Synthesis of the enantiomers of 6-deoxy-myo-inositol 1,3,4,5-Tetrakisphosphate, structural analogues of myo-inositol 1,3,4,5-Tetrakisphosphate

Article abstract of DOI:10.1002/1521-3765(20010105)7:1<80::AID-CHEM80>3.0.CO;2-B

D-myo-Inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P₄] is produced rapidly from the established second messenger D-myo-inositol 1,4,5trisphosphate [Ins(1,4,5)P₄] in stimulated cells. Despite extensive investigations, in particular

Full text of DOI:10.1002/1521-3765(20010105)7:1<80::AID-CHEM80>3.0.CO;2-B

FULL PAPER
Synthesis of the Enantiomers of 6-Deoxy-myo-Inositol 1,3,4,5-Tetrakisphos-
phate, Structural Analogues of myo-Inositol 1,3,4,5-Tetrakisphosphate
Graeme Horne and Barry V. L. Potter*[a]
Abstract: d-myo-Inositol 1,3,4,5-tetra-
kisphosphate [Ins(1,3,4,5)P4] is pro-
duced rapidly from the established sec-
ond messenger d-myo-inositol 1,4,5-tris-
phosphate [Ins(1,4,5)P4] in stimulated
cells. Despite extensive investigations, in
particular concerning its potential role
in mediating cellular Ca2‡ influx, no
exact cellular function has been de-
scribed for this inositol phosphate; how-
ever, binding sites have been identified
in a number of tissues and it has been
shown to act synergistically with
Ins(1,4,5)P3. To assist in the elucidation
of the mechanism of action and struc-
tural requirements within the Ins-
(1,3,4,5)P4 moiety that are necessary
for recognition and activation of the
receptor, structural analogues of this
tetrakisphosphate are required. Routes
for the synthesis of racemic 6-deoxy-
myo-inositol 1,3,4,5-tetrakisphosphate
[6-deoxy-dl-Ins(1,3,4,5)P4] and the chi-
ral antipodes d- and l-6-deoxy-myo-
inositol 1,3,4,5-tetrakisphosphate are de-
scribed here. The racemic tetrakisphos-
phate was synthesised from dl-1,2-O-
isopropylidene-myo-inositol in eight
steps. Deoxygenation at C-6 was ach-
ieved following the Barton ± McCombie
procedure. Both chiral tetrakisphos-
phates were synthesised through resolu-
tion of racemic cis-diol 6-deoxy-1,4,5-tri-
O-p-methoxybenzyl-myo-inositol with
the chiral auxiliary (S)-(‡)-O-acetyl-
mandelic acid. Absolute configuration
was confirmed by synthesis of the known
d-6-deoxy-myo-inositol. Both d-6-de-
oxy-Ins(1,3,4,5)P4 and its enantiomer
will be useful tools to unravel the
enigmatic role of Ins(1,3,4,5)P4 in the
polyphosphoinositide pathway of signal
transduction.
Keywords: signal transduction ´ chi-
ral resolution
´
cyclitols
´
myo-
inositol polyphosphates
R1 = R3 = R4 = PO32-, R2 = H: Ins(1,4,5)P3
R1 = R4 = PO32-, R2 = R3 = H: Ins(1,4)P2
1
2
3
4
5
6
7
Introduction
OH
R2O
3
R1 = R2 = R3 = R4 = PO3
:
Ins(1,3,4,5)P4
2-
4
2
OR1
R3O
The involvement of myo-inositol polyphosphates in signal
transduction through the phosphoinositide pathway has
stimulated the need for the synthesis of molecules that will
interfere with, or modulate, the process of cellular signalling.[1]
The process of signal transduction via the second messenger
d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3, 1] (Figure 1)
starts when a cell surface receptor activates the enzyme
phospholipase C-b via a G-protein.[2] This enzyme hydrolyses
the membrane phospholipid, phosphatidylinositol 4,5-bis-
phosphate to produce diacylglycerol and Ins(1,4,5)P3 as
signalling molecules. Ins(1,4,5)P3 interacts specifically at an
N-terminal binding site of a tetrameric Ins(1,4,5)P3 receptor-
operated Ca2‡ channel in order to release Ca2‡ from non-
mitochondrial stores.[3] After this Ca2‡ release the signal must
be removed and this is accomplished by one or more
metabolic pathways. An Ins(1,4,5)P3 5-phosphatase removes
R1 = R2 = R3 = PO32-, R4 = H: Ins(1,3,4)P3
R1 = R2 = PO32-, R3 = R4 = H: Ins(1,3)P2
R1 = R4 = H, R2 = R3 = PO32-: Ins(3,4)P2
5
R4O
6
1
HO
R1 = R2 = R3 = R4 = H:
Inositol
OR2
R1 = R3 = R4 = R6 = PO32-, R2 = R5 = H: Ins(1,3,4,6)P4
8
9
R3O
3
1 = R3 = R4 = R5 = R6 = PO32-, R2 = H: Ins(1,3,4,5,6)P5
4
R
R4O
2
OR1
2-
R1 = R2 = R3 = R4 = R5 = R6 = PO3
:
InsP6
10
5
R5O
6
R1 = R2 = H, R3 = R4 = R5 = R6 = PO3
: Ins(3,4,5,6)P4 11
1
2-
R6O
R1 = R4 = R5 = R6 = PO32-, R2 = R3 = H: Ins(1,4,5,6)P4 12
O
OH
2-O2PO
O
DL-6-deoxy Ins(1,3,4,5)P4 (racemic mixture, rac-13)
4
2-O2PO
2-O2PO
2
3
D-6-deoxy Ins(1,3,4,5)P4:
13
2-
OPO2
6
L-6-deoxy Ins(1,3,4,5)P4: ent-13
1
5
O
O
D-configuration shown
Figure 1. myo-Inositol phosphates.
[a] Prof. B. V. L. Potter, G. Horne
Wolfson Laboratory of Medicinal Chemistry
Department of Pharmacy and Pharmacology
University of Bath, Claverton Down, Bath, BA2 7AY (UK)
Fax : (‡ 44)-1225 826114
the 5-phosphate group from Ins(1,4,5)P3 to give d-myo-
inositol 1,4-bisphosphate [Ins(1,4)P2, 2] which is inactive for
Ca2‡ release.[4] Ins(1,4,5)P3 can also be phosphorylated by a
cytosolic 3-kinase to give d-myo-inositol 1,3,4,5-tetrakisphos-
E-mail: b.v.l.potter@bath.ac.uk
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Chem. Eur. J. 2001, 7, No. 1

80±87
phate[5] [Ins(1,3,4,5)P4, 3] which is subsequently degraded to
d-myo-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3, 4]. Ins(1,3,
4)P3 is subsequently metabolised by one of two possible
pathways. The products d-myo-inositol 1,3-bisphosphate [Ins-
(1,3)P2, 5] and/or d-myo-inositol 3,4-bisphosphate [Ins(3,
4)P2, 6] are subsequently converted into monophosphates
which are ultimately dephosphorylated to inositol 7. It has
now emerged that Ins(1,3,4)P3 can be phosphorylated by a
6-kinase to d-myo-inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,
4,6)P4, 8][6] which can be further phosphorylated to d-myo-
inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P5, 9]. The
latter higher polyphosphate is metabolised to three com-
pounds, phytic acid [InsP6, 10], d-myo-inositol 3,4,5,6-tetra-
kisphosphate [Ins(3,4,5,6)P4, 11] and d-myo-inositol 1,4,5,6-
tetrakisphosphate [Ins(1,4,5,6)P4, 12]. Such higher polyphos-
phates have long been known to provide phosphate storage
and to modulate the oxygen affinity of haemoglobin in plants
and avian erythrocytes, respectively.[7, 8] Recently, it was
reported that these higher inositol polyphosphates have a
direct role in the regulation of gene expression.[9, 10] Inositol
phosphate signalling has also been implicated in the induction
of genes whose proteins catabolise arginine with the effect
being mediated through Ins(1,4,5,6)P4. Ins(3,4,5,6)P4 has been
shown to inhibit Ca2‡ dependent Clꢀ currents in T84 colonic
epithelial cells[11] as well as inhibiting receptor mediated Ca2‡
dependent Clꢀ currents in CFPAC-1 cells.[12] This latter effect
identifies proteins that bind this particular polyphosphate as
potentially important therapeutic targets for pathological
conditions such as cystic fibrosis.
Ins(1,3,4,5)P4 receptor and is a member of a family of GTPase
activating proteins (GAP) designated as GAPIP4BP. The highly
specific nature of Ins(1,3,4,5)P4 binding to GAPIP4BP has been
demonstrated[26] and it is apparent that phosphorylation of the
1, 3 and 5 positions is essential for high affinity binding and
that there is some tolerance of phosphorylation of the
6-hydroxyl group, but none of phosphorylation of the
2-hydroxyl group.
Recently, it was reported that the interaction of
Ins(1,3,4,5)P4 with the pleckstrin homology (PH) domain of
Brutonꢁs tyrosine kinase (Btk) may be involved in B-cell
activation and development;[27] mutations in the Btk PH
domain causing human X-linked agammaglobulinaemia
(XLA) and murine X-linked immunodeficiency (Xid) are
associated with dramatically reduced Ins(1,3,4,5)P4-binding
activity. We also reported an X-ray crystal structure of
[28]
synthetic Ins(1,3,4,5)P4 bound to the PH domain of Btk[29]
and determination of the phosphate pKa values of
Ins(1,3,4,5)P4.[30] In order to investigate the possibility of a
second messenger function for Ins(1,3,4,5)P4 the synthesis of
structural analogues of this molecule is of continuing interest.
We have recently reported the synthesis of d-2-deoxy-myo-
inositol 1,3,4,5-tetrakisphosphate from d-glucose.[31] To fur-
ther enhance and elucidate these structure-activity studies on
the Ins(1,3,4,5)P4 specific GAPIP4BP binding protein,[32±34] and
for other applications[20] we required a synthesis of both
enantiomers of 6-deoxy-Ins(1,3,4,5)P4. Additionally, such
structurally modified Ins(1,3,4,5)P4 analogues are of consid-
erable use as head-group surrogates for inositol phospholipids
which themselves have been shown to have signalling roles.[35]
We now report here the synthesis of 6-deoxy-Ins(1,3,4,5)P4 in
racemic and chiral form by resolution of partially blocked
myo-inositol derivatives using the chiral auxiliary (S)-(‡)-O-
acetylmandelic acid.
Release of Ca2‡ from intracellular stores is one effect of the
phosphoinositide pathway, but subsequent Ca2‡ entry through
plasma membrane channels is also crucial. Experiments have
demonstrated a potential role for Ins(1,3,4,5)P4 acting syn-
[13±16]
ergistically with Ins(1,4,5)P3
and Ins(1,3,4,5)P4 has been
shown to independently mobilise intracellular Ca2‡ through
the Ins(1,4,5)P3 receptor.[17]
The exact reasons for the rapid production of Ins(1,3,4,5)P4
following receptor stimulation are currently unclear; how-
ever, essentially there are three possibilities: 1) the phosphor-
ylation of Ins(1,4,5)P3 to Ins(1,3,4,5)P4 is considered to be an
off signal terminating the release of Ca2‡ by Ins(1,4,5)P3; 2)
Ins(1,3,4,5)P4 is a metabolic intermediate with no other
function; 3) Ins(1,3,4,5)P4 has a second messenger function
itself, initiated through binding to its own specific intracellular
receptor. In particular, a role mediating Ca2‡ entry through
plasma membrane channels has been proposed. Despite
proposals that option 2) may be correct[18] the function of
Ins(1,3,4,5)P4 as another potential second messenger involved
in Ca2‡ release has not been unambiguously resolved.[19]
However, we have recently suggested a role for Ins(1,3,4,5)P4
in facilitating store-operated Ca2‡ influx in cells by inhibition
of Ins(1,4,5)P3-5-phosphatase[20] and this may ultimately prove
to be the real function of Ins(1,3,4,5)P4, with Ins(1,3,4,5)P4
acting as a long-lived bi-modal ªmemoryº molecule. An
Ins(1,3,4,5)P4-sensitive Ca2‡-permeable channel has been
characterised from endothelial cells[21] and Ins(1,3,4,5)P4
binding proteins have been identified from pig[22] and
rat[23, 24] cerebellum and porcine platelets.[25] The latter exam-
ple has been characterised and identified as a putative
Results and Discussion
Alkylation of tetraol rac-14,[36] obtained from myo-inositol,
(Scheme 1) following the stannylene acetal procedure using p-
methoxybenzyl chloride (PMBCl) in the presence of dibutyl
tin oxide (Bu2SnO) and tetrabutylammonium iodide (Bu4NI),
yielded two major products rac-15 (tR ˆ 0.19) and rac-16 (tR ˆ
0.28) [ether/hexane 2:1]. This selectivity can be explained by
considering the stannylene acetals formed and the reactivity
of the individual oxygen atoms involved. These acetals exist as
dimers in which the tin atoms are at the centre of a trigonal
bipyramid with the butyl groups occupying the equatorial
positions. The more electronegative of the two oxygen atoms
is co-ordinated with only one tin atom whereas the less
electronegative oxygen atom is co-ordinated to two tin atoms.
The regioselectivity observed in such reactions is a conse-
quence of a cascade of effects beginning with the selection of a
particular pair of hydroxyls for stannylene formation followed
by orientation of the more electronegative oxygen in an apical
position which is intrinsically the more reactive. The observed
regioselectivity reported here can be rationalised by consid-
ering the stannylene acetals that are most likely to be formed
and the reactivity of the individual oxygen atoms in each
Chem. Eur. J. 2001, 7, No. 1
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81

B. V. L. Potter and G. Horne
FULL PAPER
presence of sodium hydride (NaH) in anhydrous THF
followed by addition of methyl iodide (MeI) and work-up
yielded the corresponding S-methyl xanthate as a non-
isolated intermediate. The crude residue was then dissolved
in anhydrous toluene with tributyl tin hydride (Bu3SnH) and
2,2'-azobis(2-methylpropionitrile) (AIBN) being added. Re-
flux followed by work-up and purification yielded rac-17.
Removal of the cis-isopropylidene group was accomplished
by careful treatment of rac-17 with 80% aqueous acetic acid
(AcOH) with the resulting cis-diol being selectively alkylated
with PMBCl in the presence of Bu2SnO and Bu4NI in
refluxing toluene, yielding monol rac-19. Benzylation of rac-
19 with benzyl bromide (BnBr) in DMF at ambient temper-
ature with NaH as base yielded fully protected racemic
6-deoxy-myo-inositol derivative rac-20. The p-methoxybenzyl
protecting groups were then removed selectively using 10%
trifluoroacetic acid (TFA) in dichloromethane[42] leaving
tetraol rac-21. Initial attempts at the deprotection of the p-
methoxybenzyl ethers employed 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ) as oxidant[43] but problems associated
with isolation of the resulting tetraol from the aqueous work-
up negated the use of this procedure.
O
HO
HO
HO
O
HO
rac-14
i
O
PMBO
O
O
PMBO
PMBO
ii, iii
PMBO
PMBO
PMBO
PMBO
HO
+
O
O
O
PMBO
HO
PMBO
rac-16
rac-17
rac-15
iv
OH
OH
PMBO
PMBO
v
PMBO
PMBO
PMBO
PMBO
OH
OPMB
rac-18
rac-19
vi
OBn
OBn
OH
PMBO
HO
vii
PMBO
PMBO
HO
HO
OPMB
rac-20
rac-21
For the introduction of phosphate groups into tetraol rac-21
the phosphitylating reagent bis(benzyloxy) (diisopropylami-
no)phosphine[44, 45] was chosen. Treatment of tetraol rac-21
with this phosphitylating reagent and 1H-tetrazole followed
by oxidation of the tetrakisphosphite intermediate afforded
fully protected dl-2-O-benzyl-1,3,4,5-tetrakis-O-[bis(benzyl-
oxy)phospho]-6-deoxy-myo-inositol (rac-22). All the benzyl
protecting groups were removed from the fully blocked
compound in one step by hydrogenation. The crude residue
was purified by ion-exchange chromatography on Q-Sephar-
ose Fast Flow and, after pooling and evaporation of fractions
was accurately quantified by total phosphate assay.[46] The
final racemic dl-6-deoxy-myo-inositol 1,3,4,5-tetrakisphos-
phate (rac-13) eluted at 65 ± 85% 1m TEAB and was isolated
as its triethylammonium salt. Removal of all phosphate benzyl
groups was confirmed by 1H-coupled 31P NMR spectroscopy;
the observed sextet for each dibenzylated phosphate changed
to a doublet for each deprotected phosphate.
In order to synthesise the two diastereisomers of 6-deoxy-
1,3,4,5-tetrakisphosphate-myo-inositol 13 and ent-13 it was
necessary to resolve a suitable racemic precursor. This is best
achieved through the selective introduction of a chiral
auxiliary with subsequent separation of the two resulting
diastereoisomers. Racemic cis-diol rac-18 provided an ex-
cellent precursor for resolution since equatorial hydroxyl
groups can be acylated selectively. (S)-(‡)-O-Acetylmandelic
acid (23) was chosen for resolution of cis-diol rac-18; unlike
the enantiomers of commonly used camphanic acid chloride,
both R and S isomers are cheaply available. Additionally, (S)-
(‡)-O-acetylmandelic acid has previously been used success-
fully in this laboratory for the resolution of several blocked
myo-inositol derivatives.[45, 47, 48]
viii
O
OH
OBn
O
2-O2PO
(BnO)2(O)PO
(BnO)2(O)PO
(BnO)2(O)PO
ix
2-O2PO
2-
2-O2PO
OPO2
O
OP(O)(OBn)2
O
rac-22
rac-13
Scheme 1. i) p-methoxybenzyl chloride, Bu4NI, Bu2SnO, toluene, 1208C,
16 h (rac-15, 32%; rac-16, 41%); ii) NaH, CS2, THF, 1 h; then MeI, 1 h;
then iii) Bu3SnH, AIBN, toluene, 1208C, 1 h (83%); iv) 80% AcOH,
1008C, 1 h (77%); v) PMB-Cl, Bu4NI, Bu2SnO, toluene, 1208C, 16 h
(76%); vi) BnBr, NaH, DMF, room temperature, 4 h (84%); vii) 10% TFA
(CH2Cl2), room temperature, 30 min (71%); viii) dibenzyl diisopropyl-
phosphoroamidate, 1H-tetrazole, CH2Cl2, room temperature, 1 h; then m-
CPBA, ꢀ788C, 1 h (79%); ix) Pd/C (10%), MeOH/H2O 9:1, H2, 80 psi,
16 h, and purification by Q-Sepharose Fast Flow ion-exchange chromatog-
raphy (87%). All compounds are racemic.
cyclic system. It can be presumed that initially two acetals are
formed (1,6 and 4,5) and in these the oxygen atoms at
positions 1 and 4 will occupy the more reactive apical position
and hence be preferentially alkylated. The stannylene acetal
formed between the remaining trans diol (hydroxyl groups on
positions 5 and 6) does not result in the preferential alkylation
of one specific hydroxyl. However, the small difference
noticed in the alkylation (32% for alkylation of position 5
and 41% for position 6) can be explained through the
assumed higher electronegativity of the hydroxyl group at
position 6. Differentiation between the two monols rac-15 and
rac-16 was effected by NMR methods.
The next step of the synthesis required deoxygenation at
position 6. Deoxygenation of sugar hydroxyls is readily
performed by metal hydride reduction of halides, sulphonates
and epoxides as well as radical deoxygenation of thiocarbonyl
derivatives (Barton ± McCombie reaction).[37]-[41] Of these
methods available, radical deoxygenation was chosen because
of its suitability for the deoxygenation of secondary alcohols
and its compatibility with benzyl ethers and acetals/ketals.
Treatment of rac-15 with carbon disulfide (CS2) in the
Coupling of dl-6-deoxy-1,4,5-tri-O-p-methoxybenzyl-myo-
inositol (rac-18) with (S)-(‡)-O-acetylmandelic acid (23) at
low temperature yielded the two diastereoisomers 24 and 25
(Scheme 2), (structures determined after the determination of
the chirality of derived pentaols 26 and ent-26, respectively,
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Chem. Eur. J. 2001, 7, No. 1

Inositol Derivatives
80±87
the absolute configuration of each isomer. These values of
d ˆ 4.10 (24) and 4.37 (25) are in good agreement with H-2
chemical shifts for similar compounds previously synthesised
and resolved using (S)-(‡)-O-acetylmandelic acid as the
chiral auxiliary.[47, 48] In all cases an H-2 chemical shift of
approximately d ˆ 4.15 has been observed for the d-isomer
with an H-2 chemical shift value of approximately d ˆ 4.40
being detected for the corresponding l-isomer. Similarly, the
TLC mobilities of the diastereoisomers resulting from the use
of (S)-(‡)-O-acetylmandelic acid as chiral auxiliary also
provide for a tentative determination of the absolute config-
uration of each isomer. In all cases the tR value of the d-isomer
has been lower than that of the l-isomer.
Deacylation of isomers 24 and 25 with methanolic sodium
hydroxide solution yielded enantiomers 18 and ent-18,
respectively. The equatorial hydroxyl group of each enantio-
meric cis-diol was selectively alkylated with PMBCl yielding
the respective monols 19 and ent-19 which were subsequently
benzylated, followed by the selective removal of the p-
methoxybenzyl protecting groups affording tetraols 21 and
ent-21. These chiral tetraols were then phosphitylated,
oxidised, and the products deprotected and purified by ion-
exchange chromatography followed by accurate quantifica-
tion by total phosphate assay[46] yielding d- and l-6-deoxy-
Ins(1,3,4,5)P4 13 and ent-13, respectively, as their triethylam-
monium salts.
O
OH
PMBO
H
OAc
HO
PMBO
PMBO
+
OH
Ph
rac-18
23
i
HO
O
OH
O
O
O
OPMB
PMBO
H
AcO
H
OPMB
OPMB
PMBO
PMBO
OAc
Ph
Ph
ii
24
25
HO
OPMB
OH
PMBO
OPMB
PMBO
PMBO
HO
OH
OPMB
18
ent-18
iii
iv
v
OH
HO
PMBO
OPMB
PMBO
PMBO
OPMB
OPMB
OPMB
BMPO
19
ent-19
OBn
BnO
PMBO
OPMB
PMBO
PMBO
OPMB
OPMB
OPMB
BMPO
ent-20
20
OBn
BnO
OH
HO
HO
HO
OH
OH
OH
HO
21
ent-21
To establish the absolute configuration of antipodes 18 and
ent-18 we used d-6-deoxy-myo-inositol[49] as a reference. This
compound was readily accessible from 18, with the l-isomer
being easily obtained from ent-18. Thus, removal of the p-
methoxybenzyl groups of both enantiomers by hydrogenation
provided pentaols 26 and ent-26, respectively (Scheme 3). The
vi
BnO
OP(O)(OBn)2
OBn
(OBn)2(O)PO
OP(O)(OBn)2
OP(O)(OBn)2
(BnO)2(O)PO
(BnO)2(O)PO
(BnO)2(O)PO
OP(O)(OBn)2
22
ent-22
vii
O
O
OH
O
HO
2-O2PO
O
2-
OPO2
2-O2PO
2-O2PO
2-
OPO2
HO
OH
2-
2-O2PO
O
PMBO
OPO2
O
2-
OPMB
OPO2
OPMB
OPMB
PMBO
PMBO
O
O
HO
OH
ent-13
13
18
ent-18
Scheme 2. i) DMAP, DCC, CH2Cl2, ꢀ208C (38% for 24, 40% for 25); ii)
NaOH, MeOH, reflux, 30 min (87% for 18, 81% for ent-18); iii) PMB-Cl,
Bu2SnO, Bu4NI, toluene, 1208C, 16 h (72% for 19, 77% for ent-19); iv)
BnBr, NaH, DMF, room temperature, 4 h (67% for 20, 81% for ent-20);
v) 10% TFA (CH2Cl2), room temperature, 30 min (83% for 21, 77% for
ent-21); vi) dibenzyl diisopropylphosphoroamidate, 1H-tetrazole, CH2Cl2,
room temperature, 1 h; then m-CPBA, ꢀ788C, 1 h (74% for 22, 68% for
ent-22); vii) Pd/C (10%), MeOH/H2O 9:1, H2, 80 psi, 16 h, and purification
by Q-Sepharose Fast Flow ion-exchange chromatography (85% for 13 and
61% for ent-13). DMAP: 4-dimethylaminopyridine, DCC: dicyclohexyl-
carbodiimide.
HO
OH
OH
HO
OH
OH
HO
HO
HO
OH
ent-26
26
Scheme 3. i) Pd/C (10%), MeOH/H2O 9:1, H2, 80 psi, 16 h (quantitative
yields for both 26 and ent-26).
see below). Selectivity for the equatorial hydroxyl group was
achieved by keeping the initial temperature at ꢀ208C. No
acylation of the axial hydroxyl group was detected (by
1H NMR) with both 24 and 25 being separated by flash
chromatography. The purity of the samples was gauged by
1H NMR analysis which revealed two singlets (corresponding
to CH3CO2CH(Ph)CO2) at d ˆ 5.98 for isomer 24 and 5.99 for
isomer 25. No impurities were detected in either sample. The
distinctive chemical shift value for the H-2 proton for each
diastereoisomer was also indicative of the high purity of each
diastereoisomer and provided for a tentative determination of
specific rotation of 26 was found to be [a]D ˆ ‡6.3 and agreed
well with the literature value of ‡7. The specific rotation of
ent-26 was found as expected to be equal and opposite to the
literature value for 26. 6-Deoxy-Ins(1,4,5)P3 has been pre-
pared by several routes[50, 51] and some of its biological
properties studied.[50±52] Similarly, 3-deoxy[53] and 3-position
modified analogues[54, 55] of Ins(1,4,5)P3 have also been
synthesised and some of their biological properties stud-
ied.[53, 54] Whilst this work was in progress a route to d-
6-deoxy-Ins(1,3,4,5)P4 (13) only, starting from d-galactose
was reported.[56]
Chem. Eur. J. 2001, 7, No. 1
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0701-0083 $ 17.50+.50/0
83

B. V. L. Potter and G. Horne
FULL PAPER
with ethanolic phosphomolybdic acid, or with ethanolic sulphuric acid
followed by charring. Column chromatography was carried out under
pressure on Sorbsil C60 silica gel. NMR spectra (31P, 1H, 13C, COSY,
HETCOR) were recorded on a Varian Mercury 400 spectrometer with
signals being assigned by 1D, DEPT, and 2D spectra (COSY, HETCOR).
Chemical shifts were measured in parts per million (ppm) relative to
tetramethylsilane (TMS), deuterium oxide (D2O), D6-dimethyl sulfoxide
([D6]DMSO), or D4-methanol (CD3OD). Coupling constants are quoted in
We anticipate that the present compounds will find consid-
erable application in studies on the enigmatic nature of
Ins(1,3,4,5)P4 activity. Unlike the enantiomers of Ins(1,4,5)P3,
because of the regioisomeric arrangement of phosphate groups
(Figure 2), l-6-deoxy-Ins(1,3,4,5)P4 could also bind to Ins-
Hz and refer to JH,H unless stated otherwise. The 31P NMR shifts were
A
3
OH
OPO2
2-
2-
measured in ppm relative to external 85% phosphoric acid. Melting points
(uncorrected) were determined using a Reichert ± Jung Thermo Galen
Kofler block. Low-resolution mass spectra were recorded by the University
of Bath Mass Spectrometry Service using ‡ve and ꢀve Fast Atom
Bombardment (FAB) with 3-nitrobenzyl alcohol (NBA) as the matrix.
High resolution mass spectrometry was carried out by the University of
Bath Mass Spectrometry Service. Elemental analyses were carried out by
the Micro-Analysis Department in the School of Chemistry at the
University of Bath. Optical rotations were measured using an Optical
Activity Ltd. AA-10 polarimeter; [a]D values are given in 10ꢀ1 degcm2 gꢀ1
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.[46]
OPO2
2-O2PO
2-
OPO2
OH
OH
2-O2PO
L-Ins(1,3,4,5)P4
2-O2PO
2-O2PO
2-
OPO2
HO
2-
D-Ins(1,3,4,5)P4
OPO2
HO
2-O2PO
2-O2PO
OH 2-
OPO2
L-Ins(1,3,4,5)P4
(alternative orientation)
B
OH
OPO2
2-
2-
OPO2
2-O2PO
2-
OPO2
dl-2,3-O-Isopropylidene-1,4,5-tri-O-p-methoxybenzyl-myo-inositol (rac-
15) and dl-2,3-O-isopropylidene-1,4,6-tri-O-p-methoxybenzyl-myo-inosi-
tol (rac-16): A mixture of rac-14[36] (4.4 g, 10 mmol), Bu2SnO (22.6 g,
90 mmol), Bu4NI (18 g, 56 mmol) and p-methoxybenzyl chloride (18.48 g,
16 mL, 118 mmol) and toluene (150 mL) was heated under reflux with 4 Š
molecular sieves in a Soxhlet apparatus for 16 h. TLC (ether/hexane 2:1)
revealed two major products with tR values of 0.28 and 0.19. The solution
was cooled and washed with H2O (2 Â 100 mL), brine (100 mL), dried
(MgSO4) and concentrated in vacuo. The crude syrup was purified by flash
chromatography (ether/hexane 2:1) to provide dl-2,3-O-isopropylidene-
1,4,5-tri-O-p-methoxybenzyl-myo-inositol (rac-15) as a white crystalline
solid (3.7 g, 32%) and dl-2,3-O-isopropylidene-1,4,6-tri-O-p-methoxyben-
zyl-myo-inositol (rac-16) as a colourless syrup (4.76 g, 41%). rac-15: TLC:
6-deoxy L-Ins(1,3,4,5)P4
OH
2-O2PO
2-O2PO
2-O2PO
2-
OPO2
2-
OPO2
HO
6-deoxy D-Ins(1,3,4,5)P4
2-O2PO
2-O2PO
2-
OPO2
6-deoxy L-Ins(1,3,4,5)P4
(alternative orientation)
Figure 2. Regioisomeric arrangement of phosphate groups in Ins(1,3,4,
5)P4 and 6-deoxy-Ins(1,3,4,5)P4. A: Superimposition of phosphate groups
in both d- and l-Ins(1,3,4,5)P4. B: Superimposition of phosphate groups of
both d- and l-6-deoxy-Ins(1,3,4,5)P4. As a consequence of this regioiso-
meric overlap of phosphate groups, l-6-deoxy-Ins(1,3,4,5)P4 could also
bind to Ins(1,3,4,5)P4 receptors.
1
tR ˆ 0.19 (ether/hexane 2:1); m.p. 898C; H NMR (400 MHz, CDCl3): d ˆ
7.33 ± 6.80 (m, 12H, ArH), 4.80 ± 4.61 (m, 4H, CH2), 4.78,4.64 (AB, 2H,
CH2), 4.25 (dd, 1H, J ˆ 3.9, 5.1 Hz, H-2), 4.08 (t, 1H, J ˆ 6.6 Hz, H-3), 3.99
(t, 1H, J ˆ 9.7 Hz, H-6), 3.77 (s, 9H, OCH3), 3.65 (dd, 1H, J ˆ 6.6, 9.0 Hz,
H-4), 3.51 (dd, 1H, J ˆ 3.9, 9.8 Hz, H-1), 3.24 (t, J ˆ 9.0 Hz, H-5), 1.47, 1.33
(2s, Â3H, 2  CH3); 13C NMR (100 MHz, CDCl3): d ˆ 159.49, 159.35,
159.26 (p-Cq-Ar), 130.77, 130.68, 130.22 (Cq-Ar), 129.85, 129.81, 129.72 (o-
CH-Ar), 114.06, 113.89 (m-CH-Ar), 110.00 (Cdioxolane), 82.39 (C-4), 81.71 (C-
5), 79.67 (C-3), 76.71 (C-1), 74.98 (CH2), 74.30 (C-2), 73.59 (CH2), 72.51
(1,3,4,5)P4 receptors. We anticipate that l-6-deoxy-Ins-
(1,3,4,5)P4 (ent-13) may show unusual binding properties in
its own right. Experimental data for 6-deoxy-dl-Ins(1,3,4,5)P4
and the chiral antipodes 6-deoxy d- and l-Ins(1,3,4,5)P4
regarding binding to the Ins(1,3,4,5)P4 ªreceptorº and the
Ins(1,4,5)P3 receptor and interaction with metabolic enzymes
will be published elsewhere. Preliminary data indicate that the
d-isomer binds to GAPIP4BP, interacts potently with Ins-
(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase and facilitates Ins-
(1,4,5)P3-mediated activation[20] of the store operated Ca2‡
current ICRAC, but to a lesser extent than Ins(1,3,4,5)P4 itself.
‡
(CH2), 71.95 (C-6), 55.59 (OCH3), 28.28 (CH3), 26.27 (CH3); MS (FAB ,
‡
NBA): m/z (%): 579.3 (29) [M ꢀ H] ; MS (FABꢀ, NBA): m/z (%): 733.3
‡
‡
‡
(100) [M ‡ NBA] , 579.2 (15) [M ꢀ H] ; HRMS-FAB (m/z): [M] calcd for
C33H40O9, 580.26724; found, 580.26414; elemental analysis calcd (%) for
C33H40O9: C 68.26, H 6.94; found C 68.40, H 6.93. rac-16: TLC: tR ˆ 0.28
(ether/hexane 2:1); 1H NMR (400 MHz, CDCl3): d ˆ 7.30 ± 6.84 (m, 12H,
ArH), 4.83 ± 4.62 (m, 4H, CH2), 4.82, 4.63 (AB, 2H, CH2), 4.34 (dd, 1H, J ˆ
4.1, 5.6 Hz, H-2), 4.06 (dd, 1H, J ˆ 5.9, 6.7 Hz, H-3), 3.76 (m, 10H, H-6,
OCH3), 3.63 (m, 2H, H-1, H-4), 3.44 (brt, 1H, J ˆ 9.1 Hz, H-5), 1.51, 1.35
(2s, 2  3H, 2  CH3); 13C NMR (100 MHz, CDCl3): d ˆ 158.93, 158.87,
158.83 (p-Cq-Ar), 130.12, 130.10, 129.80 (Cq-Ar), 129.33, 129.31 (o-CH-Ar),
113.58, 113.50, 113.49 (m-CH-Ar), 109.51 (Cdioxolane), 81.27 (C-4), 80.09 (C-
6), 78.75 (C-3), 76.57 (C-1), 74.47 (C-2), 74.36 (CH2), 73.16 (C-5), 72.80
‡
(CH2), 72.47 (CH2), 55.12 (OCH3), 27.68 (CH3), 25.70 (CH3); MS (FAB ,
Experimental Section
‡
NBA): m/z (%): 579.3 (11) [M ꢀ H] ; MS (FABꢀ, NBA): m/z (%): 733.2
‡
‡
(100) [M ‡ NBA] ; HRMS-FAB (m/z): [M] calcd for C33H40O9 580.26724;
found 580.26414; elemental analysis calcd (%) for C33H40O9: C 68.26, H
6.94; found C 68.40, H 6.99.
Dry toluene and dichloromethane were distilled from calcium hydride and
stored over 4 Š molecular sieves; tetrahydrofuran was distilled from
sodium/benzophenone. Molecular sieves (4 Š) were predried in an oven
and activated for 1 h under vacuum at 2508C. Ether is diethyl ether. All
aqueous (aq) solutions were saturated unless otherwise stated. Reactions
were carried out at room temperature under nitrogen in predried glassware
unless otherwise stated. Sodium hydride (NaH) was a 60% dispersion in
mineral oil. Analytical thin-layer chromatography (TLC) was performed
on pre-coated plates (Merck TLC aluminium sheets silica 60 F254, Art. No.
5554): the products were visualised by ultraviolet radiation and staining
dl-6-Deoxy-2,3-O-isopropylidene-1,4,5-tri-O-p-methoxybenzyl-myo-
inositol (rac-17): dl-2,3-O-Isopropylidene-1,4,5-tri-O-p-methoxybenzyl-
myo-inositol (rac-15, 5 g, 8.6 mmol) was dissolved in THF (50 mL) and
stirred under nitrogen at 08C for 10 min. NaH (400 mg, 10 mmol) was
added and the solution was stirred for an additional 10 min. CS2 (723 mg,
570 mL, 9.5 mmol) was added dropwise at 08C and the mixture was stirred
at room temperature for 30 min afterwhich MeI (1.35 g, 592 mL, 9.5 mmol)
84
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0701-0084 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 1

Inositol Derivatives
80±87
was added and the reaction mixture was stirred for an additional 30 min.
TLC analysis (toluene/ethyl acetate 8:3) revealed all starting material had
been consumed and the mixture was concentrated in vacuo to yield a
yellow syrup. The crude residue was dissolved in CH2Cl2 and washed with
H2O (2 Â 50 mL), dried (MgSO4), and the solvent evaporated. The residue
was dissolved in toluene (50 mL) and Bu3SnH (5.82 g, 5.38 mL, 20 mmol)
and AIBN (40 mg) were added. The mixture was refluxed for 1 h, cooled
and concentrated in vacuo. The crude residue was purified by flash
chromatography (ether/hexane 2:1) to yield dl-6-deoxy-2,3-O-isopropyli-
dene-1,4,5-tri-O-p-methoxybenzyl-myo-inositol (rac-17) as a colourless
syrup (4.03 g, 83%): 1H NMR (400 MHz, CDCl3): d ˆ 7.31 ± 6.82 (m, 12H,
ArH), 4.79, 4.69, 4.60, 4.53, 4.58, 4.54 (3AB, 6H, CH2), 4.27 (bdd, 1H, J ˆ
3.9, 5.1 Hz, H-2), 4.00 (dd, 1H, J ˆ 5.1, 6.6 Hz, H-3), 3.58 (dd, 1H, J ˆ 6.6,
9.4 Hz, H-4), 3.52 (dt, 1H, J ˆ 3.9, 12.1 Hz, H-1), 3.24 (ddd, 1H, J ˆ 4.3, 9.4,
11.7 Hz, H-5), 2.11 (brdt, 1H, J ˆ 4.3, 12.1 Hz, H-6eq), 1.88 (q, 1H, J ˆ
12.1 Hz, H-6ax), 1.50, 1.49 (2s, 2 Â 3H, 2 Â CH3); 13C NMR (100 MHz,
CDCl3): d ˆ 159.48, 159.30, 159.20 (p-Cq-Ar), 131.14, 130.82, 130.12 (Cq-
Ar), 129.76, 129.70, 129.53 (o-CH-Ar), 114.07, 113.99, 113.84 (m-CH-Ar),
110.15 (Cdioxolane), 84.34 (C-4), 80.61 (C-3), 76.88 (C-5), 75.47 (C-2), 73.87
(CH2), 72.33 (CH2), 71.67 (C-1), 70.76 (CH2), 55.60 (OCH3), 30.17 (C-6),
08C for a further 10 min afterwhich benzyl bromide (1.54 g, 1.07 mL,
9 mmol) was added and the solution was stirred for 2 h. The reaction was
quenched by addition of MeOH and the solvent was evaporated. The crude
residue was dissolved in ether and was washed with H2O (2 Â 50 mL), dried
(MgSO4) and concentrated in vacuo. The crude residue was purified by
flash chromatography (ether/hexane 1:1 to 2:1) to yield dl-2-O-benzyl-6-
deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol (rac-20) as a colour-
less syrup (4.78 g, 84%): 1H NMR (400 MHz, CDCl3): d ˆ 7.42 ± 6.82 (m,
21H, ArH), 4.89, 4.84, 4.77, 4.74, 4.60, 4.55, 4.59, 4.58, 4.40, 4.37 (5AB, 10H,
CH2), 4.02 (brs, 1H, H-2), 3.94 (dd, 1H, J ˆ 9.1, 9.7 Hz, H-4), 3.80 ± 3.78 (3s,
12H, OCH3), 3.35 (m, 1H, H-5), 3.23 (m, 2H, H-1, H-3), 2.11 (m, 2H, H-6);
13C NMR (100 MHz, CDCl3): d ˆ 159.25, 159.18, 159.13 (p-Cq-Ar), 139.47,
131.62, 131.07, 130.96, 130.48 (Cq-Ar), 129.89, 129.45, 129.36, 129.18 (o-CH-
Ar), 128.27 (m-CH-Ar), 127.89 (o-CH-Ar), 127.35 (p-CH-Ar), 114.00,
113.95, 113.93, 113.86 (m-CH-Ar), 82.90 (C-4), 81.32 (C-3), 78.13 (C-5),
75.70 (CH2), 75.33 (C-1), 75.22 (C-2), 73.96 (CH2), 72.67 (CH2), 72.27
‡
(CH2), 70.67 (CH2), 55.63 (OCH3), 31.04 (C-6); MS (FAB , NBA): m/z
‡
(%): 733.4 (70) [M ꢀ H] ; MS (FABꢀ, NBA): m/z (%): 887.5 (100) [M ‡
‡
‡
NBA] ; HRMS-FAB: (m/z): [M] calcd for C45H50O9 734.34549; found
734.33974; elemental analysis calcd (%) for C45H50O9: C 73.97, H 6.75;
found C 73.50, H 6.88.
‡
28.44 (CH3), 26.85 (CH3); MS (FABꢀ NBA): m/z (%): 563.3 (34) [M ꢀ H] ;
‡
HRMS-FAB: (m/z): [M] calcd for C33H40O8 564.27232; found 564.26707;
dl-2-O-Benzyl-6-deoxy-myo-inositol (rac-21): dl-2-O-Benzyl-6-deoxy-
1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol (rac-20, 1.3 g, 1.7 mmol)
was dissolved in CH2Cl2/TFA (9:1, 40 mL) and stirred at room temperature
for 30 min. The reaction mixture was concentrated in vacuo and the crude
residue was redissolved in toluene. The solvent was evaporated and the
crude residue was purified by flash chromatography (ethyl acetate/acetone
1:0 to 0:1) to yield dl-2-O-benzyl-6-deoxy-myo-inositol (rac-21, 461 mg,
71%): 1H NMR (400 MHz, [D6]DMSO): d ˆ 7.41 ± 7.19 (m, 5H, ArH), 4.78,
4.76 (AB, 2H, CH2), 4.65 ± 4.55 (m, 4H, 4 Â OH), 3.65 (s, 1H, H-2), 3.52 (m,
1H, H-1), 3.32 (ddd, 1H, J ˆ 3.9, 5.1, 12.9 Hz, H-4), 3.14 (m, 2H, H-3, H-5),
1.67 (t, 2H, J ˆ 8.6 Hz, H-6eq, H-6ax); 13C NMR (100 MHz, [D6]DMSO):
d ˆ 140.63 (Cq-Ar), 128.48 (m-CH-Ar), 127.66 (o-CH-Ar), 127.44 (p-CH-
Ar), 83.11 (C-2), 75.72 (C-4), 74.66 (CH2), 73.24, 70.23 (C-3, C-5), 67.79 (C-
1), 30.41 (C-6).
elemental analysis calcd (%) for C33H40O8: C 70.19, H 7.14; found C 70.00,
H 7.01.
dl-6-Deoxy-1,4,5-tri-O-p-methoxybenzyl-myo-inositol (rac-18): dl-6-De-
oxy-2,3-O-isopropylidene-1,4,5-tri-O-p-methoxybenzyl-myo-inositol (rac-
17, 8.5 g, 15 mmol) was dissolved in 80% aqueous acetic acid (100 mL)
and heated at 508C for 30 min. The solution was allowed to cool to room
temperature before being poured into iced-water (100 mL). The aqueous
layer was extracted with ethyl acetate (4 Â 100 mL) and the combined
organic layers were washed to neutrality with NaHCO3 (6 Â 150 mL), dried
(MgSO4), and concentrated in vacuo. The crude residue was purified by
flash chromatography (toluene/ethyl acetate 8:3) to yield dl-6-deoxy-1,4,5-
tri-O-p-methoxybenzyl-myo-inositol (rac-18) (6.09 g, 77%): m.p. 125 ±
1268C; 1H NMR (400 MHz, CDCl3): d ˆ 7.29 ± 6.81 (m, 12H, ArH), 4.87,
4.63, 4.59, 4.53, 4.50, 4.46 (3AB, 6H, CH2), 4.16 (brt, 1H, J ˆ 2.3 Hz, H-2),
dl-2-O-Benzyl-1,3,4,5-tetrakis[bis(benzyloxy)phospho]-6-deoxy-myo-
inositol (rac-22): A mixture of bis(benzyloxy)(diisopropylamino)phos-
phine (1.63 g, 1.13 mL, 4.8 mmol) and dl-2-O-benzyl-6-deoxy-myo-inositol
(rac-21, 150 mg, 0.6 mmol) in CH2Cl2 (3 mL) was stirred for 15 min at 08C.
1H-Tetrazole (350 mg, 5 mmol) in CH2Cl2 (2 mL) was added and the
reaction was stirred for a further 1 h. The solution was cooled to ꢀ788C
and m-CPBA (862 mg, 5 mmol) was added and the mixture was stirred for
one further hour. The reaction mixture was diluted with CH2Cl2 (8 mL) and
washed with water (20 mL), 10% sodium metabisulfite (20 mL) and
NaHCO3 (20 mL), dried (MgSO4), and concentrated in vacuo. The
resulting syrup was purified by flash chromatography (ether/hexane 1:1
to 2:1) to give dl-2-O-benzyl-1,3,4,5-tetrakis [bis(benzyloxy)phospho]-6-
deoxy-myo-inositol (rac-22) as a colourless syrup (613 mg, 79%): 1H NMR
(400 MHz, CDCl3): d ˆ 7.33 ± 7.14 (m, 45H, ArH), 5.06 ± 4.91 (m, 17H, H-4,
CH2), 4.70, 4.65 (AB, 2H, CH2), 4.43 (brs, 1H, H-2), 4.18 (m, 2H, H-1,
H-5), 4.09 (m, 1H, H-3), 2.52 (m, 1H, H-6eq), 2.30 (q, 1H, J ˆ 12.1 Hz,
H-6ax); 13C NMR (100 MHz, CDCl3): d ˆ 139.63 (Cq-Ar), 127.46 (m-CH-
Ar), 126.66 (o-CH-Ar), 126.43 (p-CH-Ar), 78.31 (C-2), 76.06 (C-3), 75.76
(CH2), 73.67 (C-4), 72.40, 72.34 (C-1, C-5), 70.15 ± 69.73 (CH2), 32.24 (C-6);
31P NMR (162 MHz, CDCl3) [1H decoupled] d ˆ ꢀ0.35, ꢀ0.62, ꢀ1.05,
ꢀ1.07 (4s, 4  P), [1H coupled] d ˆ ꢀ0.32, ꢀ0.59, ꢀ1.01, ꢀ1.04 (4sextet,
3.79 (m, 10H, H-4, OCH3), 3.40 (m, 3H, H-1, H-3, H-5), 2.08 (m, 2H, H-6eq
,
H-6ax); 13C NMR (100 MHz, CDCl3): d ˆ 159.50, 159.35, 159.26 (p-Cq-Ar),
130.98, 130.61, 130.00 (Cq-Ar), 129.79, 129.51, 129.46 (o-CH-Ar), 114.10,
114.00 (m-CH-Ar), 81.58 (C-4), 77.47, 74.16, 72.45 (C-1, C-3, C-5), 74.99
(CH2), 71.81 (CH2), 70.68 (CH2), 69.67 (C-2), 55.62 (OCH3), 30.14 (C-6);
‡
‡
MS (FAB , NBA): m/z (%): 523.3 (30) [M ꢀ H] ; MS (FABꢀ, NBA): m/z
‡
‡
(%): 677.3 (82) [M ‡ NBA] , 523.3 (100) [M ꢀ H] ; HRMS-FAB: (m/z):
‡
[M] calcd for C30H36O8 524.24102; found 524.23801; elemental analysis
calcd (%) for C30H36O8: C 68.69, H 6.92; found C 68.90, H 6.98.
dl-6-Deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol (rac-19):
A
mixture of dl-6-deoxy-1,4,5-tri-O-p-methoxybenzyl-myo-inositol (rac-18,
5.24 g, 10 mmol), Bu2SnO (2.98 g, 12 mmol), Bu4NI (4.8 g, 13 mmol), and p-
methoxybenzyl chloride (2.03 g, 1.76 mL, 13 mmol) in toluene (100 mL)
was heated under reflux with 4 Š molecular sieves in a Soxhlet apparatus
for 16 h. The solution was cooled and washed with H2O (2 Â 50 mL), brine
(100 mL), dried (MgSO4) and concentrated in vacuo. The crude syrup was
purified by flash chromatography (ether/hexane 1:1 to 2:1) to provide dl-6-
deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol as a white crystalline
material (rac-19) (4.90 g, 76%): m.p. 115 ± 1168C; 1H NMR (400 MHz,
CDCl3): d ˆ 7.29 ± 6.81 (m, 16H, ArH), 4.79, 4.77, 4.66, 4.62, 4.61, 4.58, 4.47,
4.44 (4AB, 8H, CH2), 4.20 (brs, 1H, H-2), 3.84 (t, 1H, J ˆ 9.4 Hz, H-4),
3.81 ± 3.79 (3s, 12H, OMe), 3.33 (m, 3H, H-1, H-3, H-5), 2.14 ± 2.09 (m, 1H,
H-6eq), 1.94 (q, 1H, J ˆ 12.1 Hz, H-6ax); 13C NMR (100 MHz, CDCl3): d ˆ
159.40, 159.34, 159.20, 159.16 (p-Cq-Ar), 131.49, 130.94, 130.49, 130.10 (Cq-
Ar), 129.80, 129.63, 129.47, 129.41 (o-CH-Ar), 114.06, 113.98, 113.96, 113.88
(m-CH-Ar), 82.38 (C-4), 80.23, 77.60, 73.78 (C-1, C-3, C-5), 75.73 (CH2),
72.54 (CH2), 72.30 (CH2), 70.48 (CH2), 68.49 (C-2), 55.61 (OCH3), 30.18 (C-
‡
‡
3JP,H ˆ 7.9 Hz, 4  P); MS (FAB , NBA): m/z (%): 1295 (100) [M] ; MS
(FABꢀ, NBA): m/z (%): 1447.2 (70) [M ‡ NBA] ; HRMS-FAB: (m/z):
‡
‡
[M] calcd for C69H70O17P4 1295.35635; found 1295.36816; elemental
analysis calcd (%) for C69H70O17P4: C 63.99, H 5.45; found C 64.00, H 5.51.
dl-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate (rac-13): dl-2-O-Ben-
zyl-1,3,4,5-tetrakis[bis(benzyloxy)phospho]-6-deoxy-myo-inositol (rac-22,
120 mg, 92 mmol) was dissolved in the minimum amount of MeOH/H2O
9:1 and was hydrogenated for 16 h under 80 psi, in the presence of twice the
amount of 10% Pd/C. The catalyst was removed by filtration and the
filtrate was concentrated to give crude racemic tetrakisphosphate rac-13.
The residue was then purified by ion-exchange chromatography using a
gradient of 1m TEAB (0 ± 100%). The title compound rac-13 eluted
between 65 ± 85% TEAB and after pooling and evaporation of fractions
was obtained as the triethylammonium salt (60 mg, 87%). 1H NMR
(400 MHz, CD3OD): d ˆ 4.48 (q, 1H, J ˆ 9.0 Hz, H-4), 4.31 (brs, 1H, H-2),
‡
‡
6); MS (FAB , NBA): m/z (%): 643.3 (30) [M ꢀ H] ; MS (FABꢀ, NBA):
‡
‡
m/z (%): 797.4 (100) [M ‡ NBA] ; HRMS-FAB: (m/z): [M] calcd for
C38H44O8 644.29854; found 644.29747; elemental analysis calcd (%) for
C38H44O8: C 70.79, H 6.88; found C 70.60, H 6.85.
dl-2-O-Benzyl-6-deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol
(rac-20): dl-6-Deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol (rac-
19, 5 g, 7.7 mmol) was dissolved in DMF (70 mL) and stirred at 08C for
10 min. NaH (400 mg, 10 mmol) was added and the solution was stirred at
Chem. Eur. J. 2001, 7, No. 1
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85

B. V. L. Potter and G. Horne
FULL PAPER
4.14 (m, 2H, H-1, H-5), 4.05 (dt, 1H, J ˆ 2.7, 10.2 Hz, H-3), 2.37 (m, 1H,
H-6eq), 2.10 (q, 1H, J ˆ 12.1 Hz, H-6ax); 31P NMR (162 MHz, CD3OD) [1H
decoupled]: d ˆ 1.94, 1.31, 1.27, 0.96, (4s, 4  P); [1H coupled]: d ˆ 2.02, 1.59,
1.52, 1.17 (4d, 3JP,H ˆ 9.1 Hz, 4  P).
(100 mL), dried (MgSO4) and concentrated in vacuo. The crude syrup was
purified by flash chromatography using ether/hexane 2:1 to provide d-6-
deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol (740 mg, 72%) as a
colourless syrup; [a]D ˆ 0 ꢀ 1 (c ˆ 1 in dichloromethane); HRMS-FAB:
‡
(m/z): [M] calcd for C38H44O8 644.29854; found 644.29590; elemental
d- and l-3-O-[S-(‡)-O-Acetylmandelyl]-6-deoxy-1,4,5-tri-O-p-methoxy-
benzyl-myo-inositol (24 and 25): A mixture of dl-6-deoxy-1,4,5-tri-O-p-
methoxybenzyl-myo-inositol (rac-15) (4 g, 7 mmol), (S)-(‡)-O-acetylman-
delic acid (23) (1.55 g, 8 mmol), and DMAP (60 mg, 0.5 mmol) in CH2Cl2
(20 mL), was stirred at ꢀ208C. A solution of DCC (1.75 g, 8.5 mmol), in
CH2Cl2 (10 mL), was added dropwise over 1 h at ꢀ208C and stirring
continued overnight. TLC (ether/hexane 3:2) showed two major products
of tR ˆ 0.28 and 0.21. The reaction mixture was filtered through Celite,
which was washed thoroughly with CH2Cl2 (2 Â 100 mL). The solvent was
evaporated to give a syrup which was purified by flash chromatography
using ether/hexane (1:1 to 3:2) to provide d-3-O-[S-(‡)-O-acetylmandel-
yl]-6-deoxy-1,4,5-tri-O-p-methoxybenzyl-myo-inositol (24) (2.03 g, 38%)
and l-3-O-[S-(‡)-O-acetylmandelyl]-6-deoxy-1,4,5-tri-O-p-methoxyben-
zyl-myo-inositol (25) (2.14 g, 40%). 24: TLC: tR ˆ 0.21 (ether/hexane
3:2); m.p. 112 ± 1138C; [a]D ˆ ‡25.7 (c ˆ 1 in methanol); 1H NMR
(400 MHz, CDCl3): d ˆ 7.60 ± 6.8 (m, 17H, ArH), 5.98 (s, 1H,
O2CCH(OAc)Ph), 4.72 (dd, 1H, J ˆ 2.3, 9.8 Hz, H-3), 4.70, 4.63, 4.57,
4.53, 4.40, 4.38 (3AB, 6H, CH2), 4.10 (brs, 1H, H-2), 3.94 (t, 1H, J ˆ 9.8 Hz,
H-4), 3.38 ± 3.31 (m, 2H, H-1, H-5), 2.19 (s, 3H, OAc), 2.10 (dt, 1H, J ˆ 4.7,
12.1 Hz, H-6eq), 1.89 (q, 1H, J ˆ 12.1 Hz, H-6ax); 13C NMR (100 MHz,
CDCl3): d ˆ 170.73, 168.37 (CO), 159.47, 159.25, 159.17 (p-Cq-Ar), 133.73,
130.99, 130.75 (Cq-Ar), 129.85, 129.53, 129.46 (o-CH-Ar), 129.40 (p-CH-
Ar), 128.99 (o-CH-Ar), 127.57 (m-CH-Ar), 114.12, 113.97, 113.83 (m-CH-
Ar), 79.37 (C-4), 77.31 (C-5), 76.04 (C-3), 75.24 (CH2), 75.05
(O2CCH(OAc)Ph), 73.46 (C-1), 72.35 (CH2), 70.70 (CH2), 68.44 (C-2),
analysis calcd (%) for C38H44O8: C 70.79, H 6.88; found C 70.40, H 6.83.
NMR and mass spectrum data as for rac-19.
l-6-Deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol (ent-19) was ob-
tained in an identical fashion to that described for 19 and isolated as a
colourless syrup (791 mg, 77%). [a]D ˆ ꢀ1.4 (c ˆ 0.7 in dichloromethane);
‡
HRMS-FAB: (m/z): [M] calcd for C38H44O8 644.29854; found 644.29319;
elemental analysis calcd (%) for C38H44O8: C 70.79, H 6.88; found C 70.20,
H 6.88. NMR and mass spectrum data as for rac-19.
d-2-O-Benzyl-6-deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol (20):
d-6-Deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol (19, 500 mg,
0.8 mmol) was dissolved in DMF (10 mL) and stirred at 08C for 10 min.
NaH (50 mg, 1.2 mmol) was added and the solution was stirred at 08C for a
further 10 min afterwhich benzyl bromide (171 mg, 118 mL, 1 mmol) was
added and the solution was stirred for 2 h. The reaction was quenched by
addition of MeOH and the solvent was evaporated. The crude residue was
dissolved in ether and was washed with H2O (2 Â 20 mL), dried (MgSO4)
and concentrated in vacuo. The crude residue was purified by flash
chromatography using ether/hexane (1:1 to 2:1) to yield d-2-O-benzyl-6-
deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol (365 mg, 67%):
‡
[a]D ˆ ‡13.2 (c ˆ 1.1 in acetone); HRMS-FAB (m/z): [M] calcd for
C45H50O9 734.34549; found 734.34021. NMR and mass spectrum data as for
rac-20.
l-2-O-Benzyl-6-deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol (ent-
20) was obtained in an identical fashion as that described for 20 (475 mg,
‡
55.59 (OCH3), 30.29 (C-6), 21.16 (OAc); MS (FAB , NBA): m/z (%): 699.3
‡
81%): [a]D ˆ ꢀ7.0 (c ˆ 1 in acetone); HRMS-FAB: (m/z): [M] calcd for
‡
‡
(20) [M ꢀ H] ; MS (FABꢀ, NBA): m/z (%): 853.4 (85) [M ‡ NBA] , 699.3
C45H50O9 734.34549; found 734.34101. NMR and mass spectrum data as for
‡
‡
(100) [M ꢀ H] ; HRMS-FAB (m/z): [M] calcd for C40H44O11 700.28837;
found 700.282715. 25: TLC: tR ˆ 0.28 (ether/hexane 3:2); [a]D ˆ ‡12.0 (c ˆ
1 in methanol); 1H NMR (400 MHz, CDCl3): d ˆ 7.53 ± 6.67 (m, 17H, ArH),
5.99 (s, 1H, O2CCH(OAc)Ph), 4.72 (dd, 1H, J ˆ 2.3, 9.8 Hz, H-3), 4.56 ±
4.27 (m, 6H, CH2), 4.37 (br d, 1H, J ˆ 2.3 Hz, H-2), 3.89 (t, 1H, J ˆ 9.8 Hz,
H-4), 3.39 (m, 1H, H-1), 3.31 (m, 1H, H-5), 2.18 (s, 3H, OAc), 2.10 (dt, 1H,
J ˆ 4.7, 12.4 Hz, H-6eq), 1.92 (q, 1H, J ˆ 12.15, H-6ax); 13C NMR (100 MHz,
CDCl3): d ˆ 170.10, 168.41 (CO), 159.49, 159.20, 158.92 (p-Cq-Ar), 133.30,
130.82, 130.60 (Cq-Ar), 129.64, 129.52, 129.44 (o-CH-Ar), 129.26 (p-CH-
Ar), 129.05 (o-CH-Ar), 128.17 (m-CH-Ar), 114.14, 113.902, 113.59 (m-CH-
Ar), 79.14 (C-4), 76.92 (C-5), 76.04 (C-3), 74.92 (O2CCH(OAc)Ph), 74.58
(CH2), 73.11 (C-1), 72.05 (CH2), 70.45 (CH2), 68.11 (C-2), 55.24 (OCH3),
rac-20.
d-2-O-Benzyl-6-deoxy-myo-inositol (21): d-2-O-Benzyl-6-deoxy-1,3,4,5-
tetra-O-p-methoxybenzyl-myo-inositol (20, 250 mg, 0.35 mmol) was dis-
solved in CH2Cl2/TFA (9:1, 15 mL) and stirred at room temperature for
30 min. The reaction mixture was concentrated in vacuo and the crude
residue was redissolved in toluene. The solvent was evaporated and the
crude residue was purified by flash chromatography ethyl acetate/acetone
(1:0 to 0:1) to yield d-2-O-benzyl-6-deoxy-myo-inositol (71 mg, 83%):
[a]D ˆ ‡3.8 (c ˆ 0.76 in acetone). NMR data as for rac-21.
l-2-O-Benzyl-6-deoxy-myo-inositol (ent-21) was obtained in an identical
fashion to that for 21 (93 mg, 77%): [a]D ˆ ꢀ3.0 (c ˆ 1 in acetone). NMR
data as for rac-21.
‡
‡
29.95 (C-6), 20.66 (OAc); MS (FAB , NBA): m/z (%): 699.3 (35) [M ꢀ H] ;
MS (FABꢀ, NBA): m/z (%): 853.3 (60) [M ‡ NBA] , 699.3 (100) [M ꢀ H] ;
‡
‡
d-2-O-Benzyl-1,3,4,5-tetrakis[bis(benzyloxy)phospho]-6-deoxy-myo-
inositol (22): A mixture of bis(benzyloxy)(diisopropylamino)phosphine
(650 mg, 634 mL, 1.9 mmol) and d-2-O-benzyl-6-deoxy-myo-inositol
(60 mg, 0.24 mmol) in CH2Cl2 (4 mL) was stirred for 15 min at 08C. 1H-
Tetrazole (140 mg, 2 mmol) in CH2Cl2 (3 mL) was added and the reaction
was stirred for a further 1 h. The solution was cooled to ꢀ788C and m-
CPBA (345 mg, 2 mmol) was added and the mixture was stirred for 1 h. The
reaction mixture was diluted with CH2Cl2 (8 mL) and washed with water
(20 mL), 10% sodium metabisulfite (20 mL) and NaHCO3 (20 mL), dried
(MgSO4), and concentrated in vacuo. The remaining syrup was purified by
flash chromatography (ether/hexane 1:1 to 2:1) to give d-2-O-benzyl-
1,3,4,5-tetrakis[bis(benzyloxy)phospho]-6-deoxy-myo-inositol as a colour-
less syrup (230 mg, 74%): [a]D ˆ ‡9.0 (c ˆ 1 in methanol); HRMS-FAB:
‡
HRMS-FAB: (m/z): [M] calcd for C40H44O11 700.28837; found 700.28301.
d-6-Deoxy-1,4,5-tri-O-p-methoxybenzyl-myo-inositol (18): A mixture of
d-3-O-[S-(‡)-O-acetylmandelyl]-6-deoxy-1,4,5-tri-O-p-methoxybenzyl-
myo-inositol (24, 1.6 g, 2.3 mmol), sodium hydroxide (2 g, 50 mmol) and
methanol (50 mL) was heated at reflux temperature for 30 min. The
mixture was cooled to room temperature and neutralised by careful
addition of carbon dioxide. The resulting solid was diluted with water
(25 mL) and evaporated to dryness in vacuo. The crude product was
extracted with CH2Cl2 (4 Â 100 mL) which was then evaporated off leaving
the title compound d-6-deoxy-1,4,5-tri-O-p-methoxybenzyl-myo-inositol
‡
(1.04 g, 87%); [a]D ˆ ‡7.2 (c ˆ 0.9 in CH2Cl2); HRMS-FAB: (m/z): [M]
calcd for C30H36O8 524.24102; found 524.23549; elemental analysis calcd
(%) for C30H36O8: C 68.69, H 6.92; found C 68.40, H 6.88. NMR and mass
spectrum data as for rac-18.
‡
(m/z): [M] calcd for C69H70O17P4 1294.35635; found 1294.35745; elemental
analysis calcd (%) for C69H70O17P4: C 63.99, H 5.45; found C 63.60, H 5.48.
NMR and mass spectrum data as for rac-22.
l-6-Deoxy-1,4,5-tri-O-p-methoxybenzyl-myo-inositol (ent-18): was ob-
tained in an identical fashion to that described for 18 (600 mg, 81%).
l-2-O-Benzyl-1,3,4,5-tetrakis[bis(benzyloxy)phospho]-6-deoxy-myo-
inositol (ent-22) was obtained in an identical fashion as to that for 22 and
was isolated as a colourless syrup (281 mg, 68%): [a]D ˆ ꢀ8.3 (c ˆ 0.9 in
‡
[a]D ˆ ꢀ8.1 (c ˆ 1 in CH2Cl2); HRMS-FAB: (m/z): [M] calcd for C30H36O8
524.24102; found 524.23807; elemental analysis calcd (%) for C30H36O8: C
68.69, H 6.92; found C68.40, H 6.85. NMR and mass spectrum data as for
rac-18.
‡
methanol); HRMS-FAB: (m/z): [M] calcd for C69H70O17P4 1294.35635;
found 1294.35843; elemental analysis calcd (%) for C69H70O17P4: C 63.99 H
5.45; found C 63.50, H 5.52. NMR and mass spectrum data as for rac-22.
d-6-Deoxy-1,3,4,5-tetra-O-p-methoxybenzyl-myo-inositol (19): A mixture
of d-6-deoxy-1,4,5-tri-O-p-methoxybenzyl-myo-inositol (18, 750 mg,
1.4 mmol), Bu2SnO (400 mg, 1.6 mmol), Bu4NI (591 mg, 1.6 mmol), and
p-methoxybenzyl chloride (234 mg, 200 mL, 1.5 mmol) in toluene (30 mL)
was heated under reflux with 4 Š molecular sieves in a Soxhlet apparatus
for 16 h. The solution was cooled and washed with H2O (2 Â 50 mL), brine
d-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate (13): d-2-O-Benzyl-
1,3,4,5-tetrakis[bis(benzyloxy)phospho]-6-deoxy-myo-inositol
(80 mg,
60 mmol) was dissolved in the minimum amount of MeOH/H2O 9:1 and
was hydrogenated for 16 h under 80 psi, in the presence of twice the
amount of 10% Pd/C. The catalyst was removed by filtration and the
86
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0947-6539/01/0701-0086 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 1

Inositol Derivatives
80±87
Â
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eluted at 65 ± 85% TEAB and after pooling and evaporation of fractions
was obtained as the triethylammonium salt (40 mg, 85%). [a]D ˆ ‡9.8 (c ˆ
0.3 in water, pH approximately 4). NMR data as for rac-13.
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l-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate (ent-13) was obtained in
an identical fashion to that described for 13 (25 mg, 61%). [a]D ˆ ꢀ8.5 (c ˆ
0.35 in water, pH approximately 4). NMR data as for rac-13.
d-6-Deoxy-myo-inositol (26): d-1,4,5-Tri-O-p-methoxybenzyl-myo-inositol
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Acknowledgements
We thank the BBSRC (ICR Programme) and the Wellcome Trust
(Programme Grant to BVLP) for financial support and acknowledge
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Received: June 26, 2000 [F2569]
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