3H, s, Ins 4-CH3) ppm; dC (100 MHz,
CDCl3) 139.15, 139.04 (2 ¥ Ar C), 127.89 (2C), 127.79 (4C), 127.53
(2C), 127.13, 126.97 (10 ¥ Ar CH), 81.26, 80.72, 77.39 (3 ¥ Ins CH),
76.35 (Ins C), 75.10 (OCHPh), 74.58 (Ins CH), 74.39 (OCHPh),
72.05 (Ins CH), 15.88 (4-CH3) ppm; HRMS (ESI+) m/z (%) found
[M+Na]+ 397.1628 (100), C21H26O6Na requires 397.1627.
4-C-Methyl-myo-inositol 1,3,4,5-O-tetraphosphate (12b)
1,3,4,5-O-Tetrakis(dibenzyloxyphosphoryl)-2,6-O-dibenzyl-4-
C-methyl-myo-inositol (350 mg, 0.25 mmol) was taken up in
tBuOH-H2O (54 mL, 6:1 v/v) to which was added NaHCO3
(166 mg, 1.98 mmol) and Pd-black (527 mg, 4.95 mmol). The
solution was stirred under an atmosphere of H2 for 36 h. The
catalyst was filtered off, washed with H2O (4 ¥ 10 mL) and
the filtrate concentrated under reduced pressure. The remaining
solution was taken up in H2O, washed with CH2Cl2 (¥ 2) and
freeze dried. The powdery solid was re-dissolved in the minimum
volume of H2O and passed through DOWEX H-100 resin. Acidic
fractions of eluent were combined, neutralised with aq. NH3 and
freeze dried to yield 12b (69 mg, 100%) as a pale brown powdery
salt; dH (500 MHz, D2O) 4.23 (1H, t, J 3.4, Ins 2-H), 4.14 (1H, dd,
J 10.1, 3.4, Ins 3-H), 4.02 (1H, t, J 9.5, Ins 5-H), 3.87 (1H, ddd
J 10.1, 8.5, 3.3, Ins 1-H), 3.6 (1H, t, J 9.9, Ins 6-H), 1.44 (3H, s,
Ins 4-CH3) ppm; dC (125 MHz, D2O) 84.05 (dt, J 6.8, 4.9, Ins
4-CCH3), 81.28 (bm), 76.69 (d, J 5.7), 73.92 (d, J 5.2), 70.48 (s),
70.21 (bm) (5 ¥ Ins CH), 15.21 (s, Ins 4-CH3) ppm; dP (162 MHz,
D2O) 7.74, 1.38, 0.83, -2.35 ppm; HRMS (ESI-) m/z (%) found
[M-H]- 512.9350 (77), C7H17O18P4 requires 512.9365.
Acknowledgements
Funding from the UK Medical Research Council (PRJG, grant
no. G 120/709) and an EPSRC doctoral training award to the
Chemical Biology Centre, Imperial College (SC) are gratefully
acknowledged. GB is a postdoctoral fellow of the Research
Foundation, Flanders (F.W.O.) and would like to thank Tomas
Luyten for technical assistance.
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2,6-O-Dibenzyl-4-C-methyl-myo-inositol 1,3,5-O-orthobenzoate
(10, 200 mg, 0.43 mmol) was refluxed in conc. HCl-methanol
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1
evaporated under reduced pressure. H NMR showed that more
than one isomeric product was present, including 13a. If desired
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EtOAc-hexane (1:4 → 3:2 v/v); dH (400 MHz, CD3OD) 7.93–7.06
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C), 134.41 (2 ¥ Ar CH), 131.13 (Ar C), 130.80 (3C), 129.57 (2C),
129.17, 129.08 (3C), 128.55 (2C), 128.47, 128.36 (13 ¥ Ar CH),
80.21, 79.86, 79.12 (3 ¥ Ins CH), 77.70 (Ins 4-CCH3), 76.40, 76.17
(2 ¥ PhCH2O), 75.56, 75.30 (2 ¥ Ins CH), 16.97 (Ins 4-CCH3).
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