Design and synthesis of biotinylated inositol phosphates relevant to the biotin-avidin techniques

Article abstract of DOI:10.1039/b719938d

Six bifunctional molecules containing biotin and various inositol phosphates were synthesized. These compounds were designed on the basis of X-ray structures of the complexes of d-myo-inositol 1,4,5-triphosphates (IP ₃) and phospholipase C δ pleckstrin homology domain (PLCδ PH) considering the application to the biotin-avidin techniques. The building blocks of the inositol moiety were synthesized starting with optically resolved myo-inositol derivatives and assembled to the biotin linker through a phosphate linkage. The Royal Society of Chemistry.

Full text of DOI:10.1039/b719938d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Design and synthesis of biotinylated inositol phosphates relevant to the
biotin–avidin techniques†
Kensaku Anraku,a Teruhiko Inoue,b Kenji Sugimoto,c Takashi Morii,c Yasuo Mori,d Yoshinari Okamotoe and
Masami Otsuka*e
Received 2nd January 2008, Accepted 7th March 2008
First published as an Advance Article on the web 1st April 2008
DOI: 10.1039/b719938d
Six bifunctional molecules containing biotin and various inositol phosphates were synthesized. These
compounds were designed on the basis of X-ray structures of the complexes of D-myo-inositol
1,4,5-triphosphates (IP3) and phospholipase C d pleckstrin homology domain (PLCd PH) considering
the application to the biotin–avidin techniques. The building blocks of the inositol moiety were
synthesized starting with optically resolved myo-inositol derivatives and assembled to the biotin linker
through a phosphate linkage.
the development of inositol phosphate mimetics that control PH
domains, and search for novel PH domains without resort to
radiolabels. We report here the synthesis of biotin derivatives of
six different inositol phosphates, i.e., D-1,4,5-IP3, L-1,4,5-IP3, D-
Introduction
D-myo-Inositol 1,4,5-triphosphate (IP3) plays a key role in the sig-
naling cascade that links extracellular messengers to intracellular
Ca2+ mobilization.1 Upon stimulation of a certain receptor, the as-
2,4,5-IP3, D-1-IP1, L-1-IP1, and 2-IP1 (Fig. 1).
sociated G protein or tyrosine kinase activates a membrane-bound
phospholipase C (PLC), which hydrolyzes phosphatidylinositol
4,5-bisphosphate (PtdIns 4,5-P2) into two second messengers, IP3
and diacylglycerol (DAG), bifurcating the signaling pathway. The
hydrophilic IP3 diffuses into the cytosol and activates the receptor
of a Ca2+ channel on the endoplasmic reticulum, resulting in the
release of Ca2+ from an internal store. Pleckstrin homology (PH)
domains are structural modules of around 120 amino acids with
sequence similarity to two regions in pleckstrin, the major protein
kinase C substrate in platelets.2–4 These domains are found in
more than 100 different proteins, and appear to be important
for membrane association of proteins involved in intracellular
signaling and the cytoskeleton,5–8 in some cases by binding specific
phosphoinositides and their head groups.9–11 As an example,
PLCd PH domain is an IP3-binding region which sterospecifically
recognizes and binds to PtdIns 4,5-P2 with high affinity (the
dissociation constant Kd value is 1.7 lM).11 However, there have
been a few reports on binding analysis of the inositol phosphate
head groups and PH domains. Thus in order to study the relative
affinity and specificity in the binding of inositol phosphates and
Fig. 1 Biotin derivatives of six different inositol phosphates.
diverse PH domains, we intend to prepare biotinylated inositol
phosphates. The biotin–avidin techniques12 would address the
binding analysis of inositol phosphates and PH domains and
Results and discussion
aInstitute of Health Sciences, Kumamoto Health Science University, 325
Izumi-machi, Kumamoto 861-5598, Japan
Fig. 2 shows the design and synthetic strategy of biotinylated
inositol phosphates. The X-ray crystal structure of the IP3–
PLCd PH domain complex13 revealed that the inositol and 4,5-
phosphate groups of IP3 are accommodated in the binding pocket,
therefore we thought that a biotin linker could be introduced
at the 1-phosphate or 2-phosphate (the latter is an artificially-
created derivative) of inositol without affecting the PH domain
binding. Our synthetic strategy is to differentiate the six hydroxyl
groups of D-myo-inositol through the optical resolution of diac-
etal intermediates14 to obtain a suitably protected intermediate
that could be coupled with the biotin linker by a bifunctional
bGraduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-
8501, Japan
cInstitute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan
dLaboratory of Molecular Biology, Department of Synthetic Chemistry and
Biological Chemistry, Graduate School of Engineering, Kyoto University,
Kyoto 615-8510, Japan
eFaculty of Medical and Pharmaceutical Sciences, Kumamoto Univer-
sity, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan. E-mail: motsuka@
gpo.Kumamoto-u.ac.jp; Fax: +81-96-371-4620; Tel: +81-96-371-4620
† Electronic supplementary information (ESI) available: Further experi-
mental and characterisation details. See DOI: 10.1039/b719938d
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prepared as the starting material of the D-1-IP1 moiety by the
method of Billington et al.14 Hydrolysis of the cyclohexylidene
groups of (+)-1 with acid provided 2a, which was acetylated to
give fully protected 3a. Finally treatment with H2/10% palladium-
carbon gave the debenzylated D-1-IP1 moiety 4a in 73% yield
(for 3 steps). The enantiomeric isomer L-1-O-benzyl-2,3:5,6-di-
O-cyclohexylidene-myo-inositol (−)-1 gave the L-1-IP1 moiety 4b
by the same procedure.
The syntheses of the D-1,4,5-IP3 and L-1,4,5-IP3 moieties were
carried out as shown in Scheme 2. The starting material D-
1,2:4,5-di-O-cyclohexylidene-myo-inositol 5a was prepared by
hydrogenolysis of the benzyl group of (−)-1. Allylation of the
resulting alcohol 5a provided 6a, which was further treated
with p-toluenesulfonic acid and H2O to give deprotected 7a in
92% yield (for 2 steps). The cis-1,2-diol of 7a was regioselec-
tively p-methoxybenzylated by means of the dibutyltin oxide
procedure.16,17 Thus the tin complex of the 1,2-diol was regios-
electively reacted with p-methoxybenzyl chloride in the presence
of caesium fluoride to give 8a exclusively in 97% yield. The intro-
duction of isopropylidene acetal to the 4,5-vicinal alcohol under
the usual conditions gave 9a in 85% yield. Isomerization of the allyl
groups of 9a followed by the treatment with HgO, HgCl2 gave 10a
in 67% yield.18 Acetylation of the resulting alcohol provided 11a,
which was treated with p-toluenesulfonic acid and ethylene glycol
to give the intermediate 12a in 88% yield (for 2 steps), which
is ready for the next phosphorylation. Bis(b-cyanoethyl)-N,N-
diisopropylphosphoramidite, considered one of the most effective
phosphorylating agents, was prepared by the method of Uhlmann
and Engels.19 The 4,5-dihydroxy compound 12a was converted to
the 4,5-bisphosphonate with phosphoramidite and 1H-tetrazole
and subsequent oxidation with MCPBA to afford 13a in 93% yield.
Oxidative cleavage of the p-methoxybenzyl group with CAN20 gave
the D-1,4,5-IP3 moiety 14a in 71% yield. The enantiomeric isomer
D-2,3:5,6-di-O-cyclohexylidene-myo-inositol gave the L-1,4,5-IP3
moiety 14b by the same procedure.
Fig. 2 Design and synthetic strategy of biotinylated D-myo-inositol
1,4,5-triphosphate.
phosphorylating agent.15 As the target molecules have a sulfur in
the biotin structure, our strategy does not end up with any benzyl
or related protecting groups that require hydrogenolysis to remove
them.
The syntheses of the D-1-IP1 and L-1-IP1 moieties were carried
out as shown in Scheme 1. The optically resolved alcohol D-
1-O-benzyl-2,3:5,6-di-O-cyclohexylidene-myo-inositol (+)-1 was
D-2,4,5-IP3 moiety was prepared from the intermediate 7a as
shown in Scheme 3. The regioselective allylation16,17 at the 1-
hydroxyl group of 7a by the dibutyltin oxide method gave 15a
in 59% yield. Introduction of isopropylidene acetal to the 4,5-
position of 15a provided 16a, which was further protected with
a p-methoxybenzyl group at the 2-hydroxyl position to give fully
protected 17a in 66% yield (for 2 steps). Deprotection of the allyl
groups18 provided 18a, which was acetylated to give 19a. Finally
the isopropylidene acetal was removed to afford 20a in 42% yield
(for 3 steps). Phosphorylation19 of the 4,5-hydroxyl groups gave
21a, which was treated with CAN20 to give the D-2,4,5-IP3 moiety
22a in 59% yield (for 2 steps). The 2-hydroxy pentaacetate 24a
was prepared by exhaustive acetylation of 20a, and removal of the
p-methoxybenzyl group in 64% yield (for 2 steps).
The connection of inositol and biotin moieties prepared by the
method of Pon21 was carried out as shown in Scheme 4. The biotin-
linker moiety was reacted with bifunctional phosphorylating agent
(b-cyanoethyl)-N,N,Nꢀ,Nꢀ-tetraisopropylphosphoramidite15 and
1H-tetrazole to give a rather labile phosphoramidite which was
immediately condensed with inositol moiety 4a, 14a, 4b, 14b,
22a, or 24a. Subsequent oxidation of the condensation products
with tert-BuOOH gave the fully protected biotinylated inositol
phosphates 25a (93% yield), 26a (78% yield), 25b (83% yield), 26b
Scheme 1 Reagents and conditions: (i) TFA, MeOH, rt; (ii) Ac2O, DMAP,
Py, rt; iii) H2/10% Pd-C, CH3COOH, rt.
1
(85% yield), 27 (99% yield), and 28 (52% yield). The H NMR
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Scheme 2 Reagents and conditions: (i) allyl-Br, NaH, DMF, rt; (ii) TsOH, THF–MeOH, reflux; (iii) (a) Bu2SnO, toluene, reflux, 3 h; (b) CsF, MPM-Cl,
DMF, −40 ◦C then rt; (iv) 2-methoxypropene, TsOH, DMF, rt; (v) (a) (Ph3P)3RhCl, DABCO, EtOH–benzene–H2O, reflux, 5 h; (b) HgO, HgCl2,
acetone–H2O, rt, 5 min; (vi) Ac2O, DMAP, Py, rt; (vii) TsOH, ethylene glycol, CH2Cl2, rt, 10 min; (viii) (a) bis(b-cyanoethyl)-N,N-diisopropyl-
phosphoramidite, 1H-tetrazole, CH2Cl2, rt, 1.5 h; (b) MCPBA, CH2Cl2, rt, 5 min; (ix) CAN, CH3CN–H2O, rt, 1 h.
Scheme 3 Reagents and conditions: (i) (a) Bu2SnO, toluene, reflux, 3 h; (b) CsF, allyl-Br, DMF, −40 ◦C then rt; (ii) 2-methoxypropene, TsOH, DMF,
rt; (iii) MPM-Cl, NaH, DMF, rt; (iv) (a) (Ph3P)3RhCl, DABCO, EtOH–benzene–H2O, reflux, 5 h; (b) HgO, HgCl2, acetone–H2O, rt, 5 min; (v) Ac2O,
DMAP, Py, rt; (vi) TsOH, ethylene glycol, CH2Cl2, rt, 10 min; (vii) (a) bis(b-cyanoethyl)-N,N-diisopropylphosphoramidite, 1H-tetrazole, CH2Cl2, rt,
1.5 h; (b) MCPBA, CH2Cl2, rt, 5 min; (viii) CAN, CH3CN–H2O, rt, 1 h; (ix) Ac2O, DMAP, Py, rt; (x) H2/10% Pd-C, CH3COOH, rt.
and FABMS data of these compounds showed no oxidation of the
biotin sulfide.
against the PLCdPH domain or other PH domains will be reported
elsewhere.
The final stage of the synthesis was carried out as shown in
Scheme 5. After removal of trityl group of biotin with acid treat-
ment, all protecting groups were removed in one step by reaction
with NH3 to give water-soluble biotinylated inositol phosphates.
The biotinylated mono or polyphosphates were efficiently purified
by anion-exchange chromatography with gradients of ammonium
formate as eluent to give biotinylated D-1,4,5-IP3 (32% yield), D-
1-IP1 (30% yield), L-1,4,5-IP3 (35% yield), L-1-IP1 (46% yield),
D-2,4,5-IP3 (32% yield), and D-2-IP3 (66% yield) (for 2 steps).
Biological study of the synthetic biotinylated inositol phos-
phates using streptavidin beads was carried out. The original PLCd
PH domain bound efficiently to the prebound beads of biotiny-
lated D-1,4,5-IP3. The dissociation constant Kd of biotinylated D-
1,4,5-IP3 binding of the PLCdPH domain was 250 20 nM, which
was comparable to that of non-tethered D-1,4,5-IP3.11 The detailed
binding assay of the synthetic biotinylated inositol phosphates
Exprimental
Materials and methods
Chemicals were purchased from Aldrich, Fluka, Kanto Chemical,
Nacalai tesque, and Wako. Dehydrated CH2Cl2 was distilled
from calcium hydride. Thin layer chromatography (TLC) was
performed on precoated plates (Merck TLC sheets silica 60 F254):
products were visualized by spraying phosphomolybdic acid in
EtOH, or basic potassium permanganate and heating at high
temperature. Chromatography was carried out on Silica Gel 60 N
(40–100 mesh). Anion exchange chromatography was performed
using Dowex 1 × 8 (Cl−, 50–100 mesh), eluting with ammonium
formate containing formic acid. Column fractions containing bi-
otinylated inositol polyphosphates were assayed for phosphate by
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Scheme 4 Reagents and conditions: (i) (a) (b-cyanoethyl)-N,N,Nꢀ,Nꢀ-tetraisopropylphosphoramidite, 1H-tetrazole, CH2Cl2, rt, 1.5 h; (ii) 4a, 14a,
1H-tetrazole, CH2Cl2, rt, 2 h; (iii) tert-BuOOH, CH2Cl2, rt, 5 min; (iv) 4b or 14b, 1H-tetrazole, CH2Cl2, rt, 2 h; (v) 22a or 24a, 1H-tetrazole, CH2Cl2, rt,
2 h.
of each ester gave the corresponding enatiomeric forms of the
alcohol 1 in quantitative yield. [a]2D5 = +23.32 and −23.16 (c 0.1,
CHCl3). (Diisopropylamino)dichlorophosphine was prepared by
the method of Uhlmann and Engels.19 by adding two equivalents
of diisopropylamine to a solution of phosphorus trichloride in
dry ethyl ether at −78 ◦C. The crude product was purified by
distillation under reduced pressure and could be stored as a crys-
talline solid at −20 ◦C. Two equivalents of b-cyanoethanol in the
presence of diisopropylethylamine were reacted with the purified
product in dehydrated CH2Cl2, to afford bis(b-cyanoethyl)-N,N-
diisopropylaminophosphoramidite. The biotin-linker moiety was
prepared by the method of Pon21 N-Hydroxysuccinimide-biotin
was reacted with 6-amino-1-hexanol to give biotinyl-6-amino-1-
hexanol, which was further converted into the dimethoxytrityl
derivative by silylation of the OH group, N1-protection with
a dimethoxytrityl group, and removal of the O-silyl group.
Specific rotations of enantiopure compounds were recorded by
JASCO Dip-1000. NMR spectra (JEOL JNM-AL300MHz) were
referenced to SiMe4, or HDO. Infra-red spectra were recorded
on a JASCO FT/IR-410. The samples were prepared as KBr
discs, or thin films between sodium chloride discs. Microanalysis
was carried out by Yanaco MT-5S. Mass spectra (EI) and high
resolution mass spectra (HRMS) were recorded by a JEOL JMS-
DX303HF. HRMS were recorded by using positive and negative
fast atom bombardment (FAB) with 3-nitrobenzyl alcohol (NBA)
(containing HMPA or not) as the matrix.
The dissociation constants (Kd) of biotinylated D-1,4,5-IP3
binding of the original PLCd PH domain were estimated based on
Scheme 5 Reagents and conditions: (i) TCA, CH2Cl2, rt, 1 h; (ii) aq. NH3,
MeOH, 55 ◦C, 10 h.
SDS–acrylamide gel electrophoretic analysis. 1 ml of streptavidin
beads which has binding capacity up to 85 nmol free biotin was
incubated with 5 nmol biotinylated D-1,4,5-IP3 solution for 6 h
at 4 ◦C in 30 mM HEPES, 50 mM NaCl, 0.005% Tween20,
3 mM EDTA (pH 7.4). The beads were washed with the same
buffer and divided up into five different volumes (100, 50, 25, 10,
0 ll) in separate tubes, which volumes of beads were converted
to biotinylated D-1,4,5-IP3 concentrations (1.00, 0.50, 0.25, 0.10,
0 lM), respectively. The divided beads were incubated with 500 ll
the Briggs test.22 The optically resolved D-1-O-benzyl-2,3:5,6-di-
O-cyclohexylidene-myo-inositol (+)-1 and L-1-O-benzyl-2,3:5,6-
di-O-cyclohexylidene-myo-inositol (−)-1 were prepared by the
method of Billington et al.14 Treatment of the racemic 1 with
(R)-camphanic acid chloride gave a mixture of the diastereomeric
camphanate esters. Separation of the diastereomeric esters by
recrystallization and chromatography, and subsequent hydrolysis
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of the PLCd PH domain (0.20 lM) for 10 min at 4 ◦C in the
same buffer. The beads were separated from the supernatant (S,
nonbinding fraction) by centrifugation at 1000 g for 1 min at
room temperature, the PLCd PH domain was eluted from the
beads with 50 lM IP3 to give the binding fraction (B). The
nonbinding fraction (S) and B were precipitated by addition of
an equal volume of 20% trichloroacetic acid and centrifugation,
and were quantified by analysis of 15% SDS–polyacrylamide gel
electrophoresis followed by CBB staining. The quantification of
each band was performed by analyzing the scaled gel data with
NIH image (version 1.6) software to integrate the intensity of the
dots of which each band was composed. The binding fraction Q =
[B]/([B] + [S]) for the PLCdPH domain to biotinylated D-1,4,5-IP3
was plotted against biotinylated D-1,4,5-IP3 concentration. The
Kd was derived from the best fit for a theoretical dissociation
equation; Qfit = [biotinylated D-1,4,5-IP3]/(Kd + [biotinylated D-
1,4,5-IP3]), where [biotinylated D-1,4,5-IP3] is the concentration of
biotinylated D-1,4,5-IP3.
93%) as a white solid. H NMR (CDCl3) d 2.00 (s, 3H), 2.01 (s,
3H), 2.02 (s, 3H), 2.09 (s, 3H), 2.21 (s, 3H), 3.90 (dd, J = 10.1,
2.9 Hz, 1H), 4.97 (dd, J = 10.6, 2.7 Hz, 1H), 5.15 (t, J = 9.7 Hz,
1H), 5.31 (t, J = 9.9 Hz, 1H), 5.45 (t, J = 10.1 Hz, 1H), 5.59 (t,
J = 2.9 Hz, 1H). 13C NMR (CDCl3) d 20.5, 20.7, 20.8, 68.7, 69.2,
70.4, 70.8, 72.4, 169.7, 169.8, 169.9, 170.3, 171.0. IR (KBr) 3400,
1740, 1360, 1210, 1040 cm−1. MS (EI) m/z 391 (M + H)+. Anal.
calcd for C16H22O11: C, 49.23; H, 5.68. Found: C, 48.93; H, 5.81.
1
D-3,6-Di-O-allyl-1,2:4,5-di-O-cyclohexylidene-myo-inositol (6a)
To a solution of (−)-1 (1.94 g, 4.50 mmol) in MeOH (50 ml)
was added 10% palladium-carbon (800 mg), and the resulting
mixture was stirred at room temperature under hydrogen for
2 days. The residue was purified by recrystallization (hexane–
AcOEt = 1 : 1) to afford 5a (1.45 g, 95%) as a white solid.
To a solution of 5a (2.00 g, 5.9 mmol) in DMF (30 ml) was
added NaH (576 mg, 24.0 mmol) followed by allyl bromide
(1.04 ml, 12.0 mmol), and the resulting mixture was stirred at room
temperature under argon for 24 h. The reaction was quenched
with MeOH, and concentrated under reduced pressure, and the
residue was diluted with AcOEt. The organic phase was washed
with H2O and saturated aqueous NaCl, dried over Na2SO4, and
then concentrated under reduced pressure. The crude product was
purified by column chromatography (hexane–AcOEt = 5 : 1) to
D-1-O-Benzyl-myo-inositol (2a)
To a solution of D-1-O-benzyl-2,3:5,6-di-O-cyclohexylidene-myo-
inositol (+)-1 (861 mg, 2.0 mmol) in MeOH (30 ml) was added TFA
(30 ml), and the resulting mixture was stirred at room temperature
for 24 h. The mixture was concentrated under reduced pressure,
and the residue was washed with AcOEt to give 2a (443 mg, 82%)
as a white solid. 1H NMR (CD3OD) d 3.03–3.22 (m, 3H), 3.52 (t,
J = 9.5 Hz, 1H), 3.67 (t, J = 9.5 Hz, 1H), 4.01 (s, 1H), 4.55 (d, J =
11.7 Hz, 1H), 7.15–7.35 (m, 5H). 13C NMR (CD3OD) d 70.8, 73.0,
73.2, 73.7, 74.1, 76.5, 81.1, 128.6, 129.1, 129.3, 139.9. IR (KBr)
3350, 2850, 1730, 1360, 1200, 1060, 1030 cm−1. MS (EI) m/z 271
(M + H)+. Anal. calcd for C13H18O6: C, 57.77; H, 6.71. Found: C,
57.80; H, 6.88.
1
afford 6a (2.65 g, >99%) as a colorless oil. H NMR (CDCl3) d
1.38–1.76 (m, 20H), 3.31 (t, J = 9.9 Hz, 1H), 3.63 (dd, J = 10.6,
6.4 Hz, 1H), 3.77 (dd, J = 10.1, 4.2 Hz, 1H), 3.97 (t, J = 9.9 Hz,
1H), 4.06 (t, J = 4.9 Hz, 1H), 4.22–4.37 (m, 4H), 4.45 (t, J =
4.4 Hz, 1H), 5.17–5.36 (m, 4H), 5.89–6.05 (m, 2H). 13C NMR
(CDCl3) d 23.5, 23.7, 23.8, 23.9, 24.9, 25.0, 35.2, 36.3, 36.4, 37.6,
70.9, 71.2, 74.9, 76.0, 76.5, 78.4, 80.4, 80.8, 110.5, 112.6, 117.1,
117.8, 134.8, 134.9. IR (KBr) 2920, 2850, 1730, 1440, 1360, 1260,
1160 cm−1. MS (EI) m/z 420 (M + H)+. Anal. calcd for C24H36O6:
C, 68.54; H, 8.63. Found: C, 68.55; H, 8.77.
D-1-O-Benzyl-2,3,4,5,6-penta-O-acetyl-myo-inositol (3a)
To a solution of 2a (432 mg, 1.6 mmol) in pyridine (40 ml)
was added acetic anhydride (1.51 ml, 16.0 mmol) followed by
4-dimethylaminopyridine (100 mg, 0.8 mmol), and the resulting
mixture was stirred at room temperature for 24 h. The mixture was
concentrated under reduced pressure, and the crude product was
purified by recrystallization (hexane–AcOEt) to afford 3a (730 mg,
95%) as a white solid. 1H NMR (CDCl3) d 1.60 (s, 3H), 1.98 (s, 3H),
1.99 (s, 3H), 2.01 (s, 3H), 2.20 (s, 3H), 3.60 (dd, J = 10.1, 2.9 Hz,
1H), 4.39 (d, J = 12.1 Hz, 1H), 4.66 (d, J = 12.1 Hz, 1H), 4.94
(dd, J = 10.6, 2.7 Hz, 1H), 5.08 (t, J = 9.9 Hz, 1H), 5.39–5.52 (m,
2H), 5.76 (s, 1H), 7.22–7.34 (m, 5H). 13C NMR (CDCl3) d 20.5,
20.7, 20.8, 66.6, 69.1, 69.5, 70.9, 71.1, 71.8, 74.4, 127.8, 128.1,
128.5, 136.9, 169.6, 169.7, 169.8, 169.9, 170.0. IR (KBr) 2900,
1740, 1360, 1210, 1130 cm−1. MS (EI) m/z 480 (M + H)+. Anal.
calcd for C23H28O11: C, 57.50; H, 5.87. Found: C, 57.46; H, 5.94.
D-3,6-Di-O-allyl-myo-inositol (7a)
To a solution of 6a (2.65 g, 6.30 mmol) in a mixture of THF–
H2O (5 : 1, 60 ml) was added p-toluenesulfonic acid monohydrate
(300 mg, 1.58 mmol), and the resulting mixture was refluxed for
3 h. The mixture was neutralized with Et3N, and concentrated
under reduced pressure. The crude product was purified by column
chromatography (CH2Cl2 : MeOH = 7 : 1) to afford 7a (1.80 g,
>99%) as a white solid. 1H NMR (CD3OD) d 3.02–3.42 (m, 4H),
3.61 (t, J = 9.7 Hz, 1H), 3.98–4.13 (m, 3H), 4.22–4.24 (m, 2H),
5.02–5.25 (m, 4H), 5.82–5.99 (m, 2H). 13C NMR (CDCl3) d 71.0,
72.1, 73.1, 73.6, 75.1, 76.3, 80.7, 82.5, 116.7, 117.4, 136.6, 137.2.
IR (KBr) 3420, 2900, 1420, 1350, 1100, 1070, 930 cm−1. MS (FAB)
m/z 261 (M + H)+. Anal. calcd for C12H20O6: C, 55.37; H, 7.74.
Found: C, 55.21; H, 7.80.
D-2,3,4,5,6-Penta-O-acetyl-myo-inositol (4a)
D-3,6-Di-O-allyl-1-O-(p-methoxybenzyl)-myo-inositol (8a)
To a solution of 3a (699 mg, 1.45 mmol) in acetic acid (30 ml) was
added 10% palladium-carbon (50 mg), and the resulting mixture
was stirred at room temperature under hydrogen for 10 h. The
mixture was filtered through a pad of celite and concentrated
under reduced pressure. The crude product was purified by column
chromatography (CH2Cl2–MeOH = 14 : 1) to afford 4a (525 mg,
A mixture of 7a (624 mg, 2.40 mmol) and dibutyltin oxide
(750 mg, 3.00 mmol) in toluene (50 ml) was refluxed for 3 h
in a Dean–Stark apparatus to remove water. The mixture was
concentrated under reduced pressure. To the residue was added
caesium fluoride (760 mg, 5.00 mmol), and the mixture was
suspended in heated DMF (30 ml). To the resulting suspension was
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added p-methoxybenzyl chloride (0.47 ml, 3.00 mmol) at −78 ◦C,
and the mixture was stirred at room temperature under argon
for 24 h. The reaction mixture was diluted with CHCl3, filtered
through a pad of celite, and concentrated under reduced pressure.
The residue was purified by column chromatography (CH2Cl2 :
MeOH = 12 : 1) to afford 8a (800 mg, 88%) as a colorless oil. 1H
NMR (CDCl3) d 2.53 (bs, 1H), 2.88–2.95 (m, 2H), 3.14 (dd, J =
9.4, 2.8 Hz, 1H), 3.31–3.40 (m, 2H), 3.63–3.93 (m, 5H), 4.07–4.44
(m, 5H), 4.63 (s, 2H), 5.16–5.33 (m, 4H), 5.87–6.04 (m, 2H), 6.88
(d, J = 8.6 Hz, 2H), 7.28 (d, J = 8.6 Hz, 2H). 13C NMR (CDCl3)
d 55.3, 66.9, 71.2, 72.2, 74.0, 78.8, 79.4, 80.0, 113.9, 116.9, 117.9,
129.5, 129.9, 134.4, 135.1, 159.4. IR (KBr) 3450, 2900, 1510, 1250,
1100, 1040 cm−1. MS (FAB) m/z 381 (M + H)+. Anal. calcd for
C20H28O7: C, 63.14; H, 7.42. Found: C, 63.00; H, 7.44.
The residue was purified by column chromatography (CH2Cl2 :
MeOH = 12 : 1) to afford 10a (200 mg, 45%) as a white solid. 1H
NMR (CD3OD) d 1.31 (s, 3H), 1.35 (s, 3H), 3.16–3.21 (m, 2H),
3.54–3.86 (m, 6H), 3.98 (s, 1H), 4.48 (d, J = 11.4 Hz, 1H), 4.55 (d,
J = 11.4 Hz, 1H), 4.78 (bs, 3H), 6.77 (d, J = 8.6 Hz, 2H), 7.25 (d,
J = 8.44 Hz, 2H). IR (KBr) 3350, 2900, 1520, 1250, 1070 cm−1. MS
(FAB) m/z 340 (M + H)+. Anal. calcd for C17H23O7 − 1/2H2O: C,
58.61; H, 6.94. Found: C, 58.63; H, 7.06.
D-2,3,6-Tri-O-acetyl-1-O-(p-methoxybenzyl)-4,5-O-
isopropylidene-myo-inositol (11a)
To a solution of 10a (390 mg, 1.15 mmol) in pyridine (10 ml) was
added 4-dimethylaminopyridine (25 mg, 0.21 mmol) followed by
acetic anhydride (0.47 ml, 5.00 mmol), and the resulting mixture
was stirred at room temperature for 12 h. The mixture was diluted
with toluene, the resulting azeotropic mixture was concentrated
under reduced pressure, and then the residue was diluted with
AcOEt. The organic phase was washed with H2O and saturated
aqueous NaCl, dried over Na2SO4, and then concentrated under
reduced pressure. The crude product was purified by column
chromatography (hexane–AcOEt = 1 : 1) to afford 11a (427 mg,
>99%) as a white solid. 1H NMR (CDCl3) d 1.35 (s, 3H), 1.41 (s,
3H), 2.00 (s, 6H), 2.10 (s, 3H), 3.39–3.49 (m, 2H), 3.73 (s, 3H), 4.00
(t, J = 10.1 Hz, 1H), 4.24 (d, J = 11.7 Hz, 1H), 4.51 (d, J = 11.7 Hz,
1H), 4.93 (dd, J = 10.8, 2.7 Hz, 1H), 5.35 (t, J = 9.9 Hz, 1H), 5.74
(s, 1H), 6.80 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.2 Hz, 2H). 13C
NMR (CDCl3) d 20.4, 20.5, 20.6, 20.8, 26.5, 26.6, 55.1, 67.7, 70.0,
70.7, 71.6, 74.6, 76.5, 77.2, 112.5, 113.7, 128.9, 129.4, 159.3, 169.5,
170.0. IR (KBr) 3000, 1750, 1510, 1370, 1230, 1060 cm−1. MS
(FAB) m/z 467 (M + H)+. Anal. calcd for C23H30O10 − 1/5H2O:
C, 58.77; H, 6.52. Found: C, 58.88; H, 6.60.
D-3,6-Di-O-allyl-1-O-(p-methoxybenzyl)-4,5-O-isopropylidene-
myo-inositol (9a)
To a solution of 8a (1.43 g, 3.76 mmol) in DMF (30 ml) was added
2-methoxypropene (0.52 ml, 10.0 mmol) followed by dehydrated
p-toluenesulfonic acid (95 mg, 0.50 mmol). The resulting mixture
was stirred at room temperature under argon for 24 h. The mixture
was neutralized with Et3N and concentrated under reduced
pressure, and the residue was diluted with AcOEt. The organic
phase was washed with H2O and saturated aqueous NaCl, dried
over Na2SO4, and then concentrated under reduced pressure. The
crude product was purified by column chromatography (hexane–
1
AcOEt = 3 : 2) to afford 9a (1.23 g, 78%) as a colorless oil. H
NMR (CDCl3) d 1.38 (s, 3H), 1.39 (s, 3H), 2.73 (bs, 1H), 3.25–3.46
(m, 3H), 3.75–3.46 (m, 10H), 4.60 (d, J = 11.4 Hz, 1H), 4.67 (d,
J = 11.4 Hz, 1H), 5.13–5.31 (m, 4H), 5.83–5.99 (m, 2H), 6.83 (d,
J = 8.6 Hz, 2H), 7.25 (d, J = 8.6 Hz, 2H). 13C NMR (CDCl3) d
26.6, 26.7, 55.0, 69.3, 70.4, 72.1, 72.7, 75.9, 76.6, 77.8, 78.8, 80.4,
111.3, 113.6, 116.2, 117.2, 129.3, 134.4, 134.9, 159.1. IR (KBr)
3500, 3080, 3000, 2900, 1610, 1510, 1240, 1080 cm−1. MS (FAB)
m/z 421 (M + H)+. Anal. calcd for C23H32O7: C, 65.70; H, 7.67.
Found: C, 65.67; H, 7.76.
D-2,3,6-Tri-O-acetyl-1-O-(p-methoxybenzyl)-myo-inositol (12a)
To a solution of 11a (360 mg, 0.77 mmol) in CH2Cl2 (10 ml)
was added ethylene glycol (89 ll, 1.5 mmol) followed by p-
toluenesulfonic acid monohydrate (10 mg, 0.051 mmol). The
resulting mixture was stirred at room temperature for 10 min.
The mixture was neutralized with Et3N (0.1 ml, 0.72 mmol),
and concentrated under reduced pressure. The crude product was
purified by column chromatography (CH2Cl2 : MeOH = 12 : 1)
to afford 12a (168 mg, 77%) as a white solid. 1H NMR (CDCl3) d
2.03 (s, 3H), 2.04 (s, 3H), 2.12 (s, 3H), 3.39–3.56 (m, 2H), 3.78 (s,
3H), 3.88 (s, 1H), 4.05 (bs, 2H), 4.29 (t, J = 11.7 Hz, 1H), 4.55 (d,
J = 11.7 Hz, 1H), 4.79 (d, J = 13.0 Hz, 1H), 5.16 (t, J = 9.9 Hz,
1H), 5.69 (s, 1H), 6.85 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz,
2H). 13C NMR (CDCl3) d 20.6, 20.8, 55.0, 67.2, 70.8, 70.9, 71.0,
72.9, 73.0, 74.0, 113.6, 129.1, 129.2, 159.2, 170.1, 170.5, 170.9. IR
(KBr) 3430, 2900, 1740, 1370, 1230, 1030 cm−1. MS (FAB) m/z
427 (M + H)+. Anal. calcd for C20H26O10 − 1/2H2O: C, 55.17; H,
6.25. Found: C, 55.22; H, 6.01.
D-1-O-(p-Methoxybenzyl)-4,5-O-isopropylidene-myo-inositol
(10a)
To a solution of 9a (552 mg, 1.31 mmol) in a mixture of EtOH–
benzene–H2O (7 : 3 : 1, 22 ml) was added diazabicyclo[2.2.2]octane
(147 mg, 1.31 mmol) followed by triphenylphosphine rhodium(I)
chloride (121 mg, 0.13 mmol), and the resulting mixture was
refluxed for 5 h. The mixture was concentrated under reduced
pressure, and the residue was diluted with AcOEt. The organic
phase was washed with H2O and saturated aqueous NaCl, dried
over Na2SO4, and then concentrated under reduced pressure. To
a solution of the residue in a mixture of acetone–H2O (10 : 1,
5 ml) was added mercury(II) oxide (284 mg, 1.31 mmol). To the
resulting mixture was added dropwise a solution of mercury(II)
chloride (355 mg, 1.31 mmol) in a mixture of acetone–H2O (10 : 1,
5 ml). The resulting mixture was stirred at room temperature for
5 min. The mixture was neutralized with aqueous NaOH, filtered
through a pad of celite, and concentrated under reduced pressure,
and the residue was diluted with saturated aqueous NaCl. The
aqueous phase was extracted with CH2Cl2, and the organic phase
was dried over Na2SO4, and concentrated under reduced pressure.
D-2,3,6-Tri-O-acetyl-1-O-(p-methoxybenzyl)-4,5-di-O-[bis(b-
cyanoethyl)phosphoryl]-myo-inositol (13a)
To a solution of 12a (250 mg, 0.60 mmol) in CH2Cl2 (10 ml)
was added bis(b-cyanoethyl)-N,N-diisopropylphosphoramidite
(0.77 ml, 3.00 mmol) followed by 1H-tetrazole (210 mg,
3.00 mmol). The resulting mixture was stirred at room temperature
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for 1.5 h. To the mixture was added m-chloroperbenzoic acid
(863 mg, 5.0 mmol) in small portions, and the resulting mixture
was stirred for 5 min. The mixture was diluted with CH2Cl2, and
washed with saturated aqueous NaHCO3. The organic phase
was dried over Na2SO4, and then concentrated under reduced
pressure. The residue was purified by column chromatography
(CH2Cl2 : MeOH = 13 : 1) to afford 13a (296 mg, 68%) as a
D-1,3,6-Tri-O-allyl-(p-methoxybenzyl)-4,5-O-isopropylidene-myo-
inositol (17a)
To a solution of 16a (760 mg, 2.23 mmol) in DMF (30 ml) was
added NaH (240 mg, 3.00 mmol) followed by p-methoxybenzyl
chloride (0.44 ml, 3.00 mmol), and the resulting mixture was
stirred at room temperature for 24 h. The reaction was quenched
with MeOH, and concentrated under reduced pressure, and the
residue was diluted with AcOEt. The organic phase was washed
with H2O and saturated aqueous NaCl, dried over Na2SO4, and
then concentrated under reduced pressure. The crude product was
purified by the column chromatography (hexane–AcOEt = 3 : 1)
to afford 17a (748 mg, 73%) as a white solid. 1H NMR (CDCl3) d
1.42 (s, 3H), 1.43 (s, 3H), 3.23–3.33 (m, 2H), 3.46 (dd, J = 10.4,
2.8 Hz, 1H), 3.80 (s, 3H), 3.85–3.91 (m, 1H), 4.01–4.46 (m, 8H),
4.77 (s, 2H), 5.12–5.33 (m, 6H), 5.84–5.99 (m, 3H), 6.86 (d, J =
8.6 Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H). 13C NMR (CDCl3) d 26.9,
27.0, 55.2, 70.6, 72.3, 72.4, 74.7, 76.3, 77.1, 77.4, 78.0, 79.4, 82.0,
111.5, 113.6, 116.3, 116.6, 116.7, 129.5, 131.0, 134.8, 135.0, 135.3,
159.1. IR (film) 3030, 3000, 2900, 1610, 1460, 1370, 1220, 1100,
1030 cm−1. MS (EI) m/z 461 (M + H)+. Anal. calcd for C26H36O7 −
1/2H2O: C, 66.50; H, 7.94. Found: C, 66.80; H, 7.83.
1
colorless oil. H NMR (CDCl3) d 2.10 (s, 3H), 2.12 (s, 3H), 2.19
(s, 3H), 2.78–2.85 (m, 8H), 3.60 (dd, J = 10.2, 2.4 Hz, 1H), 3.79
(s, 3H), 4.25–4.47 (m, 9H), 4.50–4.58 (m, 2H), 4.80 (dd, J = 18.7,
9.3 Hz, 1H), 5.06 (dd, J = 10.1, 2.6 Hz, 1H), 5.38 (t, J = 4.9 Hz,
1H), 5.68 (s, 1H), 6.80 (d, J = 8.2 Hz, 2H), 7.15 (d, J = 8.2 Hz,
2H). 13C NMR (CDCl3) d 19.2, 19.3, 19.4, 19.4, 20.6, 20.9, 55.1,
62.6, 62.7, 62.8, 62.9, 66.3, 68.5, 70.4, 71.4, 73.2, 76.5, 77.3, 113.7,
116.6, 116.7, 116.9, 117.0, 128.6, 129.3, 159.3, 169.7, 169.8, 170.0.
IR (film) 2970, 2250, 1740, 1510, 1510, 1410, 1370, 1240, 1030,
940 cm−1. MS (FAB) m/z 799 (M + H)+.
D-2,3,6-Tri-O-acetyl-4,5-di-O-[bis(b-cyanoethyl)phosphoryl]-myo-
inositol (14a)
To a solution of 13a (296 mg, 0.37 mmol) in a mixture of CH3CN–
H2O (9 : 1, 10 ml) was added diammonium cerium(IV) nitrate, and
the resulting mixture was stirred at room temperature for 1.5 h. The
mixture was concentrated under reduced pressure, and the residue
was purified by column chromatography (CH2Cl2 : MeOH = 10 :
1) to afford 14a (68 mg, 27%) as a colorless oil. 1H NMR (CDCl3)
d 2.08 (s, 3H), 2.18 (s, 3H), 2.19 (s, 3H), 2.90–2.97 (m, 8H), 3.97
(dd, J = 10.1, 2.9 Hz, 1H), 4.30–4.41 (m, 8H), 4.68–4.86 (m, 2H),
5.21 (dd, J = 9.9, 2.9 Hz, 1H), 5.35 (t, J = 9.9 Hz, 1H), 5.50 (dd,
J = 5.3, 2.9 Hz, 1H). 13C NMR (CDCl3) d 20.1, 20.2, 20.2, 20.3,
20.8, 21.0, 21.5, 64.7, 64.8, 64.9, 68.1, 70.7, 71.7, 73.3, 78.4, 78.9,
118.6, 118.7, 118.8, 118.9, 171.5, 172.0, 172.2. IR (KBr) 3420,
2960, 1740, 1370, 1220, 1030 cm−1.
D-2-O-(p-Methoxybenzyl)-4,5-O-isopropylidene-myo-inositol
(18a)
17a (290 mg, 0.63 mmol) was allowed to react under the same
conditions as described for the preparation of 10a to give 18a
1
(106 mg, 49%) as a colorless oil. H NMR (CD3OD) d 1.41 (s,
6H), 3.26–3.30 (m, 2H), 3.65–3.67 (m, 1H), 3.78 (s, 3H), 3.83–3.96
(m, 2H), 4.09 (s, 1H), 4.58 (d, J = 11.4 Hz, 1H), 4.66 (d, J =
11.4 Hz, 1H), 6.88 (d, J = 8.6 Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H).
13C NMR (CD3OD) d 27.1, 27.2, 55.7, 71.2, 71.8, 72.1, 73.3, 78.8,
80.2, 83.0, 112.4, 114.7, 130.9, 131.7, 160.9. IR (KBr) 3350, 2900,
1510, 1250, 1070 cm−1. Anal. calcd for C17H24O7 − 1/3H2O: C,
58.95; H, 7.18. Found: C, 58.79; H, 7.16.
D-1,3,6-Tri-O-allyl-myo-inositol (15a)
7a (521 mg, 2.00 mmol) was allowed to react under the same
conditions as described for the preparation of 8a to give 15a
(356 mg, 59%) as a white solid. 1H NMR (CDCl3) d 2.53 (s, 1H),
2.88–2.95 (m, 2H), 3.18 (dd, J = 4.8, 2.8 Hz, 1H), 3.27 (dd, J =
9.3, 2.8 Hz, 1H), 3.37 (t, J = 9.3 Hz, 1H), 3.65 (t, J = 9.3 Hz,
1H), 3.91 (t, J = 9.3 Hz, 1H), 4.11–4.42 (m, 7H), 5.15–5.34 (m,
6H), 5.87–6.02 (m, 3H). IR (KBr) 3450, 2900, 1110, 1030, 990,
910 cm−1. Anal. calcd for C15H24O6: C, 59.98; H, 8.05. Found: C,
59.96; H, 8.09.
D-1,3,6-Tri-O-acetyl-2-O-(p-methoxybenzyl)-4,5-O-
isopropylidene-myo-inositol (19a)
18a (103 mg, 0.30 mmol) was allowed to react under the same
conditions as described for the preparation of 12a to give 19a
(140 mg, >99%) as a white solid. 1H NMR (CDCl3) d 1.35 (s, 3H),
1.41 (s, 3H), 2.00 (s, 6H), 2.10 (s, 3H), 3.39–3.49 (m, 2H), 3.73 (s,
3H), 4.00 (t, J = 10.1 Hz, 1H), 4.24 (d, J = 11.7 Hz, 1H), 4.51 (d,
J = 11.7 Hz, 1H), 4.93 (dd, J = 10.8, 2.7 Hz, 1H), 5.35 (t, J =
9.9 Hz, 1H), 5.74 (s, 1H), 6.80 (d, J = 8.2 Hz, 2H), 7.11 (d, J =
8.2 Hz, 2H). 13C NMR (CDCl3) d 20.3, 20.5, 20.6, 20.8, 26.5, 26.6,
55.1, 67.7, 70.0, 70.7, 71.6, 74.6, 76.5, 77.2, 112.5, 113.7, 128.9,
129.4, 159.3, 169.5, 170.0. IR (KBr) 3000, 1750, 1515, 1370, 1220,
1100, 1060, 1030 cm−1. Anal. calcd for C23H30O10: C, 59.22; H,
6.48. Found: C, 59.04; H, 6.57.
D-1,3,6-Tri-O-allyl-4,5-O-isopropylidene-myo-inositol (16a)
15a (330 mg, 1.10 mmol) was allowed to react under the same
conditions as described for the preparation of 9a to give 16a
1
(340 mg, 91%) as a colorless oil. H NMR (CDCl3) d 1.42 (s,
3H), 1.43 (s, 3H), 2.62 (s, 1H), 3.29–3.36 (m, 2H), 3.53 (dd, J =
9.9, 3.2 Hz, 1H), 3.82 (dd, J = 9.9, 8.6 Hz, 1H), 4.00 (t, J = 9.5 Hz,
D-1,3,6-Tri-O-acetyl-(p-methoxybenzyl)-myo-inositol (20a)
1H), 4.13–4.38 (m, 7H), 5.14–5.34 (m, 6H), 5.87–6.02 (m, 3H). 13
C
NMR (CDCl3) d 27.3, 27.4, 69.9, 71.1, 72.7, 72.8, 76.5, 77.2, 78.3,
79.3, 81.5, 112.0, 116.9, 117.9, 118.0, 134.9, 135.0, 135.5. IR (KBr)
3475, 3080, 3000, 2900, 1640, 1460, 1420, 1370, 1230, 1080, 1000,
920 cm−1. MS (FAB) m/z 341 (M + H)+. Anal. calcd for C18H28O6:
C, 63.51; H, 8.29. Found: C, 63.27; H, 8.47.
19a (115 mg, 0.27 mmol) was allowed to react under the same
conditions as described for the preparation of 13a to give 20a
(140 mg, 94%) as a white solid. 1H NMR (CDCl3) d 1.98 (s,
3H), 2.04 (s, 3H), 2.05 (s, 3H), 3.49–3.56 (m, 1H), 3.67–3.75 (m,
2H), 3.79 (s, 3H), 4.00–4.06 (m, 2H), 4.53–4.63 (m, 2H), 4.78 (dd,
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J = 10.3, 2.2 Hz, 1H), 4.92 (dd, J = 10.4, 2.6 Hz, 1H), 5.41 (t,
J = 9.7 Hz, 1H), 6.86 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.4 Hz,
2H). 13C NMR (CDCl3) d 20.6, 20.7, 20.8, 55.1, 71.2, 71.3, 72.0,
72.9, 73.1, 74.6, 113.6, 129.5, 129.8, 159.2, 169.9, 170.6, 170.8. IR
(KBr) 3450, 2900, 1740, 1510, 1360, 1230, 1030 cm−1. Anal. calcd
for C20H26O10 − 1/2H2O: C, 55.17; H, 6.25. Found: C, 55.47; H,
6.15.
hydrogen for 10 h. The mixture was filtered through a pad of celite
and concentrated under reduced pressure. The crude product was
purified by column chromatography (CH2Cl2–MeOH = 14 : 1) to
afford 24a (61 mg, 74%) as a white solid. 1H NMR (CDCl3) d 2.01
(s, 9H), 2.10 (s, 6H), 3.12 (bs, 1H), 4.33 (s, 1H), 5.03 (dd, J = 10.3,
2.4 Hz, 2H), 5.20 (t, J = 9.7 Hz, 1H), 5.60 (t, J = 10.1 Hz, 2H).
13C NMR (CDCl3) d 20.4, 20.5, 20.6, 68.2, 69.5, 70.7, 70.8, 169.7,
169.8. IR (KBr) 3420, 2950, 1740, 1370, 1230, 1040 cm−1. MS
(FAB) m/z 391 (M + H)+. Anal. calcd for C16H22O11 − 1/3H2O:
C, 48.49; H, 5.76. Found: C, 48.53; H, 5.68.
D-1,3,6-Tri-O-acetyl-2-O-(p-methoxybenzyl)-4,5-di-O-[bis(b-
cyanoethyl)phosphoryl]-myo-inositol (21a)
20a (135 mg, 0.32 mmol) was allowed to react under the same
conditions as described for the preparation of 14a to give 21a
(256 mg, >99%) as a white solid. 1H NMR (CDCl3) d 2.11 (s, 3H),
2.13 (s, 3H), 2.20 (s, 3H), 2.71–2.85 (m, 8H), 3.42–3.62 (m, 2H),
3.80 (s, 3H), 4.10–4.59 (m, 10H), 4.80 (dd, J = 18.7, 9.3 Hz, 1H),
5.05 (dd, J = 10.1, 2.6 Hz, 1H), 5.38 (t, J = 9.9 Hz, 1H), 5.68 (s,
1H), 6.86 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H). IR (film)
2960, 2250, 1740, 1610, 1510, 1410, 1360 cm−1. MS (FAB) m/z 799
(M + H)+.
D-2,3,6-Tri-O-acetyl-4,5-di-O-[bis(b-cyanoethyl)phosphoryl]-myo-
inositol 1-{[(4,4ꢀ-dimethoxytrityl)biotinyl-6-aminohexyl]
(b-cyanoethyl)phosphate} (25a)
To a solution of 1-N-(4,4-dimethoxytrityl)biotinyl-6-aminohexan-
1-ol (97 mg, 0.15 mmol) in CH2Cl2 (5 ml) was added
(b-cyanoethyl)-N,N,Nꢀ,Nꢀ-tetraisopropylphosphoramidite (48 ll,
0.15 mmol) followed by 1H-tetrazole (11 mg, 0.15 mmol), and the
resulting mixture was stirred at room temperature under argon for
1.5 h. To the mixture was added 14a (84 mg, 0.12 mmol) followed
by 1H-tetrazole (11 mg, 0.15 mmol), and the resulting mixture
was stirred at room temperature for further 2 h. To the mixture
was added tert-butylhydroperoxide (0.10 ml, 0.78 mmol), and the
resulting mixture was stirred at room temperature for a further
5 min, and concentrated under reduced pressure to half volume.
The residue was purified by column chromatography (CH2Cl2 :
MeOH = 13 : 1, 0.5%Et3N) to afford 25a (45 mg, 38%) as a
colourless oil. 1H NMR (CDCl3) d 1.36–1.67 (m, 14H), 2.03–2.29
(m, 11H), 2.43–2.45 (m, 1H), 2.70–2.80 (m, 10H), 3.08–3.20 (m,
3H), 3.79 (s, 6H), 4.02–4.29 (m, 15H), 4.67–4.81 (m, 3H), 5.22–
5.26 (m, 1H), 5.49 (t, J = 9.7 Hz, 1H), 5.63–5.73 (m, 2H), 5.99
(bs, 1H), 6.80 (d, J = 8.4 Hz, 4H), 7.11–7.29 (m, 9H). IR (KBr)
3400, 2950, 1750, 1700, 1650, 1510, 1220, 1130 cm−1. MS (FAB)
m/z 1440 (M + H)+.
D-1,3,6-Tri-O-acetyl-4,5-di-O-[bis(b-cyanoethyl)phosphoryl]-myo-
inositol (22a)
21a (240 mg, 0.30 mmol) was allowed to react under the same
conditions as described for the preparation of 14a to give 22a
1
(120 mg, 59%) as a colorless oil. H NMR (CD3OD) d 2.02 (s,
3H), 2.09 (s, 3H), 2.15 (s, 3H), 2.88–2.94 (m, 8H), 4.18–4.40 (m,
9H), 4.81–4.93 (m, 2H), 5.01 (dd, J = 10.4, 2.6 Hz, 1H), 5.14
(dd, J = 9.7, 2.6 Hz, 1H), 5.56 (t, J = 10.1 Hz, 1H). 13C NMR
(CD3OD) d 20.1, 20.2, 20.3, 20.7, 20.9, 21.2, 21.3, 64.7, 64.8, 64.9,
68.4, 70.8, 71.9, 72.1, 78.4, 78.9, 118.6, 118.8, 118.9, 119.0, 171.6,
171.8, 171.9. IR (KBr) 3400, 2950, 1740, 1360, 1220, 1030 cm−1.
1,3,4,5,6-Penta-O-acetyl-2-O-(p-methoxybenzyl)-myo-inositol
(23a)
To a solution of 20a (115 mg, 0.27 mmol) in pyridine (15 ml) was
added 4-dimethylaminopyridine (30 mg, 0.25 mmol) followed by
acetic anhydride (0.15 ml, 1.60 mmol), and the resulting mixture
was stirred at room temperature for 12 h. The mixture was
diluted with toluene, and the resulting azeotropic mixture was
concentrated, and then the residue was diluted with AcOEt. The
organic phase was washed with H2O and saturated aqueous NaCl,
dried over Na2SO4, and then concentrated under reduced pressure.
The crude product was purified by column chromatography
(hexane–AcOEt = 1 : 1) to afford 23a (118 mg, 86%) as a white
solid. 1H NMR (CDCl3) d 1.99 (s, 15H), 3.80 (s, 3H), 4.12 (s, 1H),
4.62 (s, 2H), 4.97 (dd, J = 10.3, 1.8 Hz, 2H), 5.14 (t, J = 9.5 Hz,
1H), 5.61 (t, J = 9.9 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 7.25 (d, J =
7.5 Hz, 2H). 13C NMR (CDCl3) d 20.3, 20.4, 20.4, 55.1, 70.0, 70.9,
71.1, 74.1, 74.6, 113.7, 129.4, 129.6, 159.3, 169.5, 170.0. IR (KBr)
2950, 1740, 1360, 1230, 1040 cm−1. Anal. calcd for C24H30O12: C,
56.47; H, 5.92. Found: C, 56.39; H, 5.93.
D-1-O-[(Biotinyl-6-aminohexyl) hydrogen phosphoryl]-myo-
inositol 4,5-bis(hydrogenphosphate):biotinylated D-myo-inositol
1,4,5-triphosphate
To a solution of 25a (45 mg, 0.031 mmol) in CH2Cl2 (10 ml)
was added trichloroacetic acid (30 mg), and the resulting mixture
was stirred at room temperature for 30 min. The mixture was
concentrated under reduced pressure, and the residue was purified
by column chromatography (CH2Cl2 : MeOH = 7 : 1) to afford
dimethoxytrityl-free crude compound. To a solution of crude
compound in MeOH (5 ml) was added 28% NH4OH (5 ml), and
the resulting mixture was stirred at 55 ◦C for 10 h. The mixture was
concentrated under reduced pressure, and the residue was purified
by anion-exchange chromatography to afford the ammomium salt
of biotinylated D-myo-inositol 1,4,5-triphosphate (23 mg, 32%)
as a white solid. 1H NMR (D2O) d 1.22–1.58 (m, 14H), 2.09
(t, J = 7.1 Hz, 2H), 2.63 (d, J = 13.0 Hz, 1H), 2.85 (dd, J =
13.0, 4.9 Hz, 1H), 3.02 (d, J = 6.6 Hz, 2H), 3.15–3.21 (m, 1H),
3.58 (d, J = 8.1 Hz, 1H), 3.72–3.96 (m, 5H), 4.11–4.29 (m, 3H),
4.33–4.48 (m, 1H). 13C NMR (D2O) d 27.3, 27.9, 28.4, 30.3, 30.5,
30.9, 32.4, 38.2, 41.9, 42.3, 58.0, 62.9, 64.7, 69.1, 72.8, 73.4, 78.2,
79.7, 81.1, 168.0, 179.3. IR (KBr) 3400, 3200, 2900, 1700, 1400,
1030 cm−1. MS (FAB) m/z 746 (M + H)+. FABHRMS calcd m/z
1,3,4,5,6-Penta-O-acetyl-myo-inositol (24a)
To a solution of 23a (110 mg, 0.21 mmol) in acetic acid (10 ml)
at room temperature was added 10% palladium-carbon (50 mg),
and the resulting mixture was stirred at room temperature under
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for C22H41N3O17P3S 744.1370. Found: 744.1358 (M − H+). Anal.
calcd for C22H42N6 O17P3S − 5/2NH3 − 5/2H2O: C, 31.71; H,
6.59; N, 9.25. Found: C, 31.51; H, 6.64; N, 9.29. [a]2D5 = −3.8 (c
0.1, H2O).
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Acknowledgements
The present study was financially supported in part by a Grant-in-
Aid for Scientific Research (NO.17390030 to MO), the Kumamoto
Technology and Industry Foundation (to YO), and the Shorai
Foundation for Science and Technology (to MO).
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1830 | Org. Biomol. Chem., 2008, 6, 1822–1830
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