Chemical Resolution of 1,2-O-Cyclohexylidene-3,4-O-(tetraisopropyldisiloxane-1,3-diyl)-myo-inositol and Synthesis of Phosphatidyl-D-myo-inositol 3,5-Bisphosphate from Both L- and D-Enantiomers

Article abstract of DOI:10.1002/ejoc.200300517

Chemical resolution of a versatile starting material, 1,2-O-cyclohexylidene-3,4-O-(tetraisopropyldisiloxane-1,3-diyl)-myo-inositol, which is used to access naturally occurring inositol phosphates and phosphatidylinositol phosphates, is described. Starting

Full text of DOI:10.1002/ejoc.200300517

FULL PAPER
Chemical Resolution of 1,2-O-Cyclohexylidene-3,4-O-(tetraisopropyldisiloxane-
1,3-diyl)-myo-inositol and Synthesis of Phosphatidyl-
D
-myo-inositol
3,5-Bisphosphate from Both
L
- and
D
-Enantiomers
Fushe Han,[a] Minoru Hayashi,[b] and Yutaka Watanabe*[b]
Keywords: Enantiomers / Resolution / myo-Inositol derivatives / Synthesis design / Biological activity
Chemical resolution of a versatile starting material, 1,2-O-
CH2Cl2 mixture did not proceed at all, whereas in an optim-
cyclohexylidene-3,4-O-(tetraisopropyldisiloxane-1,3-diyl)- ized solvent system, pyridine/CH2Cl2 (1.1:1, v/v), the reac-
myo-inositol, which is used to access naturally occurring
inositol phosphates and phosphatidylinositol phosphates, is
tion afforded 68% of the desired 1-O-phosphate as a single
product. Further investigation by 1H NMR spectroscopy in-
dicated that the reactivity of the three OHs on 1,2,4-triol de-
rivatives is governed by intermolecular hydrogen bonding,
which may be disrupted by an increase in the proportion of
pyridine in the reaction solvent.
described. Starting from both
D- and
L-enantiomers of the
material, the synthesis of phosphatidyl-
D-myo-inositol 3,5-
bisphosphate [PtdIns(3,5)P2] has been conveniently accomp-
lished via convergent routes. One of the key reactions in the
synthetic procedure was the regioselective phosphorylation
of suitably protected 1,2,4-triol derivatives of inositol. Phos- ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
phorylation of the triol attempted in a 1:12 (v/v) pyridine/
Germany, 2004)
1. Introduction
The biological functions of phosphatidylinositol phos-
phates PtdInsPns, such as PtdIns(3)P, PtdIns(5)P,
PtdIns(3,4)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, in intra-
cellular-signal-transduction, exocytosis and the regulation
of membrane-trafficking have been well recognized, and
have attracted considerable interest in cell biology.[1] Re-
cently, a previously unknown phosphatidylinositol 3,5-bis-
phosphate PtdIns(3,5)P2 (1) was found to occur in mam-
malian cell-lines.[2] It has been suggested that this molecule
could be a new potential member of the PI cascade.[3] Al-
though the biological role of PtdIns(3,5)P2 has not yet been
well recognized, several investigations have shown that
PtdIns(3,5)P2 is biologically important; for instance, in
sorting membrane proteins into the lumen of the yeast
vacuole and maintaining the vacuolar size.[4]
from natural sources. To date, six reports on the chemical
synthesis of PtdIns(3,5)P2 analogues have appeared, in
which glucose,[5] myo-inositol orthoacetate,[6] orthofor-
mate,[7] and camphor ketal were used.[8] The inositol ortho-
ester methods have advantages, both in terms of number of
steps and of yield. We recently communicated a convenient
method for synthesizing dipalmitoyl PtdIns(3,5)P2 1b from
1,2-O-cyclohexylidene-3,4-O-(tetraisopropyldisiloxane-1,3-
diyl)-myo-inositol (2)[9] (Scheme 1). The highlights of our
method include that it involves a convergent synthetic plan,
which enables both -and -2 to access the same target
product with natural -configuration; this being in contrast
to previous methods. This drawback of the previous meth-
ods is apparent not only in the synthesis of PtdIns(3,5)P2
(1), but in the whole field of synthetic inositol chemistry
where myo-inositol is used as the starting material. In ad-
dition, the convergent method developed in our laboratory
is general insofar as it can be applied to the synthesis of
other PtdInsPns and/or InsPn molecules. In particular, by
Due to its biological potential, PtdIns(3,5)P2 (1) is cur-
rently of keen interest to biochemists. This makes the
chemical synthesis of natural 1a and its analogues an im-
portant and attractive goal, as biological screening requires
much larger quantities of material than can be obtained
[a]
Venture Business Laboratory, Ehime University,
Matsuyama 790-8577, Japan
[b]
Department of Applied Chemistry, Faculty of Engineering,
Ehime University,
Matsuyama 790-8577, Japan
Fax: (internat.) ϩ 81-89-927-9944
E-mail: wyutaka@dpc.ehime-u.ac.jp
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DOI: 10.1002/ejoc.200300517
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Chemical Resolution of L- and D-myo-Inositol Derivatives
FULL PAPER
quired. Although - and -1,2-O-cyclohexylidene-myo-in-
ositol, the precursor of optically active 2, can be obtained
rapidly using enzyme-aided resolution,[11] these compounds
need to be converted into the corresponding optically active
2 separately. Moreover, the efficiency of this resolution was
found to vary depending on the batch of enzyme. Owing to
these shortcomings, we sought to develop an efficient and
reliable method for obtaining both of the optically pure en-
antiomers of 2. Earlier investigations in this laboratory
showed that racemic 2 could be easily transformed into the
fully protected 3 as column-separable diastereoisomers.[12]
Therefore, selective desilylation of the triethylsilyl (TES)
group on - and -3 was attempted under various con-
ditions (Table 1). Neither the tetraisopropyldisiloxane-1,3-
diyl (TIPDS) nor the TES groups were removed by treat-
ment with AcOH (Entries 1 and 2). After several experi-
ments, hydrogen fluoride (HF) was found to be effective at
selectively cleaving the TES group. 3 was then treated with
HF at different concentrations to determine the most effec-
tive conditions (Entries 3Ϫ6). Treatment with six equiva-
lents of aqueous 1% HF with 7:1 (v/v) MeCN/CHCl3 as
solvent for 36 h at room temperature produced the best re-
sult, affording 95% of the desired alcohol 4 (Entry 5). Re-
moval of the acetylmandeloyl group from 4 proceeded
smoothly by treatment with hydrazine monohydrate to give
optically pure - and -2 in quantitative yield in both cases.
In addition to - and -2, the intermediates shown in the
Scheme 1. Reagents and conditions: (a) 1% HF (6 equiv.), MeCN/
CHCl3 (7:1 v/v), room temp., 36 h, 95%; (b) NH2NH2·H2O (3.0 Scheme are also useful compounds for synthesis. -3 and
-4 could be rapidly transformed into PtdIns(3,4,5)P3[13]
equiv.), CH2Cl2, room temp., 1.5 h, quantitative
and PtdIns(5)P,[14] respectively. Their opposite -enanti-
employing the strategy of regiospecific functionalization at
the 3-position of a vicinal 3,4-diol myo-inositol deriva-
tive.[9,10] Finally, one of the key reactions, regioselective
phosphorylation of the triol derivatives, is unprecedented,
and this markedly limits the number of steps.
omers may also serve as starting materials for alternative
targets. For instance, -4 might be converted into -
PtdIns(5)P via the following sequence: (1) phosphorylation
of -4 at the 5-position; (2) desilylation of TIPDS group to
afford the vicinal 3,4-diol; (3) selective phosphorylation of
the 3,4-diol[10b] to complete the precursor of -PtdIns(5)P.
This reaction sequence has, however, not yet been carried
out.
During our synthetic studies, we found that the reactivity
of the OHs on the triol derivatives was dramatically affected
by the proportion of pyridine in the reaction solvent. Phos-
phorylation attempted in 1:12 (v/v) of pyridine/CH2Cl2 did
not proceed at all, whereas that performed in a 1.1:1 pyri-
dine/CH2Cl2 co-solvent system afforded the desired product
in 68% yield. It is important to clarify the origins of this
finding, since it may provide a new and rapid route for the
synthesis of PtdInsPns and/or InsPns through the selective
phosphorylation of a polyolic myo-inositol derivative, and
this would markedly reduce the laborious protection-depro-
tection work. The results of our investigation into this
phenomenon, along with an account of the chemical resolu-
tion of 2 and the experimental procedures for the synthesis
of dipalmitoyl PtdIns(3,5)P2 (1b) are detailed in this report.
Table 1. Selective cleavage of TES group in - and -3
Entry Acid (equiv.) Solvent (v/v)
Time (h) Yield of 2 (%)
1
2
3
4
5
6
AcOH (4Ϫ8) THF
AcOH (8) THF/H2O (8:1)
2% HF (6) MeCN/CHCl3 (7:1) overnight
1% HF (6) MeCN/CHCl3 (7:1) 20
1% HF (6) MeCN/CHCl3 (7:1) 36
1% HF (10) MeCN/CHCl3 (8:1) 28
10
10
NR[a]
NR
65
60[b]
95
91
[a]
[b]
No reaction.
33% starting material was recovered.
2.b. Synthesis of Dipalmitoyl PtdIns(3,5)P2 from both
- and -2
2. Results and Discussion
D
L
2. a. Chemical Resolution of 2
As outlined in Scheme 2, diol -2 was easily converted
All of the naturally occurring PtdInsPns and InsPns and/ into the fully protected -6,[14] which was then transformed
or their analogues might be synthesized from - and/or - into diol -7, without the migration of the phosphate func-
2. Hence, an effective preparation of optically pure 2 is re- tionality, by careful treatment with TBAF and AcOH at
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F. Han, M. Hayashi, Y. Watanabe
FULL PAPER
Scheme 2. Conditions and reagents: (a) CH3CO(CH2)2COOH, DCC, DMAP, CH2Cl2, room temp., 89%; (b) (BnO)2PN(iPr)2, 1H-tetra-
zole, CH2Cl2, room temp. then mCPBA, Ϫ78 °C to room temp., 96%; (c) TBAF·3H2O, AcOH, THF, Ϫ15 to Ϫ10 °C, 92%; (d) (BnO)3P,
pyridinium tribromide, 2,6-lutidine, CH2Cl2, Ϫ42 to 0 °C, 91%; (e) Py(HF)x, ethylene glycol, CH2Cl2, 0 °C to room temp., 84%; (f) 10,
pyridinium tribromide, 2,6-lutidine, pyridine/CH2Cl2 (v/v 1.1:1), Ϫ22 °C to room temp., 68%; (g) hydrazine monohydrate, pyridine/AcOH
(v/v 4:1), 0 °C to room temp., 89%; (h) 5% Pd/C, H2, EtOAc/MeOH (v/v 1:1), room temp., quant.
low temperature. As one of the key reactions, installation of These reported results clearly suggest that 1-OH would be
a 3-phosphate group onto -7 to give the 3,5-diphosphate 8 the most reactive of the three hydroxy groups at the 1, 2,
was carried out in high yield (91%) using the protocol for and 4 positions of the triol 9. Therefore, the selective phos-
selective phosphorylation of 3,4-diol derivatives.[10] Because phorylation of 9 is expected to occur at 1-OH. Accordingly,
of the overlap of some of the signals for the inositol protons 3,5-diphosphate derivative 8 was transformed into the triol
1
in the H NMR spectrum of 8, the determination of the 9 smoothly by the cleavage of the cyclohexylidene ketal with
exact phosphorylation site of -7 was achieved by trans- pyridinium poly(hydrogen fluoride) [Py(HF)n], which has
forming 8 into its chloroacetate. The resonances for the six been used to cleave the isopropylidene group without the
inositol methine protons of the chloroacetate derivative migration of an adjacent phosphate functionality.[15] Phos-
were clearly separated, and InsH-4 was shifted downfield phorylation of 9 with dipalmitoylglycerol phosphite 10 was
from δ ϭ 4.20 to 5.66 ppm as a triplet.
first attempted in a 1:12 (v/v) mixed solvent system of pyri-
The synthesis of a 1-phosphatidyl derivative starting from dine and CH2Cl2, and did not proceed at all (Table 2, Entry
8 might be accomplished generally via the following pro- 1). Thus, the triol 9 showed unusually low reactivity com-
cedure: (a) protection of the 4-OH in 8; (b) removal of the pared to the 1,2- and 3,4-diol derivatives we had examined
cyclohexylidene moiety, affording a 1,2-diol intermediate; up to that point. After some trials, we found that the reac-
(c) selective 1-O-phosphorylation of the vicinal 1,2-diol de- tion was dramatically affected by the ratio of pyridine to
rivative. However, we reasoned that such a circuitous pro- CH2Cl2. A trace amount of the desired 1-O-phosphatidyli-
cedure might be simplified through the direct selective nositol 11 was formed (as shown by TLC) in 1:5 pyridine/
phosphorylation of the triol 9, since regioselective 1-O- CH2Cl2 as solvent (Entries 2 and 3), and 36% of 11 was
phosphorylation of vicinal 1,2-diol derivatives of myo-inosi- isolated when the reaction was carried out in a pyridine/
tol has been well documented by our own group[13] and CH2Cl2 mixture with the ratio 1:1.8 (Entry 4).
others.[7a] On the other hand, selective functionalization
In order to investigate the reason for the low reactivity
(such as phosphorylation or acylation) at the 3-position of of triol 9, we studied the relationship between the concen-
1
3,4-diol derivatives has also been exploited very recently.[10b] tration and intra- vs. intermolecular interactions using H
560
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Chemical Resolution of L- and D-myo-Inositol Derivatives
FULL PAPER
Table 2. Phosphorylation of triol 9 with phosphite 10 under different conditions
Entry
Base
Py/CH2Cl2 (v/v)
Temperature and time
Yield (%) of 11
1
2
2,6-lutidine
2,6-lutidine
Et3N
Et3N
2,6-lutidine
Ϫ
1:12
1:5
1:5
1:1.8
1.1:1
1.1:1
1.1:1
3:1
Ϫ42 °C, 10 min; 0 °C, 2 h; then room temp., 2 h
Ϫ42 °C, 10 min; 0 °C, 2 h; then room temp., 2 h
Ϫ22 °C, 10 min; 0 °C, 2 h; then room temp., 2 h
Ϫ22 °C, 10 min; 0 °C, 2 h; then room temp., 2 h
Ϫ22 °C, 10 min; 0 °C, 1.5 h; then room temp., 1 h
Ϫ22 °C, 10 min; 0 °C, 1.5 h; then room temp., 1 h
Ϫ22 °C, 10 min; 0 °C, 2 h;
nr[a]
trace[b]
trace
36
68
61
3
4[c]
5
6
7
8
2,6-lutidine
2,6-lutidine
47
Ϫ22 °C, 10 min; 0 °C Ϫ room temp., 2 h
25[d]
[a] nr: no reaction. [b] Not isolated. [c] The molar ratio of phosphite to triol was 6:1 instead of 3:1 as in the other cases. [d] Some inseparable
unknown products included.
NMR spectroscopy. We found that as the concentration of
the solution increased in CDCl3, the chemical shifts of the
three OH protons shifted downfield, and finally merged
into a single broad peak (Figure 1). The same phenomenon
was observed in CD2Cl2 when spectra were recorded at
various concentrations. These observations indicate the for-
mation of intermolecular hydrogen bonds of the triol 9 at
high concentrations. In contrast, in the case of a 1,2-diol,
3,6-di-O-benzyl-4,5-di-O-(dibenzylphosphoryl)-myo-
inositol, intramolecular hydrogen bonding predominates
throughout the range of concentrations tested for the triol
(9) (Figure 2). It is interesting to note that the unprotected
4-OH in the triol 9 markedly changes the inherent intramol-
ecular nature of the hydrogen bonding in the 1,2-diol sys-
tem, facilitating intermolecular hydrogen bonding.
These findings directed us to increase the ratio of pyri-
dine to CH2Cl2 in the phosphorylation reaction solvent in
Figure 2. 1H NMR spectra (400 MHz) of 3,6-di-O-benzyl-4,5-di-
O-(dibenzylphosphoryl)-myo-inositol in CDCl3 at various concen-
trations
order to disrupt the intermolecular hydrogen-bonding net-
work. As expected, the yield of the desired phosphorylation
product 11 increased as the ratio of pyridine was increased,
and finally 68% of 11 was obtained in an optimized 1.1:1
ratio of the mixed solvent system at room temperature (En-
try 5). When the reaction was carried out in the absence of
2,6-lutidine or Et3N in the same solvent system, 61% of 11
was obtained (Entry 6). These results suggested that the
reaction was less affected by the type of base. In addition
to the proportion of pyridine, temperature was also shown
to be one of the factors to affect the reaction, since the yield
decreased to 47% when the reaction was conducted at 0 °C
(Entry 7). A further increase in the proportion of pyridine
lowered the yield of 11 dramatically (Entry 8), presumably
due to the decomposition of the reactive phosphorus inter-
mediate by pyridine. In all cases, the desired compound 11
was formed as the single product, as shown by TLC and
NMR spectroscopy. In order to determine the exact phos-
phorylation site, 11 was converted into the corresponding
1
1
chloroacetate. Analysis of the H NMR and H-1H COSY
spectra of this compound clearly showed that phosphoryl-
ation had occurred at the 1-OH position. The methine pro-
tons at C-2 and C-4 were shifted downfield to δ ϭ 5.88 and
5.52 ppm, respectively, from the region around δ ϭ
4.20 ppm.
In a similar manner, the opposite enantiomer -2 was
also transformed into the diol 15, the key precursor of the
final product is outlined in Scheme 3. -7, in a similar man-
Figure 1. 1H NMR spectra (400 MHz) of triol 9 in CDCl3 at
various concentrations
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F. Han, M. Hayashi, Y. Watanabe
FULL PAPER
Scheme 3. (a) 10, pyridinium tribromide, 2,6-lutidine, CH2Cl2, Ϫ42 to 0 °C, 87%; (b) Py(HF)x, ethylene glycol, CH2Cl2, 0 °C to room
temp., 89%; (c) (BnO)3P, pyridinium tribromide, 2,6-lutidine, pyridine/CH2Cl2 (v/v 1:1), Ϫ22 to 0 °C, 81%; (d) hydrazine monohydrate,
pyridine/AcOH (v/v 4:1), 0 °C to room temp., 91%; (e) 5% Pd/C, H2, EtOAc/MeOH (v/v 1:1), room temp., quant.
ner to -7, the phosphatidyl group was introduced prior to resulting in smooth regiospecific phosphorylation, to give
the 3-phosphate group, affording alcohol 13. Removal of the key intermediates (11 and 15, respectively) in the syn-
the cyclohexylidene ketal in 13 afforded the triol 14, which thesis of the target molecule -PtdIns(3,5)P2 (1b), in mod-
was then selectively phosphorylated to give 15. Finally, re- erate to good yields. Regioselective phosphorylation reac-
moval of the levulinoyl groups from 11 (Scheme 2) and 15 tions of diol and triol compounds markedly reduced the
(Scheme 3) by treatment with hydrazine monohydrate in a number of protection- and deprotection-steps, thus improv-
[13,16]
mixture of pyridine and acetic acid
gave the triol 12, ing the synthetic route. In addition to the practical appli-
and subsequent debenzylation by hydrogenolysis over 5% cation to the synthesis of PtdIns(3,5)P2 (1b), the convergent
palladium on carbon afforded the dipalmitoyl synthetic strategy and the selective phosphorylation method
PtdIns(3,5)P2 (1b) as its free acid. The structure of the final of a polyol intermediate presented in this report might be
product was confirmed by its NMR and MS spectra. applicable to the synthesis of other PtdInsPns and InsPns.
Further purification of the crude final product was not
done, because no impurities were detected in its NMR spec-
tra. No migration or decomposition of 1b in its free acid
form was seen when a sample was left standing in an open
4. Experimental Section
flask for four months at room temperature.
General Methods: All solvents were purified according to standard
procedures. Reagents were reagent grade, and were purified where
further purification was required. Pyridinium tribromide (PTB)
was recrystallized from AcOH and dried at 60 °C under reduced
pressure. 2,6-Lutidine and 1,3-dichlorotetraisopropyldisiloxane
were purified by distillation under reduced pressure. Compounds
- and -5 were prepared according to the reported method.[13]
NMR spectra were recorded with a Bruker Avance 400 spec-
trometer unless otherwise noted. Optical rotations were measured
using a JASCO P-1010 polarimeter with a 1-cm cell. Elemental
analyses were performed with a PerkinϪElmer 240C machine.
Melting points are uncorrected and were measured in open capil-
laries using a Yamato Melting Point Apparatus Model MP-21. Sil-
3. Conclusions
We have developed a convergent strategy to access
PtdIns(3,5)P2 with the natural -configuration from both
enantiomers of 2. The regioselective phosphorylation of the
3,4-diol, 5-dibenzyl 1,2-O-cyclohexylidene-6-O-levulinoyl-
myo-inositol phosphate (7), played a key role in the conver-
gent transformation of both enantiomers into the same tar-
get molecule. For the selective phosphorylation of triols 9
and 14, a modified solvent system improved their reactivity, ica gel (Fuji Silysia Chemical Ltd., 200Ϫ400 mesh) was used for
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Chemical Resolution of L- and D-myo-Inositol Derivatives
FULL PAPER
flash chromatography. Stereospecifically numbered is abbreviated
to ‘‘sn’’. ‘‘Ins’’ appearing in the text is the abbreviation of inositol.
was dried, concentrated, and purified by chromatography to give 4
(95%) as a colorless oil. Rf ϭ 0.52 (EtOAc/hexane, 1:5); L
-4: 1H
NMR (400 MHz, CDCl3): δ ϭ 7.49 (dd, J ϭ 3.9, 7.4 Hz, 2 H),
7.38 (m, 3 H), 6.05 (s, 1 H, methenyl), 5.21 (dd, J ϭ 8.0, 10.8 Hz,
1 H, InsH-6), 4.21 (t, J ϭ 4.4 Hz, 1 H, InsH-2), 3.99 (t, J ϭ 9.2 Hz,
1 H, InsH-4), 3.82Ϫ3.88 (m, 2 H, InsH-3, InsH-1), 3.44 (dd, J ϭ
9.2, 10.8 Hz, 1 H, InsH-5), 2.18 (s, 3 H, CH3), 1.76 (br., 2 H, cyclo-
hexylidene), 1.52 (br., 6 H, cyclohexylidene), 1.35 (br., 2 H, cyclo-
Preparation of
D- and L-6-O-Acetylmandeloyl-1,2-O-cyclohexyl-
idene-3,4-O-(tetraisopropyldisiloxane-1,3-diyl)-5-O-triethylsilyl-
myo-inositol (3): DMF (0.1 mL) was added dropwise to a CH2Cl2
solution (5 mL) of oxalyl chloride (1.70 g, 13.5 mmol) at 0 °C and
the mixture was stirred for 5 min. Then, (S)-(ϩ)-O-acetylmandelic
acid (2.00 g, 10.3 mmol) in 10 mL CH2Cl2 was added dropwise.
The mixture was stirred at ambient temperature for 3 h. Volatile
materials were evaporated under reduced pressure and trace
amounts of H2O in the residue were removed by azeotropic distil-
lation with benzene. The crude acetylmandeloyl chloride thus ob-
tained was dissolved in 10 mL CH2Cl2 and added to a 30 mL
CH2Cl2 solution of racemic 2 (3.75 g, 7.5 mmol) at 0 °C. Pyridine
(2.13 g, 27.0 mmol) was added and the mixture was stirred at room
temperature for 16 h. After being diluted with EtOAc, the organic
layer was washed with aqueous KHSO4, aqueous NaHCO3 and
brine, dried, concentrated and purified by chromatography to give
(Ϯ)-6-O-acetylmandeloyl1,2-O-cyclohexylidene-3,4-O-(tetraiso-
propyldisiloxane-1,3-diyl)-myo-inositol (4.71 g, 89%) as a colorless
oil. Rf ϭ 0.52 (EtOAc/hexane, 1:10). 1H NMR (400 MHz, CDCl3):
δ ϭ 7.37Ϫ7.48 (m, 10 H, aromatic), 6.10 & 6.05 (s ϫ 2, 2 H, me-
thenyl), 5.15 & 5.23 (dd ϫ 2, J ϭ 7.6, 10.4 Hz, 2 H, InsH-6), 4.21 &
4.27 (t ϫ 2, J ϭ 4.2 Hz, 2 H, InsH-2), 4.07 (dd, J ϭ 4.2, 7.6 Hz, 1
H, InsH-1), 3.93 & 4.02 (t ϫ 2, J ϭ 9.0 Hz, 2 H, InsH-4),
3.80Ϫ3.88 (m, 3 H, InsH-3, InsH-1), 3.25 & 3.44 (t ϫ 2, J ϭ
10.4 Hz, 2 H, InsH-5), 2.18 (s, 3 H, CH3), 2.16 (s, 3 H, CH3), 1.78
(br., 4 H, cyclohexylidene), 1.51Ϫ1.57 (m, 12 H, cyclohexylidene),
1.35 (br., 4 H, cyclohexylidene), 1.03 (br., 56 H, isopropyl) ppm.
The compound thus obtained (1.73 g, 2.5 mmol), ethyldiisopro-
pylamine (EDA, 2.13 mL, 12.3 mmol) and a catalytic amount of
DMAP were dissolved in CH2Cl2 (18 mL) at 0 °C, and chlorotrie-
thylsilane (TESCl, 1.64 mL, 9.8 mmol) was added dropwise. The
ice-bath was removed, and the solution was stirred at room tem-
perature for 20 h. The mixture was diluted with EtOAc and washed
with H2O, aqueous KHSO4, aqueous NaHCO3 and brine, then the
organic phase was dried, concentrated and purified by chromatog-
raphy (EtOAc/hexane, 1:50) to afford -3 (0.71 g, 38%), -3 (0.74 g,
39%) and 0.19 g mixture of - and -3. -3 Rf ϭ 0.23 (EtOAc/
hexylidene), 1.04 (m, 28 H, isopropyl) ppm.
D
-4: 1H NMR
(400 MHz, CDCl3): δ ϭ 7.48 (dd, J ϭ 3.9, 7.4 Hz, 2 H), 7.38 (m,
3 H), 6.10 (s, 1 H, methenyl), 5.14 (dd, J ϭ 8.0, 10.8 Hz, 1 H, InsH-
6), 4.28 (t, J ϭ 4.4 Hz, 1 H, InsH-2), 4.06 (dd, J ϭ 4.4, 8.0 Hz, 1
H, InsH-1), 3.92 (t, J ϭ 9.2 Hz, 1 H, InsH-4), 3.83 (dd, J ϭ 4.4,
9.2 Hz, 1 H, InsH-3), 3.24 (dd, J ϭ 9.2, 10.8 Hz, 1 H, InsH-5),
2.16 (s, 3 H, CH3), 1.74 (br., 2 H, cyclohexylidene), 1.54 (br., 6 H,
cyclohexylidene), 1.37 (br., 2 H, cyclohexylidene), 1.03 (m, 28 H,
isopropyl) ppm. C34H54O10Si2 (679.0).
D- and L-1,2-O-Cyclohexylidene-3,4-O-(tetraisopropyldisiloxane-1,3-
diyl)-myo-inositol (2). General Procedure: Hydrazine monohydrate
(20 equiv.) was added dropwise to a DMF solution of 4 at 0 °C,
and the mixture was stirred at room temperature for 1.5 h. The
mixture was diluted with EtOAc and washed with aqueous
KHSO4, aqueous NaHCO3 and brine. The organic phase was
dried, concentrated, and purified by chromatography to give op-
tically pure 2 (100%) as a white solid. -2, [α]2D6 ϭ ϩ13.2 (c ϭ 1.0,
CH2Cl2); -2, [α]2D6 ϭ Ϫ13.6 (c ϭ 1.0, CH2Cl2). For spectroscopic
data for - and -2, see ref.[13]
.
5-Dibenzyl 1,2-O-Cyclohexylidene-6-O-levulinoyl-3,4-O-(tetraisop-
ropyldisiloxane-1,3-diyl)-myo-inositol Phosphate (6). -6: Dibenzyl
D
N,N-diisopropylphosphoramidite (379.5 mg, 1.11 mmol) and 1H-
tetrazole (115.5 mg, 1.65 mmol) were added to a CH2Cl2 (10 mL)
solution of -5 (330.0 mg, 0.55 mmol). The solution was stirred at
room temperature for 1 h, then treated with 10 µL H2O over
15 min. After being cooled to Ϫ78 °C, mCPBA (236.5 mg,
1.38 mmol) was added, and the mixture was stirred at ambient tem-
perature for 1 h. The solution was diluted with EtOAc, and washed
with aqueous Na2SO3, aqueous NaHCO3, and brine. The organic
phase was dried, concentrated, and purified by chromatography
(hexane/EtOAc, 5:1) to afford -6 (454.2 mg, 96%) as a colorless
1
hexane, 1:15). H NMR (400 MHz, CDCl3): δ ϭ 7.36Ϫ7.47 (m, 5
viscous oil: [α]2D4 ϭ Ϫ17.6 (c ϭ 1.0, CHCl3). -6: Yield, 95%. [α]2D4
L ϭ
H, aromatic), 6.15 (s, 1 H, methenyl), 5.07 (dd, J ϭ 7.2, 8.4 Hz, 1
H, InsH-6), 4.12 (dd, J ϭ 4.0, 4.8 Hz, 1 H, InsH-2), 3.96 (t, J ϭ
9.2 Hz, 1 H, InsH-4), 3.75 (dd, J ϭ 4.0, 9.2 Hz, 1 H, InsH-3), 3.59
(dd, J ϭ 4.8, 7.2 Hz, 1 H, InsH-1), 3.45 (dd, J ϭ 8.4, 9.2 Hz, 1 H,
InsH-5), 2.18 (s, 3 H, CH3), 1.76 (br., 2 H, cyclohexylidene),
1.28Ϫ1.56 (br., 8 H, cyclohexylidene), 1.05 (br., 28 H, isopropyl),
ϩ17.2 (c ϭ 1.0, CHCl3); Rf ϭ 0.30 (hexane/EtOAc, 2:1). 1H NMR
(400 MHz, CDCl3): δ ϭ 7.31Ϫ7.36 (br., 10 H, aromatic), 5.26 (dd,
J ϭ 7.6, 10.0 Hz, 1 H, InsH-6), 5.00Ϫ5.05 (m, 2 H, C6H5CH2),
4.86Ϫ4.95 (m, 2 H, C6H5CH2), 4.28 (dd, J ϭ 3.6, 4.8 Hz, 1 H,
InsH-2), 4.25 (dd, J ϭ 9.0, 10.0 Hz, 1 H, InsH-5), 4.15 (dd, J ϭ
8.8, 9.0 Hz, 1 H, InsH-4), 4.02 (dd, J ϭ 4.8, 7.6 Hz, 1 H, InsH-1),
3.91 (dd, J ϭ 3.6, 8.8 Hz, 1 H, InsH-3), 2.52Ϫ2.70 (m, 4 H,
CH2CH2), 2.05 (s, CH3), 1.70Ϫ1.90 (m, 2 H, cyclohexylidene),
1.45Ϫ1.66 (m, 6 H, cyclohexylidene), 1.22Ϫ1.38 (m, 2 H, cyclohex-
ylidene), 0.96Ϫ1.08 (m, 28 H, isopropyl) ppm. 31P NMR
(162 MHz, CDCl3): δ ϭ Ϫ0.29 ppm. C43H65O12PSi2 (861.1) (for -
6): calcd. C 59.97, H 7.61; found C 59.75, H 7.53.
0.94 (t, J ϭ 7.8 Hz, 9 H, SiCH2CH3), 0.63 (q, J ϭ 7.8 Hz, 6 H,
1
SiCH2CH3) ppm.
D
-3: Rf ϭ 0.15 (EtOAc/hexane, 1:15). H NMR
(400 MHz, CDCl3): δ ϭ 7.36Ϫ7.47 (m, 5 H, aromatic), 6.10 (s, 1
H, methenyl), 5.06 (t, J ϭ 5.4 Hz, 1 H, InsH-6), 4.37 (dd, J ϭ 3.6,
5.4 Hz, 1 H, InsH-2), 4.18 (t, J ϭ 5.4 Hz, 1 H, InsH-1), 4.04 (dd,
J ϭ 6.4, 9.6 Hz, 1 H, InsH-4), 3.79 (dd, J ϭ 3.6, 9.6 Hz, 1 H, InsH-
3), 3.43 (dd, J ϭ 5.4, 6.4 Hz, 1 H, InsH-5), 2.18 (s, 3 H, CH3), 1.69
(br., 2 H, cyclohexylidene), 1.28Ϫ1.55 (br., 8 H, cyclohexylidene),
1.03 (br., 28 H, isopropyl), 0.84 (t, J ϭ 7.8 Hz, 9 H, SiCH2CH3),
0.45 (q, J ϭ 7.8 Hz, 6 H, SiCH2CH3) ppm. C40H68O10Si3 (793.2).
5-Dibenzyl 1,2-O-Cyclohexylidene-6-O-levulinoyl-myo-inositol Phos-
phate (7).
D-7: AcOH (93.4 mg, 1.56 mmol) and TBAF·3H2O
(367.9 mg, 1.17 mmol) were added to a THF (10 mL) solution of
-6 (334.8 mg, 0.39 mmol) at Ϫ15 °C. The solution was stirred at
Ϫ15 to Ϫ10 °C for 4.5 h, after which time, the solvent was removed
D- and L-6-O-Acetylmandeloyl-1,2-O-cyclohexylidene-3,4-O-(tetra-
isopropyldisiloxane-1,3-diyl)-myo-inositol (4). General Procedure: under reduced pressure and the residue purified by chromatography
Aqueous HF (6.0 equiv.) was added dropwise to a CH3CN/CHCl3
(v/v 7:1) solution of 3 at 0 °C. The solution was stirred at room
temperature for 36 h. After being diluted with EtOAc, the solution chromatography).
was washed with aqueous NaHCO3 and brine. The organic phase
(hexane/EtOAc, 1:3) to afford -7 (220.9 mg, 92%) as a white solid:
Rf ϭ 0.49 (EtOAc); m.p. 151.0Ϫ153.0 °C (sample was obtained by
D L-7: Yield
-7: [α]2D4 ϭ Ϫ17.6 (c ϭ 1.0, CHCl3).
88%. [α]2D4 ϭ ϩ17.4, (c ϭ 1.0, CHCl3). 1H NMR (400 MHz,
Eur. J. Org. Chem. 2004, 558Ϫ566
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
563

F. Han, M. Hayashi, Y. Watanabe
FULL PAPER
CDCl3): δ ϭ 7.35 (m, 10 H, 2 ϫ C6H5), 5.28 (dd, J ϭ 7.3, 9.2 Hz, (162 MHz, CDCl3):
δ
ϭ
0.11 (1 P), Ϫ0.06 (1 P) ppm.
1 H, InsH-6), 5.00Ϫ5.10 (m, 4 H, 2 ϫ C6H5CH2), 4.44 (dd, J ϭ
C39H44O14P2·0.5H2O (807.7): calcd. C 57.99, H 5.62; found C
4.0, 5.1 Hz, 1 H, InsH-2), 4.09Ϫ4.16 (m, 2 H, InsH-5, InsH-1), 57.94, H 5.87.
4.00 (dd, J ϭ 8.8, 9.1 Hz, 1 H, H-4), 3.77 (dd, J ϭ 4.0, 9.1 Hz, 1
Phosphate 11:
A
mixture of 1,2-di-O-palmitoyl-sn-glycerol
H, InsH-3), 2.48Ϫ2.58 (m, 4 H, CH2CH2), 2.08 (s, 3 H, CH3), 1.76
(m, 2 H, cyclohexylidene), 1.61 (m, 6 H, cyclohexylidene), 1.38 (m,
2 H, cyclohexylidene) ppm. 13C NMR (100 MHz, CDCl3): δ ϭ
206.81 (s), 172.12 (s), 135.99 (s), 135.93 (s), 135.92 (s), 135.85 (s),
128.25Ϫ129.11 (m), 111.93 (s), 79.84 (br.), 76.16 (d, J ϭ 7.3 Hz),
75.24 (d, J ϭ 6.0 Hz), 74.44 (d, J ϭ 5.1 Hz), 72.28 (br.),
70.10Ϫ70.33 (br.), 70.30 (s), 70.25 (s), 38.09 (s), 37.64 (s), 35.42 (s),
30.14 (s), 28.28 (s), 25.27 (br.), 24.17 (s), 23.94 (s) ppm. 31P NMR
(162 MHz, CDCl3): δ ϭ Ϫ0.47 ppm. C31H39O11P (618.6) (for -7):
calcd. C 60.19, H 6.40; found C 60.14, H 6.61.
(162.1 mg, 0.29 mmol), dibenzyl N,N-diisopropylphosphoramidite
(114.8 mg, 0.33 mmol), and 1H-tetrazole (33.3 mg, 0.48 mmol) in
CH2Cl2 (5 mL) was stirred at room temp. for 1 h. After being di-
luted with hexane, the solution was washed with brine and dried,
and the solvents were evaporated. The benzene solution of crude
10 thus obtained was concentrated under reduced pressure to re-
move traces of H2O. The residue, 10, was mixed with 9 (75.9 mg,
0.10 mmol) in the solvent mixture benzene/CH2Cl2. The solvents
were evaporated under reduced pressure, and dried in vacuo for
4 h. The resultant mixture of 9 and 10 was dissolved in pyridine
Phosphate 8: -7 (230.8 mg, 0.37 mmol), 2,6-lutidine (201.7 mg, (3.3 mL) and CH2Cl2 (3 mL). After being cooled to Ϫ22 °C, 2,6-
1.87 mmol) and tribenzyl phosphite (394.4 mg, 1.12 mmol) were lutidine (51.4 mg, 0.48 mmol) and PTB (121.7 mg, 0.38 mmol) were
dissolved in CH2Cl2 (16 mL) at Ϫ42 °C, and pyridinium tribromide added. The solution was stirred vigorously at Ϫ22 °C for 10 min,
(PTB) (478.0 mg, 1.49 mmol) was added. The solution was stirred
at Ϫ42 °C for 10 min, and for a further 1.2 h at 0 °C, then diluted
with EtOAc and washed with aqueous KHSO4, aqueous NaHCO3,
and then at 0 °C (in an ice/H2O bath containing a small amount
of NaCl) for 1.5 h. The bath was removed, and the solution was
stirred at ambient temperature for an additional 1 h. The solution
and brine. The organic phase was dried, concentrated, and purified was diluted with EtOAc, and washed with aqueous KHSO4, aque-
by chromatography (hexane/EtOAc, 1:2) to afford 8 (297.1 mg,
91%) as a white solid: Rf ϭ 0.35 (EtOAc/hexane, 4:1); m.p.
ous NaHCO3 and brine. The organic phase was dried, concen-
trated, and purified by chromatography (hexane/EtOAc, 1:2) to af-
ford 11 (97.9 mg, 68%): Rf ϭ 0.48 (EtOAc/hexane, 2:1). H NMR
155.5Ϫ157.0 °C (sample was obtained by chromatography). [α]2D5
ϭ
1
Ϫ2.9 (c ϭ 2.7, CHCl3). 1H NMR (400 MHz, CDCl3): δ ϭ 7.35 (m, (400 MHz, CDCl3): δ ϭ 7.32 (m, 25 H, 5 ϫ C6H5), 5.58 (dt, J ϭ
20 H, 4 ϫ C6H5), 5.26 (dd, J ϭ 7.4, 8.8 Hz, 1 H, InsH-6), 6.4, 9.6 Hz, 1 H, InsH-6), 5.24 (m, 1 H, glyceryl sn-2 H), 4.91Ϫ5.10
4.50Ϫ5.19 (m, 8 H, 4 ϫ C6H5CH2), 4.44Ϫ4.50 (m, 2 H, InsH-3, (m, 5 ϫ C6H5CH2), 4.08Ϫ4.47 (m, 9 H, glyceryl sn-1 H, sn-3 H,
H-2), 4.13Ϫ4.23 (m, 2 H, InsH-4, H-5), 4.04 (dd, J ϭ 4.8, 7.4 Hz,
InsH-1, -2, -3, -4 and -5), 2.24Ϫ2.61 (m, 4 H, CH2 in levulinoyl),
1 H, InsH-1), 2.44Ϫ2.57 (m, 4 H, CH2CH2), 2.06 (s, 3 H, CH3), 2.26 (m, 4 H, α-CH2 in palmitoyl), 2.01 (s, 3 H, CH3 in levulinoyl),
1.60 (br., 2 H, cyclohexylidene), 1.48 (br., 6 H, cyclohexylidene),
1.33 (br., 2 H, cyclohexylidene) ppm. 13C NMR (100 MHz,
1.55 (br., 4 H, β-CH2 in palmitoyl), 1.25 (br., 48 H, CH2 in palmi-
toyl), 0.88 (t, J ϭ 6.4 Hz, 6 H, CH3 in palmitoyl) ppm. 13C NMR
CDCl3): δ ϭ 206.52 (s), 172.17 (s), 136.16 (s), 136.08 (s), 136.01 (100 MHz, CDCl3): δ ϭ 206.51 (s), 206.45 (s), 173.84 (s), 173.71
(s), 135.94 (s), 128.26Ϫ129.50 (m), 112.01 (s), 79.04 (br.), 76.50 (s), 173.61 (s), 173.46 (s), 172.43 (s), 172.30 (s), 135.70Ϫ136.20 (m),
(br.), 75.93 (m), 74.56 (s), 73.91 (t, J ϭ 3.3 Hz), 70.28 (s), 70.23 (s,
128.25Ϫ129.01 (m), 79.38 (m), 78.94 (m), 76.57 (m), 75.12 (m),
2 C), 70.17 (s), 69.97 (d, J ϭ 5.5 Hz), 38.04 (s), 37.69 (s), 35.39 (s), 70.23Ϫ70.70 (m), 69.83 (m), 66.40 (m), 62.21 (m), 37.68 (s), 37.54
30.10 (s), 28.29 (s), 25.34 (br.), 24.15 (s), 23.98 (s) ppm. 31P NMR (s), 34.55 (s), 34.40 (s), 32.34 (s), 29.55Ϫ30.12 (m), 28.23 (s), 25.24
(162 MHz, CDCl3):
δ
ϭ
0.45 (1 P), Ϫ0.05 (1 P) ppm.
(s), 23.11 (s), 14.55 (br.) ppm.31 P NMR (162 MHz, CDCl3): δ ϭ
C45H52O14P2·0.5H2O (887.8): calcd. C 60.87, H 6.02; found C Ϫ0.02 (2 P), Ϫ0.18 (1 P), Ϫ0.28 (1 P), Ϫ1.08 (1 P), Ϫ1.14 (1 P)
61.02, H 6.24.
-6-O-Levulinoyl-myo-inositol 3,5-Bis(dibenzyl Phosphate) (9): Eth-
ppm, indicating that the phosphorylation product consisted of a
1:1 diastereomeric mixture based on the phosphorus center at the
1-position. C81H117O21P3 (1519.7): calcd. C 64.02, H 7.76; found C
64.32, H 7.69.
D
ylene glycol (33.9 mg, 0.55 mmol) and pyridinium poly(hydrogen
fluoride) (624.8 µL, 21.86 mmol) were added to a CH2Cl2 (20 mL)
solution of 8 (240.0 mg, 0.27 mmol) at 0 °C. The solution was
stirred at 0 °C to room temperature for 3 h. After being re-cooled
to 0 °C, an excess of saturated aqueous NaHCO3 was added drop-
wise and stirred for 15 min. The mixture was washed with aqueous
NaHCO3, aqueous KHSO4, and brine. The organic phase was
dried, concentrated, and purified by chromatography (hexane/
EtOAc, 1:5) to afford 9 (150.7 mg, 84%) as a colorless solid, along
with recovered starting material 8 (35 mg, 14.6%): Rf ϭ 0.37
Phosphate 13: 2,6-Lutidine (100.5 mg, 0.93 mmol) was added to a
CH2Cl2 (10 mL) solution of -7 (115.0 mg, 0.19 mmol) and 10 (3
equiv.). After being cooled to Ϫ42 °C, PTB (238.2 mg, 0.74 mmol)
was added to the solution. The mixture was stirred at Ϫ42 °C for
10 min, and 0 °C for 2 h. The solution was diluted with EtOAc,
washed with aqueous KHSO4, aqueous NaHCO3, and brine. The
organic phase was dried, concentrated, and purified by chromatog-
raphy (hexane/EtOAc, 1:2) to afford 13 (217.2 mg, 87%): Rf ϭ 0.43
1
1
(EtOAc); m.p. 179.0Ϫ181.0 °C. [α]2D4 ϭ ϩ3.3 (c ϭ 1.0, CHCl3). H
(hexane/EtOAc, 1:2). H NMR (400 MHz, CDCl3): δ ϭ 7.28 (br.,
NMR (400 MHz, CDCl3/D2O one drop): δ ϭ 7.33 (m, 20 H, 4 ϫ 15 H, 3 ϫ C6H5), 5.27 (br. dd, J ϭ 6.4, 9.6 Hz, 1 H, InsH-4), 5.19
C6H5), 5.35 (t, J ϭ 9.8 Hz, 1 H, InsH-6), 4.93Ϫ5.12 (m, 8 H, 4 ϫ (m, 1 H, glyceryl sn-2 H), 5.00Ϫ5.15 (m, 6 H, 3 ϫ C6H5CH2),
C6H5CH2), 4.23 (br. q, J ϭ 9.8 Hz, 1 H, InsH-5), 4.12Ϫ4.17 (m, 3 4.49Ϫ4.54 (m, 2 H, InsH-2, InsH-5), 4.36 (t, J ϭ 6.2 Hz, 0.5 H,
H, InsH-2, InsH-3, InsH-4), 3.57 (br. dd, J ϭ 4.6, 9.8 Hz, 1 H,
InsH-3), 4.33 (t, J ϭ 6.2 Hz, 0.5 H, InsH-3), 4.09Ϫ4.29 (m, 6 H,
InsH-1), 2.41Ϫ2.67 (m, 3 H, CH2CH2), 2.31 (t, J ϭ 6.0 Hz, 0.5 H, glyceryl sn-1 H, sn-3 H, InsH-1, InsH-6), 2.49Ϫ2.60 (m, 4 H, CH2
CH2CH2), 2.27 (t, J ϭ 6.0 Hz, 0.5 H, CH2CH2), 2.06 (s, 3 H, CH3) in levulinoyl), 2.31 (br. q, J ϭ 6.0 Hz, 4 H, α-CH2 in palmitoyl),
ppm. 13C NMR (100 MHz, CDCl3): δ ϭ 207.91 (s), 173.17 (s), 2.09 (s, 3 H, CH3 in levulinoyl), 1.78 (br., 2 H, cyclohexylidene),
136.25 (s), 136.17 (s), 136.06 (s), 135.98 (s), 128.29Ϫ128.95 (m),
1.60 (br., 10 H, β-CH2 in palmitoyl, cyclohexylidene), 1.51 (br., 2
79.58 (br.), 78.83 (br.), 73.34 (d, J ϭ 3.9 Hz), 71.16Ϫ71.30 (m), H, cyclohexylidene), 1.28 (br., 48 H, CH2 in palmitoyl), 0.91 (t, J ϭ
31
70.34 (d, J ϭ 5.6 Hz), 70.18 (d, J ϭ 5.8 Hz), 70.15 (s), 70.11 (s),
6.0 Hz, 6 H, CH3 in palmitoyl) ppm.
P NMR (162 MHz,
31
70.08 (s), 70.06 (s), 38.37 (s), 30.11 (s), 28.47 (s) ppm. P NMR CDCl3): δ ϭ 0.56 (1 P), 0.43 (1 P), Ϫ0.29 (1 P), Ϫ0.43 (1 P) ppm.
564
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 558Ϫ566

Chemical Resolution of L- and D-myo-Inositol Derivatives
FULL PAPER
C73H112O18P2 (1339.6): calcd. C 65.45, H 8.43; found C 65.66, H
8.52.
(hexane/EtOAc, 1:3) to afford 12 (68.2 mg, 89%): Rf ϭ 0.47
(EtOAc/hexane, 2:1); compound 11 was converted into 12 by the
same procedure in 91% yield. 1H NMR (400 MHz, CDCl3): δ ϭ
7.33 (br., 25 H, 5 ϫ C6H5), 5.22 (m, 1 H, glyceryl sn-2 H),
5.03Ϫ5.15 (m, 10 H, 5 ϫ C6H5CH2), 4.12Ϫ4.32 (m, 10 H, glyceryl
sn-1 H, sn-3 H, InsH-1 to -6), 2.26 (m, 4 H, α-CH2 in palmitoyl),
1.56 (br., 4 H, β-CH2 in palmitoyl), 1.25 (br., 48 H, CH2 in palmi-
toyl), 0.88 (t, J ϭ 6.4 Hz, 6 H, CH3 in palmitoyl) ppm. 13C NMR
(100 MHz, CDCl3): δ ϭ 173.85 (s), 173.77 (s), 173.54 (s, 2 C),
135.95Ϫ136.16 (m), 128.22Ϫ129.10 (m), 82.08 (m), 77.43Ϫ79.60
(m), 69.81Ϫ70.53 (m), 66.25 (m), 61.74 (m), 34.56 (s), 34.40 (s),
33.34 (s), 29.00Ϫ31.00 (m), 25.23 (s), 23.10 (s), 14.49 (m) ppm. 31P
NMR (162 MHz, CDCl3): δ ϭ 1.10 (2 P), 0.15 (1 P), Ϫ0.05 (1 P),
Ϫ0.07 (1 P), Ϫ0.54 (1 P) ppm. Compound 12 (20.0 mg) thus ob-
tained was dissolved in a 8 mL mixture of EtOAc and MeOH (v/v
1:1). The solution was flushed with N2, then 5% Pd/C (10 mg) was
added. The suspension was hydrogenated at 1 atm for 16 h at room
temperature. The catalyst was removed by filtration, and the resi-
due was washed sequentially with MeOH and CHCl3. The com-
bined solutions were concentrated in vacuo to afford 1b (13.4 mg,
quant.) as its free acid from. [α]2D4 ϭ Ϫ1.4 [c ϭ 0.27, CHCl3/MeOH,
Phosphate 14: Ethylene glycol (8.8 mg, 0.14 mmol) and pyridinium
poly(hydrogen fluoride) (80.7 µL, 2.82 mmol) were added to a
CH2Cl2 (5 mL) solution of 13 (95.0 mg, 0.071 mmol) at 0 °C. The
solution was stirred at 0 °C to room temperature for 4 h. After
being re-cooled to 0 °C, an excess of saturated aqueous NaHCO3
was added dropwise and the mixture was stirred for 15 min. The
mixture was diluted with EtOAc, and washed with aqueous
NaHCO3, aqueous KHSO4, and brine. The organic phase was
dried, concentrated, and purified by chromatography (hexane/
EtOAc, 1:3) to afford 14 (81.3 mg, 89%): Rf ϭ 0.31 (EtOAc/hexane,
1
2:1). H NMR (400 MHz, CDCl3): δ ϭ 7.32Ϫ7.38 (m, 15 H, 3 ϫ
C6H5), 5.38 (br. dd, J ϭ 6.4, 9.2 Hz, 1 H, InsH-4), 5.23 (m, 1 H,
glyceryl sn-2 H), 4.94Ϫ5.17 (m, 6 H, 3 ϫ C6H5CH2), 4.25Ϫ4.33
(m, 3 H, glyceryl sn-1 H, InsH-5), 4.10Ϫ4.22 (m, 5 H, glyceryl sn-
3 H, InsH-1, InsH-2 and InsH-6), 3.59 (br., 1 H, InsH-3), 3.20 (br.,
1 H, 3-OH), 2.46Ϫ2.59 (m, 4 H, CH2 in levulinoyl), 2.27 (m, 4 H,
α-CH2 in palmitoyl), 2.07 (s, 3 H, CH3 in levulinoyl), 1.57 (br., 4
H, β-CH2 in palmitoyl), 1.25 (br., 48 H, CH2 in palmitoyl), 0.89 (t,
J ϭ 6.6 Hz, 6 H, CH3 in palmitoyl) ppm. 13C NMR (100 MHz,
CDCl3): δ ϭ 207.84 (s), 173.89 (s), 173.77 (s), 173.62 (s), 173.58
(s), 173.12 (s), 173.08 (s), 135.85Ϫ136.04 (m), 128.47Ϫ129.05 (m),
78.04Ϫ79.60 (m), 73.27 (d, J ϭ 3.6 Hz), 69.83Ϫ71.20 (m), 65.84
1
1:1 (v/v)]. H NMR (400 MHz, CDCl3/CD3OD/D2O, 1:1:0.1): δ ϭ
5.27 (br., 1 H, glyceryl sn-2 H), 4.41 (br. s, 1 H, InsH-2), 4.30 (br.,
0.5 H, glyceryl sn-1 H), 4.20 (m, 0.5 H, glyceryl sn-1 H), 4.03Ϫ4.17
(m, 6 H, glyceryl sn-1 H, sn-3 H, InsH-1, H-3, H-5), 3.96 (br., 2 H,
InsH-4, H-6), 2.34 (m, 4 H, α-CH2 in palmitoyl), 1.60 (br., 4 H, β-
CH2 in palmitoyl), 1.28 (br., 48 H, CH2 in palmitoyl), 0.89 (br., 6
H, CH3 in palmitoyl) ppm. 31P NMR (162 MHz,CDCl3/CD3OD/
D2O, 1:1:0.1): δ ϭ 5.33 (1 P), 4.54 (1 P), 4.12 (1 P) ppm. Negative
FABMS (triethylammonium salt): m/z (%) ϭ 1008 (25) [M Ϫ 2H
ϩ K]Ϫ, 992 (35) [(M Ϫ 2H ϩ Na]Ϫ, 970 (100) [M Ϫ H]Ϫ, 648
(50) [C15H31COOCH2CH(OCOC15H31)CH2OPO3HϪ], 255 (80)
[C15H31COO]Ϫ. HRMS (FABϪ, triethanolamine) calcd. for
(m), 61.82 (m), 38.49 (s), 38.46 (s), 34.58 (s), 34.41 (s), 32.33 (s),
31
29.52Ϫ30.11 (m), 28.46 (s), 25.23 (s), 23.09 (s), 14.51 (s) ppm.
P
NMR (162 MHz, CDCl3): δ ϭ 0.30 (1 P), 0.09 (1 P), Ϫ0.16 (1 P),
Ϫ0.44 (1 P) ppm. C67H104O18P2 (1259.5): calcd. C 63.89, H 8.32;
found C 63.64, H 8.16.
Phosphate 15: 14 (65.0 mg, 0.051 mmol) was evaporated with ben-
zene and dried in vacuo for 3 h. Pyridinium tribromide (PTB)
(65.7 mg, 0.205 mmol) was added to 6 mL of a pyridine/CH2Cl2
(1:1) solution of 14, 2,6-lutidine (27.7 mg, 0.257 mmol), and triben-
zyl phosphite (54.2 mg, 0.154 mmol) at Ϫ42 °C. The mixture was
stirred at the same temperature for 15 min and for a further 2 h at
0 °C, then diluted with EtOAc, and washed with aqueous KHSO4,
aqueous NaHCO3 and brine. The organic phase was dried, concen-
trated, and purified by chromatography (hexane/EtOAc, 1:2) to af-
Ϫ
C41H80O19P3 (970.0): 969.4506; found 969.4523.
Acknowledgments
1
ford 15 (63.2 mg, 81%): Rf ϭ 0.47 (EtOAc/hexane, 2:1). H NMR
We are grateful to the Center for Cooperative Research and
Development of Ehime University for MS analysis.
(400 MHz, CDCl3): δ ϭ 7.37 (m, 25 H, 5 ϫ C6H5), 5.58 (m, 1 H,
InsH-4), 5.22 (m, 1 H, glyceryl sn-2 H), 4.98Ϫ5.16 (m, 10 H, 5 ϫ
C6H5CH2), 4.12Ϫ4.42 (m, 9 H, glyceryl sn-1 H, sn-3 H, InsH-1, -
2, -3, -5 and -6), 2.38 (m, 2 H, CH2 in levulinoyl), 2.25Ϫ2.29 (m,
6 H, CH2 in levulinoyl, α-CH2 in palmitoyl), 1.94 (s, 3 H, CH3 in
levulinoyl), 1.57 (br., 4 H, β-CH2 in palmitoyl), 1.25 (br., 48 H,
CH2 in palmitoyl), 0.89 (t, J ϭ 6.4 Hz, 6 H, CH3 in palmitoyl)
ppm. 13C NMR (100 MHz, CDCl3): δ ϭ 205.89 (s), 173.41 (s),
173.31 (s), 173.10 (s, 2 C), 171.81 (s, 2 C), 135.49Ϫ135.74 (m),
127.92Ϫ129.07 (m), 79.16 (m), 78.93 (m), 77.76 (m), 75.08 (m),
69.33Ϫ70.20 (m), 65.96 (m), 61.78 (s), 37.31 (s), 34.14 (s), 34.00 (s),
31.92 (s), 28.84Ϫ29.68 (m), 27.81 (s), 24.81 (s), 22.69 (s), 14.11 (s)
ppm. 31P NMR (162 MHz, CDCl3): δ ϭ 0.50 (1 P), 0.23 (1 P),
Ϫ0.07 (1 P), Ϫ0.50 (1 P), Ϫ0.94 (1 P), Ϫ1.04 (1 P) ppm.
C81H117O21P3·2.5H2O (1564.7): calcd. C 62.17, H 7.85; found C
62.15, H 7.70.
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