Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes

Article abstract of DOI:10.1002/chem.200500833

The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholi-pase C (PI-PLC) and the glycosylphos

Full text of DOI:10.1002/chem.200500833

FULL PAPER
DOI: 10.1002/chem.200500833
Phospholipase Cleavage of d- and l-chiro-Glycosylphosphoinositides
Asymmetrically Incorporated into Liposomal Membranes
Julia B. Bonilla,[a] M. BelØn Cid,[b] F.-Xabier Contreras,[c] FØlix M. GoÇi,[c] and
Manuel Martín-Lomas*[a]
Abstract: The nature of chiro-inositol-
containing inositolphosphoglycans
pholipid bilayers in the form of large
unilamellar vesicles (LUVs). Similarly,
2-O-a-d-glucosaminyl- (5) and -galac-
tosaminyl-1-phosphatidyl-d-chiro-inosi-
tol (6), which differ from the corre-
sponding pseudodisaccharide motif of
the GPI anchors only in the axial ori-
entation of the phosphatidyl moiety,
were also synthesized and asymmetri-
cally inserted into LUVs. The cleavage
of these synthetic molecules in the lipo-
somal constructs by PI-PLC from Ba-
cillus cereus and by GPI-PLD from
bovine serum was studied with the use
of 6-O-a-d-glucosaminylphosphatidyl-
inositol (7) and the conserved GPI
anchor structure (8) as positive con-
trols. Although PI-PLC cleaved 3 and 4
with about the same efficiency as 7 and
8, this enzyme did not accept 5 or 6.
GPI-PLD accepted both the l-chiro- (3
and 4) and the d-chiro- (5 and 6) glyco-
sylinositolphosphoinositides. Therefore,
IPGs containing l-chiro-inositol only
are expected to be released from chiro-
inositol-containing GPIs if the cleavage
is effected by a PI-PLC, whereas GPI-
PLD cleavage could result in both l-
(IPGs), reported to be putative insulin
mediators, was studied by examination
of the substrate specificities of the
phosphatidylinositol-specific phospholi-
pase C (PI-PLC) and the glycosylphos-
phatidylinositol-specific
phospholi-
pase D (GPI-PLD) by using a series of
synthetic d-and l-chiro-glycosylphos-
phoinositides. 3-O-a-d-Glucosaminyl-
(3) and -galactosaminyl-2-phosphatid-
yl-l-chiro-inositol (4), which show the
maximum stereochemical similarity to
the 6-O-a-d-glucosaminylphosphatidyl-
inositol pseudodisaccharide motifs of
GPI anchors, were synthesized and
asymmetrically incorporated into phos-
Keywords: glycolipids · glycophos-
phoinositides · inositols · liposomes ·
phospholipases
chiro-and
d-chiro-inositol-containing
IPGs.
Introduction
Ins1-OPO3–lipid, which may appear in forms modified by
the presence of branching groups at specific positions.[2]
Other GPIs serve to attach extracellular non-protein glyco-
conjugates to cell membranes, particularly in the case of
protozoa. These non-protein GPI anchors share the
common core structure Mana(1!4)GlcNH2a(1!6)-myo-
Ins1-OPO3–lipid.[3] In addition, the existence of free GPIs
(GPI-like molecules without anchoring functions, which do
not seem to be derived from hydrolysis of GPI-anchored
proteins or glycoconjugates) has also been described.[4] The
structures of free GPIs are much less defined than those of
the GPI anchors. It has been postulated that they may be in-
volved in an intracellular signaling system that may operate
for insulin and for several growth factors and cytokines. Ac-
cording to this proposal, free GPIs at the cell surface would
be cleaved by specific phospholipases (PLs) to give water-
soluble second messengers that have been termed inositol-
phosphoglycans (IPGs).[5] Two different families of IPGs
have been proposed on the basis of chemical composition
and biological activity data: type A, which inhibit cAMP-de-
The biology and chemistry of glycosylphosphatidylinositols
(GPIs) have received a great deal of attention.[1] Some GPIs
serve to attach proteins to the outer faces of cellular mem-
branes through covalent linkages. These protein GPI an-
chors present the conserved core structure NH2EtOPO3-
6Mana(1!2)Mana(1!6)Mana(1!4)GlcNH2a(1!6)-myo-
[a] Dr. J. B. Bonilla, Prof. M. Martín-Lomas
Grupo de Carbohidratos, Instituto de Investigaciones Químicas
CSIC, AmØrico Vespucio 49, 41092 Sevilla (Spain)
Fax : (+34)95-4460-565
E-mail: manuel.martin-lomas@iiq.csic.es
[b] Dr. M. B. Cid
Departamento de Química Orgµnica
Universidad Autónoma de Madrid, 28094 Cantoblanco (Spain)
[c] Dr. F.-X. Contreras, Prof. F. M. GoÇi
Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU)
and Departamento de Bioquímica
Universidad del País Vasco, Aptdo. 644, 48080 Bilbao (Spain)
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pendent protein kinase (PKA) and contain myo-inositol or
1d-chiro-inositol and glucosamine,[6] and type P, which acti-
vate pyruvate dehydrogenase phosphatase (PDHPase) and
contain 1d-chiro-inositol and galactosamine.[7] Neither the
IPG mediators nor the free GPI precursors have been fully
characterized, with the sole exception of a biologically
active IPG isolated from beef liver, the structure of which
has recently been elucidated to be GalNH2b(1!4)-d-chiro-
Ins-3Me.[8] According to these results, the GPI precursors of
these IPG mediators would not share the minimum consen-
sus structure of GPI anchors (Mana(1!4)GlcNH2a(1!6)-
myo-Ins), although they may at least contain a GlcNH2 (or
GalNH2) 1!x-myo-(or chiro-)Ins pseudodisaccharide struc-
tural motif.
branes. In biomembranes, glycolipids and cholesterol (Ch)
assemble in microdomains (“rafts”) that sometimes cluster
and self-stabilize in flask-shaped invaginations of the cell
plasma membrane, known as caveolae.[19] These microdo-
mains have an asymmetric disposition in which the glyco-
lipids and GPI-linked proteins are located on the outer leaf-
let.[19a,c,20,21]
Some of us have described the preparation of large unila-
mellar vesicles (LUVs) of approximately 100 nm in diame-
ter, incorporating natural GPIs preferentially located in the
outer monolayer. It was shown that these GPIs could be
cleaved by specific PLs.[22] Because of the structural and
functional importance of rafts and caveolae, these synthetic
vesicles with asymmetric distributions of glycophosphoinosi-
tides constitute a highly attractive and relatively simple arti-
ficial system with which to study PL-mediated enzymatic
cleavage of GPI-like structures. By using this system, we
have investigated the enzymatic hydrolysis of some synthetic
d-and l-chiro-inositol phosphoinositides as part of a pro-
gram on the synthesis, structures, and biological activities of
IPG-like molecules as putative insulin mediators.[23] In the
context of this program, we have previously synthesized a
variety of IPG-like structures and have explored their bio-
logical activities as potential insulin mimetics. The main aim
of the current work was to investigate whether PI-PLC or
GPI-PLD could accept unusual chiro-glycophosphoinosi-
tides in membrane-like environments to produce IPG-like
structures capable of displaying insulin mimetic activity.
Although the presence of chiro-inositol in GPIs was first
reported in 1985,[9] no PLs specifically involved in the cleav-
age of chiro-inositol-containing GPIs are currently known.
Phosphatidylinositol-specific (PI-specific) PLs (PI-PLs) con-
stitute a broad family of proteins of various sizes and se-
quences. Some of them have been used as tools for the
structural study of GPIs; these include PI-PLC from Bacil-
lus cereus,[10a,b] GPI-PLC from Trypanosoma brucei,[10b,c] and
mammalian bovine GPI-PLD.[11] PI-PLCs play a central role
in signal transduction, cleaving specialized PIs to form
second messengers.[12] Bacterial PI-PLCs cleave GPIs if no
acyl group is present at the 2-position in the myo-inositol
unit, but show no hydrolytic activity towards phosphorylated
forms of PI, phosphatidylcholine (PtdCho), or phosphatidyl-
ethanolamine (PtdEth).[13] GPI-PLC from Tripanosoma
cleaves GPIs that do not bear an acyl group at the 2-posi-
tion in the myo-inositol ring.[10b,14] It could also cleave PI,
but only if presented in detergent-based micelles.[15] Mam-
malian GPI-PLD does not exhibit activity against PI or its
phosphorylated forms and seems to be specific for GPIs
with or without acyl groups at the 2-position in the myo-ino-
sitol moiety.[16] Importantly, in the only case in which the en-
zymatic hydrolysis of pure synthetic chiro-inositol-contain-
ing lipids was investigated, it was shown that bacterial PI-
PLC from Bacillus thuringiensis cleaves 1l-chiro-PI at a rate
10ꢀ3 times that attained with the natural substrate (PI), and
in addition, that synthetic 1d-chiro-PI is resistant to PI-
PLC.[17] This result suggests that the natural chiro-inositol-
containing GPIs should have the 1l configuration rather
than the 1d configuration if the chiro-inositol-containing
IPGs are produced after PI-PLC cleavage of a chiro-inosi-
tol-containing free GPI. In addition, it has also been shown
that isomerization of myo-inositol to d-chiro-inositol may
occur during acidic hydrolysis of GPIs.[18] Taken together,
these results seem to suggest that the detection of d-chiro-
inositol in GPI structures could be due either to incorrect
assignment or to isomerization.
Results and Discussion
As indicated above, the only available data on the enzymat-
ic hydrolysis of chiro-inositol-containing lipids were report-
ed more than ten years ago and involved bacterial PI-
PLC.[17] On stereochemical grounds (Figure 1), it was pre-
dicted that PI-PLC would be more likely to accept the 1l-
chiro-PI (1) than the 1d-chiro-PI diastereomer (2), and it
was found experimentally that 1 was accepted at a reduced
rate and 2 was not a substrate.[17] In the absence of structural
information on chiro-inositol-containing phospholipids,
these structures (2-phosphatidyl-chiro-inositols) were select-
ed on the basis of the greatest stereochemical similarity to
myo-inositol-containing phospholipids.
Also as a result of the absence of structural information,
we have previously synthesized different types of IPG-like
molecules containing a variety of GlcNH2 and GalNH2 a-
and b(1!x)-myo- , d- -chiro-, and -l-chiro-inositol structural
motifs.[23] These include GlcNH2-and GalNH 2a(1!3)-l-
chiro-inositol (I and II) and GlcNH2-and GalNH a(1!2)-
2
d-chiro-inositol (III and IV).[23i] The former pair would
permit access to 3-O-glycosaminyl-2-phosphatidyl-l-chiro-
inositols (such as 3 and 4), showing the maximum stereo-
chemical similarity to the GlcNH2a(1!6)-myo-Ins structural
motif of the GPI anchors, and the latter pair would allow 2-
O-glycosaminyl-1-phosphatidyl-d-chiro-inositols (such as 5
and 6)—differing from the GPI anchor motif only in the ori-
Under physiological conditions, PLs recognize their sub-
strates in the form of bilayers; therefore, studies with these
enzymes must be carried out on detergent-based micelles or
artificial lipidic vesicles (liposomes) rather than on lipid
monomers. In natural systems, the enzymatic cleavage of
GPIs takes place during attachment to biological mem-
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Phospholipase Cleavage of d-and l-chiro-Glycosylphosphoinositides
FULL PAPER
entation of the phosphatidyl
moiety—to be obtained. We
synthesized these glycolipids
and studied their behavior
when asymmetrically incorpo-
rated in a liposomal membrane,
versus PI-PLC from Bacillus
cereus and GPI-PLD from
bovine serum. For comparison,
we similarly studied the behav-
ior of the naturally occurring
myo-inositol glycophosphoino-
sitide structures 7 and 8 under
the same conditions, as positive
controls, together with that of
the rearranged l-chiro-inositol
glycophosphoinositides
9 and
10, obtained from secondary in-
termediates in the synthesis of
3 and 4, as predictable negative
substrates.
Figure 1. Structural relationship of 2-phosphatidyl-l-chiro-inositol (1), 2-phosphatidyl-d-chiro-inositol (2), PI,
and GPI anchors.
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M. Martín-Lomas et al.
We had already developed a short synthesis of l-chiro-
inositol-containing pseudodisaccharides 11 and 12
pH 7.5, in the presence of 0.1% bovine serum albumin
(BSA) with continuous stirring. The final concentrations of
PI-PLC from Bacillus cereus and bovine serum GPI-PLD
were 0.16 and 0.5 UmLꢀ1, respectively. Aliquots were re-
moved from the reaction mixture at regular intervals and
extracted with chloroform/methanol/hydrochloric acid
(66:33:1).[22a] The course of the enzymatic cleavage was fol-
lowed by evaluation of aminosugar free-amino groups in the
aqueous phase by fluorescamine assay,[30] which has previ-
ously been utilized for the quantitative evaluation of lyso-
phosphatidylethanolamine in trace amounts and has proven
to be a very sensitive and reliable method for the nanomo-
lar-range quantification of amine-containing lipids.[31] In our
hands, this was a more reliable and sensitive assay than
phosphorus determination.[32] The time-courses of PI-PLC
and GPI-PLD hydrolysis for selected synthetic glycophos-
phoinositides are shown in Figures 2 and 3, and more com-
prehensive data are collected in Table 1. The plots in
Figure 2 show that PI-PLC accepts a “natural” lipid (8) as a
substrate, whereas the “rearranged” molecule 9 is inactive
in this respect. The synthetic l-chiro-glycoinositol phospho-
lipid 3 permits an intermediate enzyme activity. The data in
Table 1 demonstrate that the “natural” substrates 7 and 8,
together with the l-chiro-compound 4, produce the highest
rates and greatest extents of hydrolysis. The d-chiro 5 and 6
and the “rearranged” lipids 9 and 10 are totally inactive as
PI-PLC substrates. The quantitative data in Table 1 also con-
firm that compound 3 is a PI-PLC substrate, but, under our
conditions, not as good a one as 7 or 8.
(Scheme 1) as key intermediates for the preparation of IPG-
like molecules with the structural motifs GlcNH2(1!3)-l-
chiro-Ins and GalNH2(1!3)-l-chiro-Ins, respectively.[23i] Al-
though protecting group manipulation was required to
obtain the suitably protected derivatives 19 and 20, these in-
termediates were chosen as starting materials for the synthe-
sis of 3 and 4. Attempted p-methoxybenzylation or allyla-
tion of 11 and 12 under mild acidic conditions[24] and
p-methoxybenzylation with p-methoxybenzyl trichloroacet-
imidate[25] did not give satisfactory results. Allylation of 11
and 12 with methyl allylcarbonate/Pd[26] afforded the rear-
ranged 1-O-allyl derivatives 13 and 14, respectively. Howev-
er, methoxymethylation in the presence of iPr2EtN[17] afford-
ed the 2-O-methoxymethyl derivatives 15 and 16 in good
yields. Debenzoylation of 15 and 16 followed by convention-
al benzylation gave 17 and 18, from which the methoxy-
methyl groups were removed to yield 19 and 20, respective-
ly. Treatment of 19 and 20 with benzyl-1,2-di-O-miristoyl-
sn-glycero-N,N-diisopropyl phosphoramidite[27] followed by
m-chloroperoxybenzoic acid (m-CPBA) under the usual
conditions gave the fully protected glycolipids 21 and 22,
which were subjected to hydrogenation in an AcOEt/THF/
EtOH/H2O solvent mixture and in the presence of 10% Pd/
C catalyst to yield the l-chiro-inositol glycophosphoinosi-
tides 3 and 4.
The 1-O-allyl derivatives 13 and 14 were debenzoylated
to yield 23 and 24, which were benzylated to give 25 and 26,
and these were in turn deallylated[28] to afford the key inter-
mediates 27 and 28, respectively. These compounds were
treated in the same way as 19 and 20 to give 29 and 30. The
free glycolipids 9 and 10 were obtained after hydrogenation
of 29 and 30, as described for 21 and 22.
Table 1. Initial rates and maximum extent of hydrolysis of compounds 3–
10 by PI-PLC and GPI-PLD. LUVs composition: PtdCho/PtEth/Ch
(2:1:1 mole ratio). Substrate conc.: 0.03 mm. Enzyme units: PI-PLC
(0.16 UmLꢀ1), GPI-PLD (0.5 UmLꢀ1).
We had also already reported the synthesis of the d-chiro-
inositol-containing pseudodisaccharides 31 and 32.[23i] These
were transformed into glycophosphoinositides 5 and 6, re-
spectively (Scheme 2), via the intermediates 33 and 34, by
using the same chemistry described above for the prepara-
tion of 3 and 4 from 21 and 22.
PI-PLC
GPI-PLD
Compound
Rate
Extent[a]
[nmol]
Rate
Extent[a]
[nmol]
[mol”10ꢀ10 sꢀ1
]
[mol”10ꢀ10 sꢀ1
]
3
4
5
6
7
8
9
10
4.5ꢁ0.3
8.2
<0.1
15ꢁ0.0
4.2ꢁ0.5
2.0
1.9
27ꢁ0.03
16
23
<1
<1
29
24ꢁ0.9
22ꢁ3.2
25ꢁ0.6
28ꢁ0.0
4.2ꢁ1.0
3.9ꢁ0.9
<0.1
2.1
The synthesis of the myo-inositol glycophosphoinositide 7
through regioselective phosphorylation of diol 35 by phos-
phite–phosphonium salt methodology[29] was reported previ-
ously by us.[23b] As an alternative, 7 was also prepared from
35 (Scheme 3) by dibutyltin-mediated allylation[30] to afford
36 (68%) and 37 (20%), benzylation of 36 (!38), deallyla-
tion[28] (!39), phosphorylation with benzyl 1,2-dimyristoyl-
sn-glycero-N,N-diisopropyl phosphoramidite[27] (!40), and
final hydrogenation, as above. Finally, the conserved GPI
structure 8 was synthesized as reported recently by us.[23m]
Each of the synthetic glycoinositol phospholipids 3–8 was
asymmetrically incorporated (0.03 mm final concentration)
into LUVs consisting of phosphatidylcholine(PtdCho)/egg
phosphatidylethanolamine(PtdEth)/cholesterol(Ch) (molar
ratio 2:1:1) at 0.3 mm total lipid concentration[22] and treated
with PI-PLC and GPI-PLD. Assays of enzymatic hydrolysis
were conducted at 398C in 10 mm HEPES, 50 mm NaCl
8.9ꢁ0.3
4.7ꢁ0.3
9.6ꢁ0.1
0.93
8.2
29ꢁ0.0
<0.1
<0.1
<1
<1
0.48
[a] Nanomoles of substrate processed after apparent equilibrium was
reached (15 min).
Data for GPI-PLD are given in Figure 3, and also in
Table 1. All eight lipids tested are accepted as substrates by
GPI-PLD, but the hydrolysis rates and extents differ vastly.
Figure 3 shows representative time-courses for the hydroly-
ses of a good (7) and a poor (10) substrate. The highest ini-
tial rates under our conditions are found for the “natural”
lipid 8. This is followed by a group of molecules that are hy-
drolyzed at lower rates, such as the “natural” lipid 7, the l-
chiro-compounds 3 and 4, and the d-chiro 5 and 6. The “re-
arranged” molecules 9 and 10 are distinctly poorer GPI-
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Phospholipase Cleavage of d-and l-chiro-Glycosylphosphoinositides
FULL PAPER
Scheme 1. Abbreviations: [Pd2(dba)3]: tris(dibenzylideneacetone)dipalladium; dppb: 1,4-bis(diphenylphosphino)butane; MOM: CH3OCH2. Reagents and
conditions: a) AllCO2Me (”2.0), [Pd2(dba)3] (”0.05), dppb (”0.2), THF, 808C, 2 h, 80%; b) MOMCl (”52), iPr2EtN, DMF, 52 h; c) MeONa (”1.0),
MeOH, 16 h, 100%; d) BnBr (”8.0), NaH (”6.0), DMF, overnight; e) PhSH (”1.2), BF3·Et2O (”1.0), CH2Cl2, 2 h; f) 1,2-di-O-myristoyl-sn-glycero-3-yl
benzyl N,N-diisopropyl phosphoramidite (”2.0), 1H-tetrazole (”2.2), CH2Cl2, RT, 2 h; g) m-CPBA (”1.0), CH2Cl2, 20 min; h) H2, Pd/C (”3.0), AcOEt/
THF/EtOH/H2O 2:1:1:0.1, RT, 6 h, Sephadex LH-20; i) [{Ir(cod)Ph2PMe}2]PF6, THF and then NBS (”1.5), H2O, 72%.
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1517

M. Martín-Lomas et al.
panosoma.[37] GPI-PLD is also active against 5 and 6 in spite
of the axial orientations of the phosphatidyl moieties in
these glycophosphoinositides (Table 1).
Conclusion
After almost twenty years of intensive chemical and biologi-
cal research, the structures of chiro-inositol-containing IPGs
proposed as putative insulin mediators remain controversial.
It has been postulated that these IPGs are generated after
PL cleavage of free GPIs at the cell surface, but neither the
structures of these GPIs nor the nature of the specific PL in-
volved in GPI hydrolysis is presently known. These issues
have now been addressed through the synthesis of several
d-and l-chiro-glycophosphoinositides and investigation of
their enzymatic hydrolysis by PLs in membrane-like envi-
ronments. Here we demonstrate for the first time that 3-O-
a-d-glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-l-
chiro-inositol (4)—showing the maximum stereochemical
similarity to the 6-O-a-d-glucosaminylphosphatidylinositol
structural motif of the GPI anchors—and 2-O-a-d-glucosa-
minyl-( 5) and galactosaminyl-1-phosphatidyl-d-chiro-inosi-
tol (6)—which differ from the corresponding structural
motif of the GPI anchors only in the orientation of the PI
moiety—can be synthesized in a straightforward manner. To
the best of our knowledge, these compounds constitute the
first synthetic d-and l-chiro-glycosylphosphoinositides ever
reported in the literature. These compounds, as well as the
myo-glycophosphoinositide (7) and the conserved GPI
structure (8), can be asymmetrically incorporated into
LUVs to mimic the natural membrane environment. These
liposomal constructs constitute a convenient system for
studying the cleavage of synthetic phosphoinositide mole-
cules by PLs, which is necessary to provide insight into the
structure–activity relationship of these important enzymes.
Investigation of the cleavage of compounds 2–8, asymmetri-
cally incorporated into these LUVs, by a bacterial PI-PLC
and a mammalian GPI-PLD has provided interesting results.
The configuration of the chiro-inositol unit in a chiro-inosi-
tol-containing naturally occurring IPG could be either d or
l, depending on the nature of the PL responsible for IPG
Scheme 2. Reagents and conditions: a) 1,2-di-O-myristoyl-sn-glycero-3-yl
benzyl N,N-diisopropyl phosphoramidite (”2.0), 1H-tetrazole (”2.2),
CH2Cl2, RT, 2 h; b) m-CPBA (”1.0), CH2Cl2, 20 min; c) H2, Pd/C (”3.0),
AcOEt/THF/EtOH/H2O 2:1:1:0.1, RT, 6 h, Sephadex LH-20.
PLD substrates. Overall, both PI-PLC and GPI-PLD show a
preference for the “natural” glycophosphoinositides, fol-
lowed by the l-chiro molecules. The “rearranged” lipids 9
and 10 are poor or very poor substrates for both enzymes.
The d-chiro-glycophosphoinositides 5 and 6 are hydrolyzed,
albeit at a low rate, by GPI-PLD, but cannot be processed
by PI-PLC.
The observation that bacterial PI-PLC hydrolyzes both 3-
O-glucosaminyl- (3) and 3-O-galactosaminyl-2-phosphatidyl-
l-chiro-inositol (4) is in agreement with previous results on
the hydrolysis of 2-O-phosphatidyl-l-chiro-inositol (1).[17]
This enzyme cleaves neither the 2-O-glycosaminyl-1-phos-
phatidyl-d-chiro-inositols 5 and 6 (Table 1) nor the rear-
ranged glycolipids 9 and 10 (Figures 2 and 3), in which the
phosphatidyl moiety is axially oriented. However, in con-
trast to previous findings[17] that bacterial PLC cleaved 1 at
a rate 10ꢀ3 times that attained with the natural substrate PI,
this enzyme hydrolyzes 3 and 4 with efficiency similar to
that for the myo-inositol lipid 7 and the GPI-anchor struc-
ture 8 (Figure 2 and Table 1), in which the glycophosphoino-
sitides presented are incorporated asymmetrically in a lipo-
somal membrane.
generation. Whereas
a 3-O-glycosyl-2-O-phosphatidyl-l-
chiro-inositol structural motif seems to be required for PI-
PLC cleavage, mammalian GPI-PLD accepts 2-O-glycosyl-
1-phosphatidyl-d-chiro-inositols, despite the axial orienta-
tions of the phosphatidyl moieties in these compounds.
Therefore, until new PI-PLs or GPI-PLs specific for chiro-
inositol-containing PIs or GPIs are discovered, the uncer-
tainty as to whether GPI precursors of IPG mediators may
be composed of either d-or l-chiro-inositol will remain. On
the other hand, neither of the enzymes utilized in this study
accepts exotic GPI structures such as 9 and 10, and for none
of them does the nature of the glycan chain beyond the glu-
cosamine unit linked to the inositol ring seem to play a
major role in enzymatic cleavage.
Both bacterial PI-PLC and mammalian GPI-PLD accept
both 7 and 8, but this is not the case for GPI-PLC from Try-
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Phospholipase Cleavage of d-and l-chiro-Glycosylphosphoinositides
FULL PAPER
Scheme 3. Reagents and conditions: a) Bn2SnO (”1.2), toluene, reflux, 24 h; b) AllBr, TBAI (”1.1), reflux, 2 h, 36 (68%), 37 (20%); c) BnBr (”8.0),
NaH (”6.0), DMF, overnight, 68%; d) [{Ir(cod)Ph2PMe}2]PF6, THF and then NBS (”1.5), H2O, 1 h 30 min, 72%; e) 1,2-di-O-myristoyl-sn-glycero-3-yl
benzyl N,N-diisopropyl phosphoramidite (”2.0), 1H-tetrazole (”2.2), CH2Cl2, RT, 2 h; f) m-CPBA, CH2Cl2, 20 min, 82%; g) H2, Pd/C (”3.0), AcOEt/
THF/EtOH/H2O, 2:1:1:0.1, RT, 16 h, Sephadex LH-20, 98%.
Figure 2. Time-courses of the enzymatic hydrolysis of compounds 3, 8,
Figure 3. Time-courses of the enzymatic hydrolysis of compounds 7 and
and 9, asymmetrically incorporated into LUVs, by PI-PLC from Bacillus
cereus under the experimental conditions indicated in Table 1.
10, asymmetrically incorporated into LUVs, by GPI-PLD from bovine
serum under the experimental conditions indicated in Table 1.
solutions were saturated unless otherwise stated. All reactions were con-
ducted under dry argon in oven-dried glassware and in freshly distilled
and dried solvents unless otherwise stated. Purification by flash chroma-
Experimental Section
tography was performed by using Merck 60 silica gel (15–200 and 230–
400 mesh) and eluents are given as volume ratios (v/v). Preparative thin-
layer chromatography (PLC) was performed by using Merck silica gel
(60 F254). Preparative gel chromatography was performed by using Amer-
sham Bioscience Sephadex LH-20. Analytical thin-layer chromatography
(TLC) was performed by using Merck silica gel (60 F254) with detection
by UV light (l=254 nm) and heating with phosphomolybdic acid/EtOH
or Ce(SO4)2/phosphomolybdic acid/H2SO4/H2O, and with Mo/MoO4/
General methods: All organic solvents used were dried by established
procedures.[33] Dichloromethane was distilled from calcium hydride,
whilst hexane, diethyl ether, and THF were distilled from sodium/benzo-
phenone. iPr2EtN was distilled from ninhydrin and KOH, whilst DMF
and MeOH were dried from molecular sieves (4 Š) unless otherwise
stated (for the preparation of 15 and 16, DMF was distilled from TsCl
and BaO). Molecular sieves (4 Š, powdered) were predried in an oven
and then activated for 10 min under vacuum at 5008C. All aqueous (aq)
Chem. Eur. J. 2006, 12, 1513 – 1528
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1519

M. Martín-Lomas et al.
H2SO4 for glycophospholipids 3–7, 9, and 10. Chemical shifts are given in
ppm and coupling constants are reported in Hz. Resonance signals were
assigned with the aid of 2D spectra (COSY, COSY-dqf, HMQC, HSQC,
TOCSY, NOESY, and ROESY). 1H NMR and 13C NMR were recorded
at 298 K by using Bruker Avance DRX 500, DRX 400, and DPX 300
spectrometers with TMS as internal reference signal. Microanalyses were
determined by using a Leco CHNS-932 instrument, high-resolution mass
spectra were recorded by using a Micromass Autospec apparatus, optical
rotations were measured by using a Perkin–Elmer 341 polarimeter, and
pH was measured by using a Cole Parmer apparatus.
(C5’), 68.9 (C6), 67.5 (C6’), 60.0 ppm (C2’); FAB HRMS: m/z calcd for
[C64H59N3O14+Na]+: 1116.3895; found: 1116.3965.
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-glucopyranosyl-a-(1!3)-2-methoxy-
methyl-1,4,5,6-tetra-O-benzoyl-l-chiro-inositol (15): MOMCl (MOM=
CH3OCH2) (866 mL, 11.48 mmol, 52 equiv) was added dropwise under Ar
to a solution of compound 11 (242 mg, 0.23 mmol, 1 equiv) and iPr2EtN
(4 mL) in dry DMF (0.3 mL). After the reaction mixture had been stirred
at RT for 52 h, NH4OH (1 mL) was added and the mixture was washed
successively with a solution of saturated NH4Cl, H2O, and brine. The or-
ganic layer was dried (MgSO4) and concentrated, and the crude product
was purified by flash chromatography (n-hexane/AcOEt 2:1) to give 15
as a syrup (250 mg, 0.23 mmol, 99%). [a]2D0 =+46 (c=0.51 in CHCl3); Rf
(n-hexane/AcOEt 2:1): 0.43; 1H NMR (300 MHz, CDCl3, 258C, TMS):
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-glucopyranosyl-a-(1!3)-1-O-allyl-
2,4,5,6-tetra-O-benzoyl-l-chiro-inositol (13): A solution of [Pd2(dba)3]
(dba=tris(dibenzylideneacetone)dipalladium)
(8 mg,
0.009 mmol,
d=8.16 (d, 3J(H,H)=7.5 Hz, 2Hortho
;
Bz), 8.08 (d, 3J(H,H)=7.5 Hz,
0.05 equiv) and 1,4-bis(diphenylphosphino)butane (dppb) (16 mg,
0.04 mmol, 0.2 equiv) in dry THF (1 mL) was added under argon to a so-
lution of 11 (196 mg, 0.19 mmol, 1 equiv) and AllCO2Me (42 mL,
0.1 mmol, 2 equiv) in dry THF (9 mL). After the solution had been stir-
red at 808C for 2 h, the solvent was evaporated and the crude product
was purified by column chromatography (n-hexane/AcOEt 8:1) to give
13 as a white foam (161 mg, 0.15 mmol, 80%). [a]2D0 =+32 (c=0.4 in
CHCl3); Rf (n-hexane/AcOEt 3:1): 0.31; 1H NMR (500 MHz, CDCl3,
258C, TMS): d=8.11 (d, 3J(H,H)=6.5 Hz, 4Hortho; Bz), 7.92 (d, 3J-
(H,H)=8.0 Hz, 2Hortho; Bz), 7.82 (d, 3J(H,H)=7.5 Hz, 2Hortho; Bz), 7.62
(t, 3J(H,H)=7.5 Hz, 1Hpara; Bz), 7.58 (t, 3J(H,H)=7.5 Hz, 1Hpara; Bz),
2Hortho; Bz), 7.93 (d, 3J(H,H)=7.5 Hz, 2Hortho; Bz), 7.76 (d, 3J(H,H)=
7.8 Hz, 2Hortho; Bz), 7.66 (t, 3J(H,H)=7.2 Hz, 1Hpara; Bz), 7.63 (t, 3J-
3
(H,H)=6.9 Hz, 1Hpara; Bz), 7.53 (t, J(H,H)=7.5 Hz, 2Hmeta; Bz), 7.51 (t,
3J(H,H)=7.5 Hz, 2Hmeta; Bz), 7.42 (t, 3J(H,H)=6.9 Hz, 1Hpara; Bz), 7.40
(t, J=7.5 Hz, 1Hpara; Bz), 7.36–7.18 (m, 4H; Bz, 13H; Bn), 6.92 (m, 2H;
Bn), 6.05 (t, 3J(H,H)=9.3 Hz, 1H; H4), 5.99 (t, 3J(H,H)=3.8 Hz, 1H;
H6), 5.89 (t, 3J(H,H)=3.8 Hz, 1H; H1), 5.81 (dd, 3J(H5,H4)=9.3 Hz, 3J-
3
3
(H5,H6)=3.8 Hz, 1H; H5), 5.52 (d, J(H,H)=3.6 Hz, 1H; H1’), 4.96 (d, J-
(H,H)=7.0 Hz, 1H; CH3OCH2), 4.79 (app.s, 2H; CH2Ph), 4.78 (d, 3J-
3
ꢀ
(H,H)=7.0 Hz, 1H; CH3OCH2), 4.62 (d, J(H,H)=11.1 Hz, 1H; CH
7.52 (t, 3J(H,H)=7.5 Hz, 2Hmeta; Bz), 7.45 (t, 3J(H,H)=6.5 Hz, 2Hmeta
;
Ph), 4.52 (t, 3J(H,H)=9.3 Hz, 1H; H3), 4.46 (d, 3J(H,H)=11.7 Hz, 1H;
Bz), 7.41 (t, 3J(H,H)=6.5 Hz, 2Hpara; Bz), 7.29–7.18 (m, 4H; Bz, 11H;
Bn), 7.12 (m, 2H; Bn), 6.97 (m, 2H; Bn), 6.08 (t, 3J(H,H)=9.5 Hz, 1H;
H4), 5.98–5.85 (m, 2H; H5, CH=CH2), 5.91 (app.s, 1H; H6), 5.80 (app.d,
3J(H,H)=10.0 Hz, 1H; H2), 5.34 (app.d, 3J(H,H)=17.0 Hz, 1H; CH=
CH Ph), 4.43 (dd, 3J(H2,H3)=9.3 Hz, 3J(H2,H1)=3.3 Hz, 1H; H2), 4.27
ꢀ
3
3
ꢀ
ꢀ
(d, J(H,H)=11.1 Hz, 1H; CH Ph), 4.20 (d, J(H,H)=11.7 Hz, 1H; CH
Ph) 3.83 (m, 1H; H3’), 3.62 (m, 2H; H4’, H5’), 3.39 (dd, 3J(H2’,H3’)=
10.5 Hz, 3J(H2’,H1’)=3.6 Hz, 1H; H2’), 3.31 (s, 3H; CH3OCH2), 3.18 (m,
1H; H6a’), 3.02 ppm (m, 1H; H6b’); 13C NMR (125 MHz, CDCl3, 258C,
TMS): d=165.8, 165.6, 165.2, 165.0 (4”COPh), 138.4, 138.1, 137.8 (3C;
3
3
CH2), 5.29 (d, J(H,H)=2.8 Hz, 1H; H1’), 5.20 (app.d, J(H,H)=10.5 Hz,
ꢀ
1H; CH=CH2), 4.74–4.58 (m, 5H; 4”CH Ph, H3), 4.34–4.18 (m, 3H;
CH Ph, CH2 CH=CH2), 4.20 (s, 1H; H1), 4.05 (d, 3J(H,H)=12.0 Hz,
ꢀ
ꢀ
Bn), 133.8, 133.4, 133.3 (4CHpara
; Bz), 130.2, 130.1, 129.94, 129.87
1H; CH Ph), 3.81 (brt, 3J(H,H)=10.0 Hz, 1H; H3’), 3.57 (m, 2H; H4’,
ꢀ
(8CHortho; Bz), 129.5–127.5 (23”CH, 4”C; Bz, Bn), 98.5 (CH2OCH3,
C1’), 80.0 (C3’), 77.9 (C2), 76.5 (C4’), 74.5 (CH2Ph), 73.6 (C3), 71.3, 70.7
(2”CH2Ph), 70.6 (C4, C5’), 70.3 (C5, C1), 68.3 (C6), 67.6 (C6’), 63.7 (C2’),
56.5 ppm (CH2OCH3); FAB HRMS: m/z calcd for [C63H59N3O15+Na]+:
1120.3844; found: 1120.3969.
H5’), 3.19 (dd, 3J(H2’,H3’)=10.0 Hz, 3J(H2’,H1’)=2.8 Hz, 1H; H2’),
2.99 ppm (m, 2H; H6a’, H6b’); 13C NMR (125 MHz, CDCl3, 258C, TMS):
d=165.7, 165.6, 165.5, 165.4 (4”COPh), 138.3, 138.0, 137.6 (3C; Bn),
133.7, 133.4, 133.23, 133.16 (4CHpara; Bz, CH=CH2), 130.0, 129.8, 129.6
(8”CHortho; Bz), 129.0–127.3 (23”CH; Bz, Bn), 118.7 (CH=CH2), 99.2
(C1’), 80.1 (C3’), 77.6 (C4’), 76.5 (C3), 75.4 (CH2Ph), 74.7 (C1), 74.4
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-galactopyranosyl-a-(1!3)-2-meth-
oxymethyl-1,4,5,6-tetra-O-benzoyl-l-chiro-inositol (16): Compound 16
was obtained as a colorless syrup (50 mg, 0.045 mmol, 58%, no optimized
yield) from 12 (83 mg, 0.079 mmol, 1 equiv) by the same experimental
procedure as that used in the preparation of 15. Compound 16 was puri-
fied by flash chromatography (n-hexane/AcOEt 2:1). [a]2D0 =+47 (c=
0.32 in CHCl3); Rf (n-hexane/AcOEt 2:1): 0.40; 1H NMR (500 MHz,
CDCl3, 258C, TMS): d=8.12 (d, 3J(H,H)=7.5 Hz, 2Hortho; Bz), 8.06 (d,
ꢀ
(CH2Ph), 74.4 (C2, CH2Ph), 73.2 (CH2 CH=CH2), 71.4 (C5’), 71.2 (C4),
70.6 (C5), 68.8 (C6), 67.1 (C6’), 63.6 ppm (C2’); FAB HRMS: m/z calcd for
[C64H59N3O14+Na]+: 1116.3895; found: 1116.3992.
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-galactopyranosyl-a-(1!3)-1-O-
allyl-2,4,5,6-tetra-O-benzoyl-l-chiro-inositol (14): Compound 14 (161 mg,
0.15 mmol, 80%) was obtained as
a white foam from 12 (196 mg,
0.19 mmol) by the same experimental procedure as that used for the
preparation of compound 13. The product was purified by flash chroma-
tography (n-hexane/AcOEt 10:1); Rf (n-hexane/AcOEt 3:1): 0.23;
1H NMR (500 MHz, CDCl3, 258C, TMS): d=8.11 (d, 3J(H,H)=8.0 Hz,
2Hortho; Bz), 8.08 (d, 3J(H,H)=8.5 Hz, 2Hortho; Bz), 7.99 (d, 3J(H,H)=
7.5 Hz, 2Hortho; Bz), 7.79 (d, 2H; 3J(H,H)=7.5 Hz, 2Hortho; Bz), 7.63 (t,
3J(H,H)=7.5 Hz, 1Hpara; Bz), 7.58 (t, 3J(H,H)=7.5 Hz, 1Hpara; Bz), 7.52
(t, 3J(H,H)=8.0 Hz, 2Hmeta; Bz), 7.47 (t, 3J(H,H)=7.5 Hz, 2Hmeta; Bz),
7.32–7.21 (m, 7H; Bz, 8H; Bn), 7.15 (m, 3H; Bn), 7.07 (m, 4H; Bn), 6.08
(t, 3J(H,H)=10.0 Hz, 1H; H4), 5.96–5.89 (m, 2H; H6, CH=CH2), 5.86
(dd, 3J(H5,H4)=10.0 Hz, 3J(H5,H6)=3.5 Hz, 1H; H5), 5.80 (dd, 3J-
(H2,H3)=9.5 Hz, 3J(H2,H1)=3.0 Hz, 1H; H2), 5.32 (dd, 3J(H,H)=
17.0 Hz, 3J(H,H)=1.5 Hz, 1H; CH=CH2), 5.30 (d, 3J(H,H)=4.0 Hz, 1H;
3
3J(H,H)=7.5 Hz, 2Hortho; Bz), 8.00 (d, J(H,H)=7.5 Hz, 2Hortho; Bz), 7.78
(d, 3J(H,H)=7.0 Hz, 2Hortho; Bz), 7.63 (t, 3J(H,H)=7.5 Hz, 1Hpara; Bz),
7.60 (t, 3J(H,H)=7.5 Hz, 1Hpara; Bz), 7.50 (t, 3J(H,H)=7.5 Hz, 2Hmeta
;
Bz), 7.44 (t, 3J(H,H)=7.5 Hz, 2Hmeta; Bz), 7.42–7.18 (m, 6H; Bz, 13H;
Bn), 7.14 (m, 2H; Bn), 6.05 (t, 3J(H,H)=9.5 Hz, 1H; H4), 6.00 (app.s,
1H; H6), 5.91 (app.s, 1H; H1), 5.83 (dd, 3J(H5,H4)=9.5 Hz, 3J(H5,H6)=
3.5 Hz, 1H; H5), 5.53 (app.s, 1H; H1’), 4.89 (d, 3J(H,H)=7.0 Hz, 2H;
CH3OCH2), 4.77 (d, 3J(H,H)=7.0 Hz, 1H; CH3OCH2), 4.72 (d, 3J-
(H,H)=11.0 Hz, 1H; CH Ph), 4.57 (t, 3J(H,H)=9.5 Hz, 1H; H3), 4.53
ꢀ
3
(app.s, 2H; CH2Ph), 4.45 (dd, 3J(H2,H3)=9.5 Hz, J(H2,H1)=3.5 Hz, H2),
3
4.32 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.24 (d, 3J(H,H)=11.5 Hz, 1H;
ꢀ
3
3
ꢀ
ꢀ
CH Ph), 4.14 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 3.95 (app.t, J(H,H)=
7.0 Hz, 1H; H5’), 3.90 (dd, 3J(H2’,H3’)=10.5 Hz, 3J(H2’,H1’)=3.5 Hz, 1H;
H2’), 3.83 (dd, 3J(H3’,H4’)=10.5 Hz, 3J(H3’,H4’)=2.5 Hz, 1H; H3’), 3.62
(app.s, 1H; H4’), 3.18 (t, 3J(H,H)=8.5 Hz, 1H; H6a’), 3.31 ppm (m, 3H;
CH3OCH2, 1H; H6b’); 13C NMR (125 MHz, CDCl3, 258C, TMS): d=
165.7, 165.5, 165.2, 165.0 (4”COPh), 138.4, 138.0, 137.7 (3C; Bn), 133.8,
133.7, 133.5, 133.4 (4CHpara; Bz), 130.1, 130.0, 129.8, (8CHortho; Bz),
129.2–127.8 (23”CH, 4C; Bz, Bn), 98.3 (C1’), 98.0 (CH2OCH3), 77.4 (C2),
76.7 (C3’), 74.8 (CH2Ph), 74.1 (C3), 73.3, 73.2 (CH2Ph, C4’), 72.2 (CH2Ph),
70.9 (C4), 70.6 (C5), 70.0, 69.8 (C1, C5’), 68.2 (C6, C6’), 59.9 (C2’), 56.5 ppm
(CH2OCH3); FAB HRMS: m/z calcd for [C63H59N3O15+Na]+: 1120.3844;
found: 1120.3807.
H1’), 5.18 (dd, 3J(H,H)=10.5 Hz, 3J(H,H)=1.5 Hz, 1H; CH=CH2), 4.68
3
(t, 3J(H,H)=9.5 Hz, 1H; H3), 4.64 (d, J(H,H)=11.0 Hz, 1H; CH Ph),
ꢀ
3
ꢀ
4.54 (AB, 2H; CH2Ph), 4.29 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.25–
4.20 (m, 3H; H1, CH2 CH=CH2), 3.95 (AB, 2H; CH2Ph), 3.88–3.80 (m,
ꢀ
3H; H3’, H4’, H5’), 3.68 (dd, 3J(H2’,H3’)=10.5 Hz, 3J(H2’,H1’)=4.0 Hz; H2’),
3.36 (t, 3J(H,H)=8.5 Hz, 1H; H6a’), 3.00 ppm (m, 1H; H6b’); 13C NMR
(125 MHz, CDCl3, 258C, TMS): d=165.8, 165.7, 165.6, 165.5 (4”COPh),
138.4, 138.0, 137.9 (3C; Bn), 133.9–133.3 (CH=CH2, 4”CHpara; Bz,),
130.2–128.6 (31”CH, Bz, Bn), 118.8 (CH=CH2), 99.4 (C1’), 77.5–76.7
(C3’), 75.1 (CH2Ph), 74.8 (C3), 74.6 (C1), 74.0 (C2), 73.3, 73.22, 73.17
ꢀ
(CH2Ph, CH2 CH=CH2, C4’), 72.3 (CH2Ph), 71.5 (C4), 70.9 (C5), 69.7
1520
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 1513 – 1528

Phospholipase Cleavage of d-and l-chiro-Glycosylphosphoinositides
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-glucopyranosyl-a-(1!3)-2-methoxy-
FULL PAPER
1
[a]2D0 =+44 (c=0.65 in CHCl3); Rf (n-hexane/AcOEt 3:1): 0.19; H NMR
methyl-1,4,5,6-tetra-O-benzyl-l-chiro-inositol
(17):
MeONa/MeOH
(500 MHz, CDCl3, 258C, TMS): d=7.38–7.07 (m, 35H; Ph), 5.43 (d, 3J-
3
ꢀ
(1.0m, 0.23 mL, 0.23 mmol, 1 equiv) was added under Ar to a solution of
compound 15 (242 mg, 0.23 mmol, 1 equiv) in dried MeOH (6 mL). The
solution was stirred for 20 h and was then concentrated to dryness and
treated directly with NaH (74 mg, 1.84 mmol, 8 equiv) in dried DMF
(1.5 mL). After the mixture had been cooled to ꢀ158C in an ice/salt
bath, BnBr (218 mL, 1.84 mmol, 8 equiv) was added dropwise. The reac-
tion mixture was stirred overnight, NH4OH was then added, and the mix-
ture was washed successively with a solution of saturated NH4Cl, H2O,
and brine. The organic layer was dried (MgSO4) and evaporated, and the
crude product was purified by flash chromatography (n-hexane/AcOEt
10:1) to provide 17 as a syrup (181 mg, 0.17 mmol, 89%). [a]2D0 =+54
(c=0.38 in CHCl3); Rf (n-hexane/AcOEt 3:1): 0.28; 1H NMR (500 MHz,
(H,H)=3.5 Hz, 1H; H1’), 5.04 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.90,
3
3
ꢀ
4.86 (AB, J(H,H)=11.0 Hz, 2H; 2”CH Ph), 4.73 (d, J(H,H)=11.0 Hz,
1H; CH Ph), 4.70 (d, J(H,H)=12.5 Hz, 1H; CH Ph), 4.63 (d, 3J-
3
ꢀ
ꢀ
3
ꢀ
ꢀ
(H,H)=11.0 Hz, 1H; CH Ph), 4.584 (d, J(H,H)=11.5 Hz, 1H; CH
Ph), 4.578 (d, 3J(H,H)=12.0 Hz, 1H; CH Ph), 4.52 (d, 3J(H,H)=
ꢀ
3
ꢀ
ꢀ
12.5 Hz, 1H; CH Ph), 4.46 (d, J(H,H)=12.0 Hz, 1H; CH Ph), 4.44 (d,
3
3
3
ꢀ
J(H,H)=11.5 Hz, 1H; CH Ph), 4.41 (d, J(H,H) J(H,H)=11.0 Hz, 1H;
CH Ph), 4.36 (d, J(H,H)=12.0 Hz, 1H; CH Ph), 4.14 (d, 3J(H,H)=
3
ꢀ
ꢀ
12.0 Hz, CH Ph), 4.08 (m, 1H; H2), 3.99 (t, 3J(H,H)=10.0 Hz, 1H; H3’),
ꢀ
3.96 (app.d, 3J(H,H)=8.5 Hz, 1H; H5’), 3.84–3.77 (m, 3H; H1, H4, H5),
3.76–3.71 (m, 2H; H3, H4’), 3.69 (m, 1H; H6), 3.53 (dd, 3J(H2’,H3’)=
10.5 Hz, 3J(H2’,H1’)=3.5 Hz, 1H; H2’), 3.29 (d, 3J(H,H)=6.0 Hz, 1H;
C2OH), 3.26 (dd, 3J(H6a’,H6b’)=10.5 Hz, 3J(H6a’,H5’)=2.0 Hz; H6a’),
3.06 ppm (dd, 3J(H6b’,H6a’)=11.0 Hz, 3J(H6b’,H5’)=1.5 Hz; H6b’); 13C NMR
(125 MHz, CDCl3, 258C, TMS): d=138.8, 138.4, 138.2, 138.1, 137.99,
137.96 (7C; Ph), 128.5–127.3 (35”CH; Ph), 98.5 (C1’), 81.2, 80.4, 79.1 (C1,
C4, C5), 81.0 (C3’), 78.2, 77.5 (C4’, C3), 75.5, 75.2, 74.8, 73.9, 73.6, 73.4,
73.2, 73.0 (7”CH2Ph, C6), 72.7 (C2), 70.8 (C5’), 67.6 (C6’), 64.4 ppm (C2’);
FAB HRMS: m/z calcd for [C61H63N3O10+Na]+: 1020.4411; found:
1020.4455.
CDCl3, 258C, TMS): d=7.38–7.00 (m, 35H; Ph), 5.52 (d, 3J(H,H)=
3
ꢀ
4.0 Hz, 1H; H1’), 4.99 (d, J(H,H)=10.5 Hz, 1H; CH Ph), 4.87 (s, 2H;
CH2Ph), 4.82, 4.714 (2”d, 3J(H,H)=6.5 Hz, 2H; CH3OCH2), 4.711 (d,
3
3
ꢀ
ꢀ
J(H,H)=10.5 Hz, 1H; CH Ph), 4.67 (d, J(H,H)=12.5 Hz, 1H; CH
Ph), 4.62 (d, J(H,H)=10.5 Hz, 1H; CH Ph), 4.58 (d, 3J(H,H)=12.0 Hz,
3
ꢀ
3
ꢀ
ꢀ
1H; CH Ph), 4.57 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.50, 4.48, 4.40
(3”d, J(H,H)=12.0 Hz, 3H; 3”CH Ph), 4.39 (d, 3J(H,H)=11.0 Hz,
3
ꢀ
3
ꢀ
ꢀ
1H; CH Ph), 4.30, 4.20 (2”d, J(H,H)=12.0 Hz, 2H; 2”CH Ph), 4.08
(app.d, 3J(H,H)=9.5 Hz, 1H; H5’), 4.06 (t, 3J(H,H)=10.0 Hz, 1H; H3),
3.99 (dd, 3J(H2,H3)=10.0 Hz, 3J(H2,H1)=2.5 Hz, 1H; H2), 3.94 (t,
3J(H,H)=9.5 Hz, 1H; H3’), 3.80 (m, 2H; H1, H5), 3.78 (t, 3J(H,H)=
9.5 Hz, 1H; H4), 3.70 (t, 3J(H,H)=3.0 Hz, 1H; H4’), 3.64 (t, 3J(H,H)=
2.5 Hz, 1H; H6), 3.35 (m, 4H; CH3OCH2, H2’), 3.20 (app.d, 3J(H,H)=
10.5 Hz, 1H; H6a’), 3.14 ppm (app.d, 3J(H,H)=9.0 Hz, 1H; H6b’);
13C NMR (125 MHz, CDCl3, 258C, TMS): d=138.7, 138.6, 138.3, 138.2
(7C; Ph), 128.6–127.4 (35”CH; Ph), 98.5 (CH2OCH3), 97.6 (C1’), 81.1
(C2), 80.6, 80.5 (C3’, C5), 79.6 (C4), 78.5 (C4’), 77.2 (C1), 75.8, 75.7, 75.5,
74.9 (3”CH2Ph, C3), 73.9, 73.45, 73.38, 73.2, 73.1 (4”CH2Ph, C6), 70.3
(C5’), 67.9 (C6’), 63.9 (C2’), 56.0 ppm (CH2OCH3); FAB HRMS: m/z calcd
for [C63H67N3O11+Na]+: 1064.4673; found: 1064.4720.
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-galactopyranosyl-a-(1!3)-1,4,5,6-
tetra-O-benzyl-l-chiro-inositol
(20):
Compound
20
(15 mg,
0.01503 mmol, 68%) was obtained as
a syrup from 18 (23 mg,
0.0221 mmol) by the same experimental procedure as that described for
the preparation of 19. Compound 20 was purified by PLC (n-hexane/
AcOEt 3:1); Rf (n-hexane/AcOEt 3:1): 0.17; [a]2D0 =+91 (c=0.20 in
CHCl3); 1H NMR (500 MHz, CDCl3,258C, TMS): d=7.38–7.16 (m, 35H;
Ph), 5.38 (d, 3J(H,H)=3.5 Hz, 1H; H1’), 4.98 (d, 3J(H,H)=11.5 Hz, 1H;
CH Ph), 4.80 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.73 (d, 3J(H,H)=
3
ꢀ
ꢀ
3
ꢀ
ꢀ
11.5 Hz, 1H; CH Ph), 4.66 (d, J(H,H)=12.0 Hz, 1H; CH Ph), 4.65
(AB, 2H; CH2Ph), 4.62 (d, J(H,H)=12.0 Hz, 1H; CH Ph), 4.513 (d, 3J-
3
ꢀ
3
ꢀ
ꢀ
(H,H)=12.0 Hz, 1H; CH Ph), 4.508 (d, J(H,H)=11.5 Hz, 1H; CH
3
Ph), 4.46 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.39 (d, 3J(H,H)=12.0 Hz,
ꢀ
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-galactopyranosyl-a-(1!3)-2-meth-
oxymethyl-1,4,5,6-tetra-O-benzyl-l-chiro-inositol (18): Compound 18
(29 mg, 0.028 mmol, 74%) was obtained from 16 (42 mg, 0.04 mmol) by
the same experimental procedure as that used for the preparation of 17
1H; CH Ph), 4.36 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.36 (d, 3J-
3
ꢀ
ꢀ
(H,H)=12.0 Hz, 1H; CH Ph), 4.19 (t, 3J(H,H)=6.0 Hz, 1H; H5’), 4.16,
ꢀ
3
ꢀ
4.11 (AB, J(H,H)=11.5 Hz, 2H; 2”CH Ph), 4.06 (m, 1H; H2), 4.03
and was purified by flash chromatography (n-hexane/AcOEt 6:1). [a]D20
=
(dd, J(H,H)=11.0 Hz, J(H,H)=3.5 Hz, 1H; H2’), 3.94–3.90 (m, 2H; H3,
H4’), 3.79–3.84 (m, 3H; H4, H5, H3’), 3.75 (t, 3J(H,H)=4.0 Hz, 1H; H1),
3.68 (m, 1H; H6), 3.61 (d, 3J(H,H)=5.5 Hz, 1H; C2OH), 3.44 (dd,
3J(H6a’,H6b’)=8.8 Hz, 3J(H6a’,H5’)=6.0 Hz, 1H; H6a’), 3.26 ppm (dd,
3J(H6b’,H6a’)=8.8 Hz, 3J(H6b’,H5’)=6.0 Hz; H6b’); 13C NMR (125 MHz,
CDCl3, 258C, TMS): d=138.6, 138.5, 138.4, 138.3, 138.2, 137.8 (7C; Ph),
128.6–127.2 (35”CH; Ph), 98.9 (C1’), 81.6, 80.4, 79.4 (C3’, C4, C5), 78.2
(C3), 77.5 (C1), 75.0, 74.7 (2”CH2Ph), 74.2 (C6), 73.8 (CH2Ph), 73.4 (C4’),
73.3, 73.2, 73.1 (3”CH2Ph), 72.6 (C2), 72.1 (CH2Ph), 69.6 (C5’), 68.3 (C6’),
61.3 ppm (C2’); FAB HRMS: m/z calcd for [C61H63N3O10+Na]+:
1020.4411; found: 1020.4438.
3
3
+97 (c=0.9 in CHCl3); Rf (n-hexane/AcOEt 3:1): 0.22; 1H NMR
(500 MHz, CDCl3, 258C, TMS): d=7.40–7.12 (m, 35H; Ph), 5.52 (d, 3J-
3
ꢀ
(H,H)=3.0 Hz, 1H; H1’), 5.99 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.794
(d, 3J(H,H)=6.5 Hz, 1H; CH3OCH2), 4.788 (d, 3J(H,H)=11.5 Hz, 1H;
CH Ph), 4.681 (d, J(H,H)=12.5 Hz, 1H; CH Ph), 4.677 (d, 3J(H,H)=
3
ꢀ
ꢀ
3
ꢀ
6.5 Hz, 1H; CH3OCH2), 4.673 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.602
3
ꢀ
(d, J(H,H)=12.0 Hz, 1H; CH Ph), 4.598 (app.s, 2H; CH2Ph), 4.50 (d,
3
3
ꢀ
ꢀ
J(H,H)=12.0 Hz, 1H; CH Ph), 4.49 (d, J(H,H)=12.5 Hz, 1H; CH
Ph), 4.44 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.34 (d, 3J(H,H)=12.0 Hz,
3
ꢀ
3
ꢀ
ꢀ
1H; CH Ph), 4.33 (m, 1H; H5’), 4.31 (d, J(H,H)=12.0 Hz, 1H; CH
Ph), 4.26, 4.21 (AB, J(H,H)=11.5 Hz, 2H; 2”CH Ph), 4.11 (t, 3J-
3
ꢀ
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-glucopyranosyl-a-(1!3)-2-O-(1’,2’-
di-O-myristoyl-sn-glycero-3’-((R,S))-benzyl-phosphatidyl)-1,4,5,6-tetra-
O-benzyl-l-chiro-inositol (21): Compound 19 (73 mg, 0.073 mmol) and
1H-tetrazole (11 mg, 0.161 mmol, 2.2 equiv) were coevaporated three
times with toluene and dried under vacuum overnight in the presence of
P2O5. The mixture was dissolved in CH2Cl2 (4 mL) and a solution of 1,2-
di-O-myristoyl-sn-glycero-3-yl benzyl N,N-diisopropyl phosphoramidite
(0.5m, 292 mL, 2 equiv, 0.146 mmol) in CH2Cl2 was added under Ar.
After 1 h 30 min, the mixture was cooled to ꢀ408C and a solution of m-
CPBA (18 mg, 0.073 mmol, 1 equiv) in CH2Cl2 (3.2 mL) was added. The
reaction mixture was neutralized with Et3N (1 mm in CH2Cl2) and puri-
fied on PLC plates (n-hexane/AcOEt 4:1) previously treated with Et3N,
to give a colorless syrup 21 as a 1:1 mixture of diastereomers (77 mg,
0.046 mmol, 63%). [a]2D0 =+25 (c=1.5 in CHCl3); Rf (n-hexane/AcOEt
3:1): 0.37; 1H NMR (500 MHz, CDCl3, 258C, TMS): d=7.44–7.01 (m,
80H; Ph), 5.44 (d, 3J(H,H)=3.5 Hz, 1H; H1’), 5.42 (d, 3J(H,H)=4.0 Hz,
(H,H)=9.5 Hz, 1H; H3), 4.01 (dd, 3J(H2,H3)=9.5 Hz, 3J(H2,H1)=2.5 Hz,
1H; H2), 3.87–3.82 (m, 2H; H4, H2’), 3.81–3.77 (m, 3H; H1, H5, H3’), 3.72
(app.s, 1H; H4’), 3.65 (t, 3J(H,H)=3.0 Hz, 1H; H6), 3.46 (t, 1H; J=
9.0 Hz, J=6.0 Hz, H6a’), 3.39 (m, 1H; H6b’), 3.36 ppm (m, 3H;
CH3OCH2); 13C NMR (125 MHz, CDCl3, 258C, TMS): d=139.2, 138.8,
138.6, 138.5, 138.4, 138.0 (7C; Ph), 128.8–127.0 (35”CH; Ph), 98.0
(CH2OCH3), 97.4 (C1’), 80.6, 80.5, 80.1, 79.7 (C2, C3’, C4, C5), 77.2 (C1),
75.7, 74.9 (2”CH2Ph), 74.3 (C3), 74.1 (C6), 73.8 (C4’), 73.38, 73.26, 73.1,
72.1 (5”CH2Ph), 69.0 (C5’), 68.7 (C6’), 60.3 (C2’), 56.1 ppm (CH2OCH3);
FAB HRMS: m/z calcd for [C63H67N3O11+Na]+: 1064.4673; found:
1064.4656.
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-glucopyranosyl-a-(1!3)-1,4,5,6-
tetra-O-benzyl-l-chiro-inositol (19): PhSH (2.2 mL, 0.0218 mmol,
1.2 equiv) and BF3·Et2O (2.3 mL, 0.0218 mmol, 1.0 equiv) were added
under Ar to a solution of 17 (19 mg, 0.0182 mmol) in CH2Cl2. After 2 h,
the mixture was washed with solutions of saturated NaHCO3 and NaCl,
the organic layer was dried (MgSO4), the solvent was evaporated, and
the crude residue was purified by preparative chromatography (n-
hexane/AcOEt 6:1) to give 19 as a syrup (14 mg, 0.01402 mmol, 77%).
1H; H1’), 5.24–5.19 (m, 2H; CH Ph, C2aHglycerol), 5.16 (d, 3J(H,H)=
ꢀ
ꢀ
ꢀ
10.5 Hz, 1H; CH Ph), 5.11–5.07 (m, 3H; 2”CH Ph, C2bHglycerol), 4.98
3
ꢀ
ꢀ
(app.d, J(H,H)=10.0 Hz, 2H; 2”CH Ph), 4.87 (m, 4H; 4”CH Ph),
4.83 (m, 2H; 2”H2), 4.74 (d, J(H,H)=11.0 Hz; CH Ph), 4.73 (d, 3J-
3
ꢀ
Chem. Eur. J. 2006, 12, 1513 – 1528
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1521

M. Martín-Lomas et al.
3
ꢀ
ꢀ
(H,H)=10.5 Hz, 1H; CH Ph), 4.65 (m, 2H; 2”CH Ph), 4.58–4.49 (m,
H1), 3.72 (app.d, J(H,H)=6.5 Hz, 1H; H5), 3.71–3.66 (m, 1H; H6a’), 3.62
(dd, 3J(H2’,H3’)=9.7 Hz, 3J(H2’,H1’)=3.6 Hz, 1H; H2’), 3.55 (m, 2H; H3,
H4), 3.49 (m, 1H; H6b’), 3.44 ppm (t, 3J(H,H)=9.7 Hz, 1H; H4’);
13C NMR (100 MHz, CDCl3, 258C, TMS): d=137.5, 137.3 (3C; Ph), 134.8
(CH=CH2), 128.7–127.3 (15”CH; Ph), 117.3 (CH=CH2), 99.1 (C1’), 87.5
(C3), 81.6 (C3’), 78.8 (C4’), 77.4 (C1), 76.0, 75.2, 73.8 (3”CH2Ph), 72.8
ꢀ
ꢀ
3H; 3”CH Ph), 4.42–3.95 (m, 14H; 14”CH Ph), 4.367, 4.32–4.26 (m,
3H; H1, C1aH, C3bHglycerol), 4.26 (m, 1H; H1), 4.22–4.14 (m, 4H; 2”H3,
3
C1aH, C3bHglycerol), 4.11–4.04 (m, 5H; 2”H5’, C3aH2, C1bHglycerol), 4.00 (t, J-
(H,H)=9.0 Hz, 1H; H3’), 3.98 (t, 3J(H,H)=9.0 Hz, 1H; H3’), 3.97 (m,
1H; C1bHglycerol), 3.88 (m, 4H; 2”H5, 2”H4), 3.71 (t, 3J(H,H)=9.5 Hz,
1H; H4’), 3.70 (t, 3J(H,H)=10.0 Hz, 1H; H4’), 3.60 (m, 2H; 2”H6), 3.29
ꢀ
(CH2 CH=CH2), 72.4 (C4), 72.0, 69.2 (C5’, C6), 71.5 (C5), 70.5 (C2), 68.9
3
3
3
(C6’), 65.0 ppm (C2’); FAB HRMS: m/z calcd for [C36H43N3O10+Na]+:
(dd, J(H2’,H3’)=10.5 Hz, J(H2’,H1’)=4.0 Hz, 2H; 2”H2’), 3.24 (app.d, J-
(H,H)=11.0 Hz, 2H; 2”H6a’), 3.18 (dd, 3J(H6b’,H6a’)=11.0 Hz, 3J-
(H6b’,H5’)=2.5 Hz; 2”H6b’), 2.35 (m, 4H; 2”CH2CO), 2.23 (m, 4H; 2”
CH2CO), 1.65–1.50 (m, 8H; 4”CH2CH2CO), 1.34–1.20 (m, 80H; 40”
CH2), 0.89 ppm (t, 3J(H,H)=7.0 Hz, 4H; 4”CH3); 13C NMR (125 MHz,
CDCl3, 258C, TMS): d=173.3, 173.2, 173.0, 172.8 (4”CO), 138.6, 138.3,
138.25, 138.20, 138.13, 138.10, 137.9 (16C; Ph), 129.2–127.4 (80”CH;
Ph), 97.8, 97.6 (2”C1’), 80.2, 80.1, 80.0, 79.2 (2”C3’, 2”C2, 2”C4, 2”C5),
78.4 (2”C4’), 76.4, 76.3 (2”C1), 75.9, 75.4 (4”CH2Ph), 74.9 (2”C3), 74.0,
73.8, 73.4 (6”CH2Ph), 73.2, 72.9 (2”C6), 72.9, 72.7 (4”CH2Ph), 70.4 (2”
700.2846; found: 700.2892.
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-galactopyranosyl-a-(1!3)-1-allyl-l-
chiro-inositol (24): Compound 24 (53 mg, 0.08 mmol) was obtained as a
white foam from 14 (83 mg, 0.08 mmol) by the same experimental proce-
dure as that used to obtain 23. [a]2D0 =+2 (c=0.2 in CHCl3); Rf (n-
hexane/AcOEt 1:3): 0.33; 1H NMR (300 MHz, CDCl3, 258C, TMS): d=
7.44–7.20 (m, 15H; Ph), 5.88 (m, 1H; CH=CH2), 5.26 (dd, 3J(H,H)=
3
3
3
17.2 Hz, J(H,H)=1.8 Hz, 1H; CH=CH2), 5.16 (dd, J(H,H)=17.2 Hz, J-
(H,H)=1.2 Hz, 1H; CH=CH2), 5.05 (d, 3J(H,H)=3.9 Hz, 1H; H1’), 4.86
3
C5’), 70.0, 69.7 (2”d, J(H,H)=5.4 Hz; 2”C2Hglycerol), 69.6, 69.5 (2”d, J=
(d, J(H,H)=11.7 Hz, 1H; CH Ph), 4.74 (s, 2H; CH2Ph), 4.50 (d, 3J-
3
ꢀ
7.9 Hz; 2”POCH2Ph), 67.9 (2”C6’), 66.1, 65.7 (2”d, 3J(H,H)=5.1 Hz;
2”C3H2glycerol), 63.52, 63.47 (2”C2’), 62.2, 61.7 (2”C1H2glycerol), 34.4, 34.3,
34.2, 34.1 (4”CH2CO), 32.0 (4”CH2CH2CH3), 29.8–29.2 (32”CH2),
25.08, 25.02, 24.94, 24.91 (4”CH2CH2CO), 22.8 (4”CH2CH2CH3),
12.2 ppm (4CH3); 31P NMR (202 MHz, CDCl3, 258C, TMS): d=ꢀ2.87,
ꢀ2.98 ppm; FAB HRMS: m/z calcd for [C99H129N3O17P+Na]+: 1685.8957;
found: 1684.8963.
3
ꢀ
ꢀ
(H,H)=11.7 Hz, 1H; CH Ph), 4.48 (d, J(H,H)=11.7 Hz, 1H; CH Ph),
4.40 (d, J(H,H)=11.7 Hz, 1H; CH Ph), 4.24 (dd, 3J(H,H)=13.2 Hz, 3J-
3
ꢀ
ꢀ
ꢀ
(H,H)=5.4 Hz; CH2 CH=CH2), 4.16–4.05 (m, 4H; H2’, H5’, H6, CH2
CH=CH2), 3.94 (m, 1H; H2), 3.91–3.85 (m, 2H; H3’, H4’), 3.84 (app.s, 1H;
H1), 3.72 (dd, 3J(H5,H4)=9.4 Hz, 3J(H5,H6)=3.0 Hz, 1H; H5), 3.62 (t, 3J-
(H,H)=9.4 Hz, 1H; H6a’), 3.53 (m, 1H; H3), 3.50 (m, 1H; H4), 3.27 ppm
(dd, 3J(H6b’,H6a’)=9.4 Hz, 3J(H6b’,H5’)=3.3 Hz, 1H; H6b’); 13C NMR
(75 MHz, CDCl3, 258C, TMS): d=137.9, 137.3 (3C; Ph), 134.9 (CH2
CH=CH2), 128.8–128.0 (15”CH; Ph), 117.2 (CH=CH2), 99.4 (C1’), 87.2
(C3), 79.0 (C3’), 77.0 (C4’), 74.5, 73.9 (2”CH2Ph), 73.1 (C1), 72.8 (CH2Ph),
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-galactopyranosyl-a-(1!3)-2-O-
(1’,2’-di-O-myristoyl-sn-glycero-3’-(R,S)-benzyl-phosphatidyl)-1,4,5,6-
tetra-O-benzyl-l-chiro-inositol (22): Compound 22 was obtained as a col-
ꢀ
orless syrup and as
a 1:1 mixture of two diastereomers (15 mg,
ꢀ
72.6 (CH2 CH=CH2), 72.3 (C4), 71.7 (C5), 71.1 (C5’), 70.5 (C2), 70.2 (C6’),
0.009 mmol, 60%) from 20 (15 mg, 0.015 mmol) by the procedure de-
scribed for the preparation of 21. The crude product was fractionated on
PLC plates (Hex/AcOEt 4:1) previously treated with Et3N to give 22 as a
colorless syrup. [a]2D0 =+34 (c=0.8 in CHCl3 diastereomers 1/1); Rf (n-
hexane/AcOEt 3:1): 0.27; 1H NMR (500 MHz, CDCl3, 258C, TMS): d=
69.3 (C6), 61.4 ppm (C2’); FAB HRMS: m/z calcd for [C36H43N3O10+Na]+:
700.2846; found: 700.2861.
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-glucopyranosyl-a-(1!3)-1-allyl-
2,4,5,6-tetra-O-benzyl-l-chiro-inositol (25): A mixture of compound 23
(141 mg, 0.21 mmol) and NaH (50 mg, 1.26 mmol, 6 equiv) in dried DMF
(8 mL) was stirred under Ar for 10 min. After the mixture had been
cooled to ꢀ158C in an ice/salt bath, BnBr (200 mL, 1.68 mmol, 8 equiv)
was added dropwise. The reaction mixture was stirred overnight, NH4OH
was added, and the mixture was then successively washed with a solution
of saturated NH4Cl, H2O, and brine. The organic layer was dried
(MgSO4) and evaporated, and the crude product was purified by flash
chromatography (Hex/AcOEt 20:1, AcOEt) to provide 25 as a syrup
(145 mg, 0.14 mmol, 68%). [a]2D0 =+32 (c=0.4 in CHCl3); Rf (n-hexane/
3
7.40–7.07 (m, 80H; Ph), 5.44 (m, J(H,H)=3.5 Hz, 2H; 2”H1’), 5.21–5.12
ꢀ
(m, 1H; C2aHglycerol), 5.13–5.04 (m, 3H; 2”CH Ph, C2bHglycerol), 4.81 (m,
3
ꢀ
1H; H2), 4.78, 4.77 (2”d, J(H,H)=11.0 Hz, 2H; 2”CH Ph), 4.69–4.63
ꢀ
ꢀ
(m, 3H; 3”CH Ph), 4.60–4.14 (m, 35H; 25”CH Ph, 2”H5’, 2”H1, 2”
H3, C1aH, C1aH, C3bH, C3aHglycerol), 4.13–3.95 (m, 4H; C3aH, C3bH,
C1bH2glycerol), 3.89–3.84 (m, 2H; 2”H5), 3.84–3.75 (m, 6H; 2”H4, 2”H2’,
2”H3’), 3.67, 3.65 (2 brs, 2H; 2”H4’), 3.60 (m, 2H; 2”H6), 3.48 (dd,
3J(H6b’,H6a’)=9.5 Hz, J(H6b’,H5’)=9.0 Hz, 2H; 2”H6a’), 3.36 (t, J(H,H)=
8.0 Hz, 2H; 2”H6b’), 2.35 (m, 4H; 2”CH2CO), 2.23 (m, 4H; 2”
CH2CO), 1.65–1.50 (m, 8H; 4”CH2CH2CO), 1.34–1.20 (m, 80H; 40”
3
3
1
AcOEt 3:1): 0.33; H NMR (500 MHz, CDCl3, 258C, TMS): d=7.37–7.01
(m, 35H; Ph), 5.73–5.64 (m, 2H; H1’, CH=CH2), 5.07–5.01 (m, 2H; CH=
CH2), 0.89 ppm (t, J(H,H)=7.0 Hz, 12H; 4”CH3); 13C NMR (125 MHz,
3
3
ꢀ
CH2), 4.99 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.88 (s, 2H; CH2Ph), 4.75
CDCl3, 258C, TMS): d=173.7–172.9 (4”CO), 139.0, 138.6, 138.4, 137.9
(16C; Ph), 128.8–127.5 (80”CH; Ph), 97.6 (2”C1’), 80.1 (2”C2), 79.7,
79.8 (2”C4, 2”C5), 77.0 (2”C3’), 76.3 (2”C1), 74.6 (2”CH2Ph), 74.1–72.4
(10”CH2Ph), 73.6 (C4’), 73.8 (2”CH2Ph), 73.5 (2”C6), 73.4 (2”C3), 73.0
(2”CH2Ph), 69.3 (C5’), 69.7, 69.5 (2”C2H2glycerol), 68.9 (2”C6’), 65.6, 65.3
(2”C3H2glycerol), 61.62 (2”C1H2glycerol), 60.0 (2”C2’), 34.2, 34.1 (4”
CH2CO), 32.1 (4”CH2CH2CH3), 29.8–29.3 (32”CH2), 24.9 (4”
CH2CH2CO), 22.8 (4”CH2CH2CH3), 14.3 ppm (4”CH3); 31P NMR
(202 MHz, CDCl3, 258C, TMS): d=ꢀ2.74, ꢀ2.91 ppm; FAB HRMS: m/z
calcd for [C99H128N3O17P+Na]+: 1684.8879; found: 1684.8948.
(d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.70 (d, 3J(H,H)=11.5 Hz, 2H; 2”
3
ꢀ
CH Ph) 4.64 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.62 (d, 3J(H,H)=
3
ꢀ
ꢀ
3
ꢀ
ꢀ
11.5 Hz, 1H; CH Ph), 4.58 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.50 (d,
3
3
ꢀ
ꢀ
J(H,H)=12.0 Hz, 1H; CH Ph), 4.44 (d, J(H,H)=11.5 Hz, 1H; CH
Ph), 4.42 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.38 (d, 3J(H,H)=11.0 Hz,
3
ꢀ
3
ꢀ
ꢀ
1H; CH Ph), 4.20 (d, J(H,H)=12.0 Hz, 1H; CH Ph), 4.08 (m, 1H;
3
ꢀ
H5’), 4.03 (t, J(H,H)=9.5 Hz, 1H; H3), 4.00–3.93 (m, 2H; H3’, CH2 CH=
CH2), 3.91 (dd, 3J(H2,H3)=9.5 Hz, 3J(H2,H1)=3.0 Hz, 1H; H2), 3.85–3.77
3
ꢀ
(m, 2H; H4, H5), 3.78 (app.d, J(H,H)=6.0 Hz, 1H; CH2 CH=CH2), 3.68
(t, 1H, 3J(H,H)=9.5 Hz, 1H; H4’), 3.67 (app.s, 1H; H6), 3.56 (t, 3J-
(H,H)=3.0 Hz, 1H; H1), 3.26 (dd, 3J(H2’,H3’)=10.5 Hz, 3J(H2’,H1’)=
4.0 Hz, 1H; H2’), 3.21 (app.d, 3J(H,H)=10.0 Hz, 1H; H6a’), 3.15 ppm
(app.d, 3J(H,H)=10.0 Hz, 1H; H6b’); 13C NMR (125 MHz, CDCl3, 258C,
TMS): d=138.7–138.2 (7C; Ph), 135.0 (CH=CH2), 128.6–127.3 (35”CH;
Ph), 117.1 (CH=CH2), 97.6 (C1’), 81.4 (C2), 80.8 (C3’), 80.6 (C5), 79.7 (C4),
78.6 (C4’), 75.8 (CH2Ph), 75.6 (C3), 75.4 (CH2Ph), 74.8 (C6, CH2Ph), 73.8
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-glucopyranosyl-a-(1!3)-1-allyl-l-
chiro-inositol (23): Compound 13 (432 mg, 0.40 mmol) was dissolved
under Ar in dried MeOH (7 mL), treated with MeONa/MeOH (1.0m,
3.2 mL, 3.2 mmol, 8 equiv), and stirred for 16 h. The solvent and BzOMe
obtained as byproduct were evaporated by heating (508C) under vacuum
to give 23 quantitatively as a syrup (270 mg, 0.40 mmol). [a]2D0 =ꢀ11 (c=
0.1 in CHCl3); Rf (n-hexane/AcOEt 1:3): 0.49; 1H NMR (500 MHz,
CDCl3, 258C, TMS): d=7.59–7.19 (m, 15H; Ph), 5.88 (m, 1H; CH=CH2),
5.26 (app.d, 3J(H,H)=17.0 Hz, 1H; CH=CH2), 5.17 (app.d, 3J(H,H)=
ꢀ
(C1), 73.5 (2”CH2Ph), 73.4, 72.5 (2”CH2Ph), 72.4 (CH2 CH=CH2), 70.1
(C5’), 67.9 (C6’), 63.7 ppm (C2’); FAB HRMS: m/z calcd for
[C64H67N3O10+Na]+: 1060.4724; found: 1060.4765.
10.0 Hz, 1H; CH=CH2), 5.09 (d, 3J(H,H)=3.6 Hz, 1H; H1’), 4.88 (s, 2H;
3
ꢀ
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-galactopyranosyl-a-(1!3)-1-allyl-
2,4,5,6-tetra-O-benzyl-l-chiro-inositol (26): Compound 26 (151 mg,
0.15 mmol, 68%) was obtained as a syrup from 24 (147 mg, 0.22 mmol)
by the experimental procedure described above for the preparation of
CH2Ph), 4.82 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.63–4.53 (m, 3H;
CH2 Ph, CH Ph), 4.24 (dd, 3J(H,H)=12.5 Hz, 3J(H,H)=5.0 Hz, 1H;
ꢀ
ꢀ
ꢀ
ꢀ
CH2 CH=CH2), 4.17–4.07 (m, 3H; H5’, H6, CH2 CH=CH2), 3.98 (m, 1H;
H2), 3.94 (t, 3J(H,H)=9.7 Hz, 1H; H3’), 3.90 (t, 3J(H,H)=3.5 Hz, 1H;
1522
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 1513 – 1528

Phospholipase Cleavage of d-and l-chiro-Glycosylphosphoinositides
FULL PAPER
25. The crude product was purified by flash chromatography (n-hexane/
AcOEt 20:1, AcOEt) and preparative chromatography (n-hexane/AcOEt
18:1). [a]2D0 =+53 (c=1.3 in CHCl3); Rf (n-hexane/AcOEt 6:1): 0.21;
1H NMR (500 MHz, CDCl3, 258C. TMS): d=7.40–7.17 (m, 35H; Ph),
(H,H)=9.3 Hz, 1H; H6b’); 13C NMR (75 MHz, CDCl3, 258C, TMS): d=
139.1–137.5 (7C; Ph), 128.8–127.4 (35”CH; Ph), 97.3 (C1’), 81.9, 80.5,
79.5 (3Cinositol), 77.0 (C3’), 75.6, 75.5 (2Cinositol), 74.9, 74.2, 73.8, (3”
CH2Ph), 73.7 (C4’), 73.2, 73.1, 72.5, 72.1 (4”CH2Ph), 68.9 (C5’), 68.8 (C6’),
67.4 (C1), 60.1 ppm (C2’); elemental analysis calcd (%) for
C61H63N3O10·3H2O: C 69.63, H 6.61, N 3.99; found: C 70.00, H 6.99, N
3.68; FAB HRMS: m/z calcd for [C61H63N3O10+Na]+: 1020.4411; found:
1020.4352.
5.73–5.63 (m, 1H; CH=CH2), 5.66 (d, 3J(H,H)=3.0 Hz, 1H; H1’), 5.07–
3
ꢀ
ꢀ
5.01 (m, 2H; CH2 CH=CH2), 4.99 (d, J(H,H)=11.5 Hz, 1H; CH Ph),
4.77 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.73 (d, 3J(H,H)=12.5 Hz, 1H;
CH Ph), 4.68 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.61 (m, 4H; 4”CH
3
ꢀ
3
ꢀ
ꢀ
ꢀ
3
ꢀ
ꢀ
Ph), 4.52 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.44 (m, 3H; 3”CH Ph),
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-glucopyranosyl-a-(1!3)-1-O-(1’,2’-
di-O-myristoyl-sn-glycero-3’-(R,S)-benzylphosphatidyl)-2,4,5,6-tetra-O-
benzyl-l-chiro-inositol (29): Compound 29 was obtained as a colorless
syrup from 27 (39 mg, 0.039 mmol) by the procedure described for the
preparation of 21. The crude product was fractionated on PLC plates (n-
hexane/AcOEt 3:1) previously treated with Et3N to give 29 as a mixture
of two diastereomers (1:1, 53 mg, 0.032 mmol, 82%). [a]2D0 =+15 (c=0.47
in CHCl3, diastereomers 3/1); Rf (n-hexane/AcOEt 3:1): 0.29; 1H NMR
(500 MHz, CDCl3, 258C, TMS): d=7.43–7.02 (m, 80H; Ph), 5.66 (d, 3J-
(H,H)=3.5 Hz, 1H; H1’), 5.64 (d, 3J(H,H)=4.0 Hz, 1H; H1’), 5.13 (m,
3
3
4.32 (app.t, J(H,H)=7.0 Hz, 1H; H5’), 4.30, 4.24 (AB, J(H,H)=11.5 Hz,
2H; 2”CH Ph), 4.08 (t, 3J(H,H)=9.0 Hz, 1H; H3), 3.98 (dd, 3J(H,H)=
ꢀ
12.5 Hz, 3J(H,H)=5.0 Hz, 1H; CH=CH2), 3.90 (m, 1H; CH=CH2), 3.87–
3.79 (m, 4H; H4, H3’, H5, H2’), 3.76 (dd, 3J(H,H)=10.5 Hz, 1H; H2), 3.72
(s, 1H; H4’), 3.69 (app.s, 1H; H6), 3.58 (app.s, 1H; H1), 3.46 (app.t, 3J-
(H,H)=9.0 Hz, 1H; H6a’), 3.38 ppm (app.t, 3J(H,H)=9.0 Hz, 1H; H6b’);
13C NMR (75 MHz, CDCl3, 258C, TMS): d=139.2–138.0 (7C; Ph), 134.8
(CH=CH2), 128.6–127.3 (35”CH; Ph), 116.9 (CH=CH2), 97.4 (C1’), 81.2,
80.6, 80.1 (C2, C4, C5), 77.4 (C3’), 75.6 (CH2Ph), 75.1 (C6), 74.8 (2”
ꢀ
CH2Ph), 74.2 (C3), 73.8 (C1, C4’), 73.5, 73.6, 73.1 (3”CH2Ph), 72.4 (CH2
CH=CH2), 72.0 (CH2Ph), 71.1 (C5’), 68.8 (C6’), 60.2 ppm (C2’); FAB
ꢀ
1H; C2aHglycerol), 5.01 (m, 3H; 2”CH Ph, C2bHglycerol), 4.94–4.81 (m, 12H;
10”CH Ph, 2”H1), 4.77–4.42 (m, 16H; 16”CH Ph), 4.38 (d, 3J(H,H)=
ꢀ
ꢀ
HRMS: m/z calcd for [C64H67N3O10+Na]+: 1060.4724; found: 1060.4793.
3
ꢀ
ꢀ
11.5 Hz, 1H; CH Ph), 4.37 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.24 (dd,
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-glucopyranosyl-a-(1!3)-2,4,5,6-
3J(H,H)=12.0 Hz, 3J(H,H)=4.0 Hz, 1H; C1aHglycerol), 4.20 (d, 3J(H,H)=
ꢀ
tetra-O-benzyl-l-chiro-inositol (27):
A
solution (6 mm) of [{Ir-
12.0 Hz, 2H; 2”CH Ph), 4.09–3.82 (m, 21H; 2”H2, 2”H3, 2”H4, 2”H5,
(cod)Ph2PMe}2]PF6 (cod=cyclooctadiene) (84 mL, 2.3 mmol, 0.03 equiv)
in THF was added to a solution of 25 (80 mg, 0.077 mmol, 1 equiv) in
freshly distilled THF (2.2 mL). After 1 h, the solvent was evaporated
with a stream of Ar and a freshly prepared solution (6 mm) of [{Ir-
(cod)Ph2PMe}2]PF6 in THF (384 mL) was added. After 30 min, N-bromo-
succinimide (NBS) (21 mg, 0.116 mmol, 1.5 equiv) and H2O (232 mL,
12.94 mmol, 168 equiv) were added and the mixture was stirred for
10 min. AcOEt was added, and the organic layer was separated and
washed with a solution of cold saturated NaHCO3 (2”5mL) and NaCl
(3”5mL), and dried with MgSO4. The crude product was purified by
flash chromatography (n-hexane/AcOEt 3:1) to provide 27 as a colorless
syrup (55 mg, 0.055 mmol, 72%). [a]2D0 =+10 (c=0.15 in CHCl3); Rf (n-
2”H6, 2”H3’, 2”H5’, C1aH, C3aH, C1bH, C3aH, C1bH, C3bH2glycerol), 3.71 (t,
3J(H,H)=9.5 Hz, 2H; 2”H4’), 3.30 (dd, 3J(H2’,H3’)=10.5 Hz, 3J(H2’,H1’)=
3.7 Hz, 2H; 2”H2’), 3.21–3.12 (m, 4H; 2”H6a’, 2”H6b’), 2.26, 2.20 (2”m,
8H; 4”CH2CO), 1.60–1.50 (m, 8H; 4”CH2CH2CO), 1.33–1.20 (m, 80H;
40”CH2), 0.89 ppm (t, 3J(H,H)=7.0 Hz, 12H; 4”CH3); 13C NMR
(75 MHz, CDCl3, 258C, TMS): d=173.6, 173.2 (4”CO), 138.8, 138.6,
138.5, 138.43, 138.35, 138.2, 137.6, 135.9 (16C; Ph), 129.1–127.8 (80”CH;
Ph), 99.4, 98.0 (2”C1’), 80.8, 80.4, 79.5, 78.6 (2”C3, 2”C4, 2”C6, 2”C3’),
78.4 (2”C4’), 77.6 (2”CH2Ph), 76.2, 75.8, 75.4, 75.1, 74.4, 73.8, 73.3, 72.7
(12”CH2Ph, 2”C2, 2”C5), 73.2 (m; POCH2Ph), 73.1 (m; POCH2Ph),
70.5 (2”C5’), 70.2 (d, 3J(H,H)=5.4 Hz; 2”C2Hglycerol), 69.65, 69.55 (2”
C1), 68.0 (2”C6’), 66.1 (m; C3H2glycerol), 65.6 (d, 3J(H,H)=5.4 Hz, C3H2gly-
cerol), 63.7 (2”C2’), 62.0 (2”C1H2glycerol), 34.5, 34.4 (4”CH2CO), 32.4 (4”
CH2CH2CH3), 30.1–29.6 (32”CH2), 25.2 (4”CH2CH2CO), 23.1 (4”
CH2CH2CH3), 14.6 ppm (4”CH3); 31P NMR (202 MHz, CDCl3, 258C;
TMS): d=ꢀ1.96, ꢀ2.1 ppm; elemental analysis calcd (%) for
C99H128N3O17·2H2O: C 69.98, H 7.83, N, 2.47; found: C 70.13, H 8.09, N
2.26; FAB HRMS: m/z calcd for [C99H128N3O17P+Na]+: 1684.8879;
found: 1684.9006.
1
hexane/AcOEt 4:1): 0.20; H NMR (500 MHz, CDCl3,): d=7.37–7.01 (m,
3
35H; Ph), 5.62 (d, J(H,H)=3.5 Hz, 1H; H1’), 4.99 (d, 3J(H,H)=11.0 Hz,
1H; CH Ph), 4.88 (AB, 2H; CH2Ph), 4.79 (d, 3J(H,H)=12.0 Hz, 1H;
ꢀ
3
CH Ph), 4.70 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.68 (d, 3J(H,H)=
ꢀ
ꢀ
3
ꢀ
ꢀ
11.0 Hz, 1H; CH Ph), 4.67, 4.62 (AB, J(H,H)=10.5 Hz, 2H; 2”CH
3
Ph), 4.61 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.52 (d, 3J(H,H)=12.0 Hz,
ꢀ
3
1H; CH Ph), 4.48 (d, J(H,H)=12.0 Hz, 1H; CH Ph), 4.46 (d, 3J-
ꢀ
ꢀ
3
ꢀ
ꢀ
(H,H)=11.0 Hz; CH Ph), 4.36 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.19
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-galactopyranosyl-a-(1!3)-1-O-
(1’,2’-di-O-myristoyl-sn-glycero-3’-(R,S)-benzylphosphatidyl)-2,4,5,6-
tetra-O-benzyl-l-chiro-inositol (30): Compound 30 was obtained as a
mixture of two diastereomers (1:1, 29 mg, 0.017 mmol, 78%) from 28
(22 mg, 0.022 mmol) by the procedure described for the preparation of
29. The crude product was fractionated on PLC plates (n-hexane/AcOEt
3
3
ꢀ
(d, J(H,H)=12.0 Hz, 1H; CH Ph), 4.08 (app.d, J(H5’,H4’)=9.5 Hz, 1H;
H5’), 3.99–3.84 (m, 7H; H1, H2, H3, H4, H5, H6, H3’), 3.68 (t, 3J(H,H)=
9.5 Hz, 1H; H4’), 3.32 (dd, 3J(H2’,H3’)=10.0 Hz, 3J(H2’,H1’)=3.5 Hz, 1H;
H2’), 3.19–3.10 (m, 2H; H6a’, H6b’), 2.29 ppm (s, 1H; C1OH); 13C NMR
(125 MHz, CDCl3, 258C, TMS): d=138.8–137.6 (7C; Ph), 128.8–127.4
(35”CH; Ph), 97.6 (C1’), 81.9 (Cinositol), 80.7 (C3’), 80.5, 79.0 (2”Cinositol),
78.5 (C4’), 75.7 (2”Cinositol, 2”CH2Ph), 74.8, 73.8, 73.5, 73.2, 72.5 (5”
CH2Ph), 70.2 (C5’), 67.8 (C6’), 67.3 (C1), 63.6 ppm (C2’); elemental analysis
calcd (%) for C61H63N3O10·H2O: C 72.10, H 6.45, N 4.14; found: C 72.40,
3:1) previously treated with Et3N to give 30 as a colorless syrup. [a]D20
=
+22 (c=0.7 in CHCl3); Rf (n-hexane/AcOEt 2:1): 0.44; 1H NMR
(500 MHz, CDCl3, 258C, TMS): d=7.44–7.12 (m, 80H; Ph), 5.66 (d, 3J-
(H,H)=3.5 Hz, 1H; H1’), 5.63 (d, 3J(H,H)=3.0 Hz, 1H; H1’), 5.11 (m,
6.85, N
4.06; FAB HRMS: m/z calcd for [C61H63N3O10+Na]+:
3
H
1H; C2aHglycerol), 5.03 (m, 1H; C2bHglycerol), 4.99 (d, J(H,H)=11.5 Hz, 1H;
3
1020.4411; found: 1020.4386.
ꢀ
ꢀ
CH Ph), 4.98 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.90–4.73 (m, 10H;
3
2”H1, 8”CH Ph), 4.68 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.66 (d, 3J-
(H,H)=11.5 Hz, 1H; CH Ph), 4.63–4.38 (m, 16H; 16”CH Ph), 4.30 (m,
2H; 2”H5’), 4.28–4.18 (m, 3H; C1aHglycerol, 2”CH Ph), 4.07–3.94 (m, 8H;
2”H2, C1aH, C3aH2, C1bHglycerol, 2”CH Ph), 3.93–3.75 (m, 13H; 2”H3,
ꢀ
ꢀ
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-galactopyranosyl-a-(1!3)-2,4,5,6-
tetra-O-benzyl-l-chiro-inositol (28): Compound 28 (22 mg, 0.021 mmol,
72%) was prepared from 26 (30 mg, 0.029 mmol) by the same procedure
as that described for the preparation of 27. Syrup 28 was purified by
flash chromatography (n-hexane/AcOEt 4:1). [a]2D0 =+31 (c=0.2 in
CHCl3); Rf (n-hexane/AcOEt 4:1): 0.21; 1H NMR (300 MHz, CDCl3,
258C, TMS): d=7.41–7.16 (m, 35H; Ph), 5.62 (d, 3J(H,H)=2.4 Hz, 1H;
ꢀ
ꢀ
ꢀ
ꢀ
2”H4, 2”H6, 2”H3’, 2”H2’, C1bH, C3bH2), 3.72 (app.s, 2H; 2”H4’), 3.68
(m, 2H; 2”H5’), 3.46–3.40 (m, 2H; 2”H6a’), 3.34 (m, 2H; 2”H6b’), 2.25
(m, 4H; 2”CH2CO), 2.18 (m, 4H; 2”CH2CO), 1.57–1.40 (m, 8H; 4”
CH2CH2CO), 1.33–1.20 (m, 80H; 40”CH2), 0.90 ppm (t, 3J(H,H)=
7.0 Hz, 12H; 4”CH3); 13C NMR and HSQC (125 and 500 MHz, CDCl3,
258C, TMS): d=138.0–137.2 (16C; Ph), 129.1–127.5 (80”CH; Ph), 97.4
(2”C1’), 80.2 (2”C6), 79.8 (2”C4), 77.2 (2”C3), 75.8–72.1 (14”CH2Ph,
2”C2, 2”C5, 2”C3’, 2”C4’), 70.0–68.7 (2”C5’, 2”C1, 2”C6’, 2”POCH2Ph,
2”C2Hglycerol), 65.3, 66.0 (2”C3H2glycerol), 61.8 (2”C1H2glycerol), 60.0 (2”
C2’), 34.3, 34.1 (4”CH2CO), 32.1 (4”CH2CH2CH3), 29.8–29.3 (32”CH2),
25.0 (4”CH2CH2CO), 22.8 (4”CH2CH2CH3), 14.3 ppm (4”CH3);
3
3
ꢀ
H1’), 5.01 (d, J(H,H)=11.7 Hz, 1H; CH Ph), 4.80 (d, J(H,H)=12.1 Hz,
1H; CH Ph), 4.78 (d, J(H,H)=11.2 Hz, 1H; CH Ph), 4.67 (d, 3J-
(H,H)=11.7 Hz, 1H; CH Ph), 4.65 (d, J(H,H)=11.0 Hz, 1H; CH Ph),
3
ꢀ
ꢀ
3
ꢀ
ꢀ
3
ꢀ
4.63–4.59 (m, 4H; 2”CH2Ph), 4.55 (d, J(H,H)=12.1 Hz, 1H; CH Ph),
4.51 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.44 (d, 3J(H,H)=11.2 Hz, 1H;
3
ꢀ
ꢀ
CH Ph), 4.32 (m, 1H; H5’), 4.25 (AB, 2H; CH2Ph), 4.06–3.88 (m, 6H;
H1, H2, H3, H4, H5, H6), 3.82 (s, 2H; H2’, H3’), 3.68 (s, 1H; H4’), 3.44 (dd,
3J(H6a’,H6b’)=9.3 Hz, 3J(H6a’,H5’)=9.0 Hz, 1H; H6a’), 3.32 ppm (t, 3J-
Chem. Eur. J. 2006, 12, 1513 – 1528
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1523

M. Martín-Lomas et al.
31P NMR (202 MHz, CDCl3, 258C, TMS): d=ꢀ2.16, ꢀ2.36 ppm; FAB
HRMS: m/z calcd for [C99H128N3O17P+Na]+: 1684.8879; found:
1684.9006.
4.6 Hz; 2”C3H2glycerol), 62.3, 62.1 (2”C1H2glycerol), 59.92, 59.76 (2”C2’),
34.2, 34.1 (4”CH2CO), 32.1 (4”CH2CH2CH3), 29.8–29.2 (32”CH2), 24.9
(4”CH2CH2CO), 23.4 (4”CH2CH2CH3), 14.2 ppm (4”CH3); 31P NMR
(202 MHz, CDCl3, 258C, TMS): d=ꢀ1.78, ꢀ2.21 ppm; FAB HRMS: m/z
calcd for [C99H128N3O17P+Na]+: 1684.8879; found: 1684.8856.
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-glucopyranosyl-a-(1!2)-1-O-(1’,2’-
di-O-myristoyl-sn-glycero-3’-(R,S)-benzylphosphatidyl)-3,4,5,6-tetra-O-
benzyl-d-chiro-inositol (33): Compound 33 was obtained as a colorless
syrup and as a mixture of two diastereomers (1:1, 43 mg, 0.026 mmol,
42% with 24% of starting material 31) from 31 (82 mg, 0.015 mmol), by
the procedure described for the preparation of 29. The crude product
was fractionated on PLC plates (n-hexane/AcOEt 4:1) previously treated
with Et3N to provide 33. [a]D20 =+42 (c=1.0 in CHCl3); Rf (n-hexane/
O-(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1!6)-1-O-
allyl-3,4,5-tri-O-benzyl-d-myo-inositol (36) and O-(2-azido-3,4,6-tri-O-
benzyl-2-deoxy-a-d-glucopyranosyl)-(1!6)-2-O-allyl-3,4,5-tri-O-benzyl-
d-myo-inositol (37): Dibutyltin oxide (30 mg, 0.121 1.2 equiv) was added
to a solution of 35 (90 mg, 0.099 mmol, 1 equiv) in dry toluene (10 mL)
and the mixture was heated at reflux overnight under a Dean–Stark ap-
paratus. The reaction mixture was then evaporated to dryness and the
residue was dissolved in AllBr (3 mL). Tetra-n-butylammonium iodide
(TBAI) (41 mg, 0.109 mmol, 1.1 equiv) was added and the mixture was
heated at reflux for 2 h. The mixture was treated with NH4OH (3.7 mL)
in an ice/water bath, diluted with AcOEt, washed with brine (3”20 mL),
and dried over MgSO4. The crude product was purified by flash chroma-
tography (n-hexane/AcOEt 6:1, 4:1, 2:1) to give 36 (54 mg, 0.057 mmol,
68%) and 37 as syrups (19 mg, 0.020 mmol, 20%).
1
AcOEt 3:1): 0.28; H NMR (500 MHz, CDCl3, 258C, TMS): d=7.38–7.01
(m, 80H; Ph), 5.200 (m, 1H; C2aHglycerol), 5.198, 5.15 (2”d, 3J(H,H)=
ꢀ
3.5 Hz, 2H; 2”H1’), 5.12 (m, 2H; 2”CH Ph), 5.09 (m, 1H; C2bHglycerol),
ꢀ
ꢀ
4.97 (m, 2H; 2”CH Ph), 4.91–4.81 (m, 4H; 2”CH Ph, 2”H1), 4.81–
ꢀ
ꢀ
4.62 (m, 16H; 16”CH Ph), 4.60–4.43 (m, 8H; 8”CH Ph), 4.34–4.28 (m,
ꢀ
3H; 2”CH Ph, C1aHglycerol), 4.28–4.21 (m, 2H; C1bH, C3aHglycerol), 4.19 (t,
3J(H,H)=3.6 Hz, 1H; H6), 4.18–4.06 (m, 5H; C1aH, C3aH, H6, 2”H2),
4.04–3.94 (m, 7H; 2”H3’, 2”H5’, C1bH, C3bH2glycerol), 3.92 (t, 3J(H,H)=
9.3 Hz, 1H; H4), 3.88 (t, J(H,H)=9.3 Hz, 1H; H3), 3.83 (dd, 3J(H5,H4)=
3
Data for 36: [a]2D0 =+43 (c=0.50 in CHCl3); Rf (n-hexane/AcOEt 2:1):
0.27; 1H NMR (500 MHz, CDCl3, 258C, TMS): d=7.40–7.05 (m, 30H;
Ph), 5.98 (ddd, 3J(H,H)=16.5 Hz, 3J(H,H)=10.5 Hz, 3J(H,H)=6.0 Hz,
1H; CH=CH2), 5.67 (d, 3J(H,H)=3.5 Hz, 1H; H1’), 5.30 (app.d, 3J-
9.6 Hz, 3J(H5,H6)=3.1 Hz, 1H; H5), 3.79–3.70 (m, 5H; H5, H3, H4, 2”
H4’), 3.59 (dd, 3J(H6a’,H6b’)=11.0 Hz, 3J(H6a’,H5’)=2.9 Hz, H6a’), 3.56 (dd,
3J(H6a’,H6b’)=10.6 Hz, 3J(H6a’,H5’)=2.9 Hz, 1H; H6a’), 3.45 (app.d,
3J(H6b’,H6a’)=11.0 Hz, 1H; H6b’), 3.42 (app.d, 3J(H6b’,H6a’)=10.6 Hz, 1H;
H6b’), 3.37 (dd, 3J(H2’,H3’)=10.1 Hz, 3J(H2’,H1’)=3.5 Hz, 1H; H2’), 3.34
(H,H)=16.5 Hz, 1H; CH=CH2), 5.22 (app.d, 3J(H,H)=10.5 Hz, 1H;
3
CH=CH2), 4.99 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.97 (d, 3J(H,H)=
ꢀ
3
3
(dd, J(H2’,H3’)=10.3 Hz, J(H2’,H1’)=3.5 Hz, 1H; H2’), 2.30–2.13 (m, 8H;
4”CH2CO), 1.61–1.48 (m, 8H; 4”CH2CH2CO), 1.32–1.18 (m, 80H; 40”
10.5 Hz, 1H; CH Ph), 4.88 (s, 2H; CH2Ph), 4.83 (d, 3J(H,H)=10.5 Hz,
ꢀ
3
ꢀ
ꢀ
1H; CH Ph), 4.75 (s, 2H; CH2Ph), 4.72 (d, J(H,H)=11.0 Hz, 1H; CH
3
CH2), 0.86 ppm (t, J(H,H)=7.1 Hz, 12H; 4”CH3); 13C NMR (125 MHz,
Ph), 4.67 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.53 (d, 3J(H,H)=12.0 Hz,
3
ꢀ
CDCl3, 258C, TMS): d=173.3, 173.2, 173.0, 172.8 (4”CO), 138.97,
138.93, 138.49, 138.44, 138.29, 138.0, 135.7 (16C; Ph), 128.9–127.6 (80”
CH; Ph), 94.4, 94.1 (2”C1’), 82.0 (C3, C4), 80.1 (2”C3’), 79.6 (C5), 80.4
(C3, 2”C4’), 78.2 (C4, C5), 76.5, 76.2, (6”CH2Ph), 74.4 (C6), 74.2 (2”C2),
73.9, 73.6, 73.1, 72.8 (8”CH2Ph), 72.8 (C6), 71.13 (2”C1), 71.1 (2”C5’),
70.0, 69.8 (2”d, 3J(H,H)=5.8 Hz, 2”C2Hglycerol), 69.5, 69,4 (d, 3J(H,H)=
1H; CH Ph), 4.41 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.26 (d, 3J-
3
ꢀ
ꢀ
ꢀ
(H,H)=12.0 Hz, 1H; CH Ph), 4.23 (app.s, 1H; H2), 4.21–4.01 (m, 5H;
H4, H5’, H6, CH2 CH=CH2), 3.94 (t, 3J(H,H)=9.2 Hz, 1H; H3’), 3.72 (t,
ꢀ
3J(H,H)=9.2 Hz, 1H; H4’), 3.48–3.38 (m, 3H; H1, H3, H5), 3.32 (dd, 3J-
(H2’,H3’)=10.5 Hz, 3J(H2’,H1’)=3.5 Hz, 1H; H2’), 3.24 (app.d, 3J(H,H)=
3
11.0 Hz, 1H; H6a’), 3.16 (app.d, J(H,H)=11.0 Hz, 1H; H6b’), 2.41 ppm (s,
3
7.7 Hz, 2”POCH2Ph), 68.2 (2”C6’), 66.0, 65.8 (2”d, J(H,H)=4.8 Hz, 2”
1H; C2OH); 13C NMR (125 MHz, CDCl3, 258C, TMS): d=138.7, 138.6,
138.21, 138.18, 138.1, 138.0 (6C; Ph), 134.2 (CH=CH2), 128.6–127.4
(30CH, Ph), 117.9 (CH=CH2), 97.7 (C1’), 81.6 (C4), 81.3, 81.0, 80.3, 79.8
(C1, C3, C5, C3’), 78.4 (C4’), 76.0, 75.9, 75.4, 75.0, 74.8, 73.5, 72.9 (6”
C3H2glycerol), 63.53, 63.31 (2”C2’), 61.95, 61.92 (2”C1H2glycerol), 34.3, 34.2
34.13, 34.10 (4”CH2CO), 32.1 (4”CH2CH2CH3), 29.8–29.2 (32”CH2),
25.0 (4”CH2CH2CO), 22.8 (4”CH2CH2CH3), 14.3 ppm (4”CH3);
31P NMR (202 MHz, CDCl3, 258C, TMS): d=ꢀ1.56, ꢀ1.98 ppm; FAB
HRMS: m/z calcd for [C99H128N3O17P+Na]+: 1684.8879; found:
1684.9002.
ꢀ
CH2Ph, C6), 71.2 (CH2 CH=CH2), 67.8 (C5’, C2), 66.6 (C6’), 63.6 ppm
(C2’); FAB HRMS: m/z calcd for [C57H61N3O10+Na]+: 970.4255; found:
970.4313.
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-galactopyranosyl-a-(1!2)-1-O-
(1’,2’-di-O-myristoyl-sn-glycero-3’-(R,S)-benzylphosphatidyl)-3,4,5,6-
tetra-O-benzyl-d-chiro-inositol (34): Compound 34 was obtained as a
colorless syrup and as a mixture of two diastereomers (1:1, 86 mg,
0.052 mmol, 91% with 8% of starting material 32) from 32 (70 mg,
0.070 mmol) by the procedure described for the preparation of 29. The
crude product was fractionated on PLC plates (n-hexane/AcOEt 4:1)
previously treated with Et3N to give 34. [a]2D0 =+42 (c=0.9 in CHCl3); Rf
(n-hexane/AcOEt 3:1): 0.23; 1H NMR (500 MHz, CDCl3, 258C, TMS):
d=7.38–7.20 (m, 80H; Ph), 5.22 (m, 1H; C2aHglycerol), 5.20, 5.15 (2”s,
Data for 37: [a]2D0 =+4.3 (c=1.1 in CHCl3); Rf (n-hexane/AcOEt 2:1):
0.38; 1H NMR (500 MHz, CDCl3, 258C, TMS): d=7.37–7.04 (m, 30H;
Ph), 5.96 (ddd, 3J(H,H)=17.0 Hz, 3J(H,H)=11.0 Hz, 3J(H,H)=6.0 Hz,
1H; CH=CH2), 5.46 (d, 3J(H,H)=3.5 Hz, 1H; H1’), 5.30 (dd, 3J(H,H)=
3
3
17.0 Hz, J(H,H)=1.5 Hz, CH=CH2), 5.19 (app.d, J(H,H)=11.0 Hz, 1H;
3
CH=CH2), 5.02 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.93 (d, 3J(H,H)=
ꢀ
3
ꢀ
ꢀ
10.5 Hz, 1H; CH Ph), 4.88, 4.84 (AB, J(H,H)=11.0 Hz, 2H; 2”CH
Ph), 4.78 (d, J(H,H)=10.5 Hz, 1H; CH Ph), 4.71 (d, 3J(H,H)=11.0 Hz,
3
ꢀ
3
ꢀ
ꢀ
1H; CH Ph), 4.70 (s, 2H; CH2Ph), 4.64 (d, J(H,H)=11.0 Hz, 1H; CH
3
ꢀ
ꢀ
Ph), 4.47 (d, J(H,H)=11.8 Hz, 1H; CH Ph), 4.47–4.40 (m, 1H; CH2
ꢀ
2H; 2”H1’), 5.14–5.05 (m, 3H; 2”CH Ph, C2bHglycerol), 4.98 (m, 2H; 2”
3
CH=CH2), 4.40 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.24 (dd, 3J(H,H)=
ꢀ
3
CH Ph), 4.58 (d, J(H,H)=11.7 Hz, 1H; CH Ph), 4.55 (d, 3J(H,H)=
ꢀ
ꢀ
12.5 Hz, J(H,H)=6.0 Hz, CH2 CH=CH2), 4.15 (d, 3J(H,H)=11.8 Hz,
3
ꢀ
3
ꢀ
ꢀ
11.7 Hz, 1H; CH Ph), 4.53 (d, J(H,H)=11.4 Hz, 1H; CH Ph), 4.96–
4.73 (m, 12H; 10”CH Ph, 2”H1), 4.73–4.65 (m, 3H; 3”CH Ph), 4.49–
4.37 (m, 9H; 9”CH Ph), 4.38–4.28 (m, 3H; 2”CH Ph C1aHglycerol), 4.28–
4.06 (m, 10H; CH Ph, 2”H6, H2, 2”H5’, C1aH, C1bH, C3aH2glycerol), 4.07–
1H; CH Ph), 4.04 (t, 3J(H,H)=9.5 Hz, 1H; H4), 3.98–3.89 (m, 4H; H2,
ꢀ
ꢀ
ꢀ
H3’, H5’, H6), 3.72 (t, 3J(H,H)=9.5 Hz, 1H; H4’), 3.60 (m, 1H; H1), 3.50
(dd, 3J(H2’,H3’)=10.5 Hz, 3J(H2’,H1’)=3.5 Hz, 1H; H2’), 3.44 (dd, 3J-
(H3,H4)=9.5 Hz, 3J(H3,H2)=2.0 Hz, 1H; H3), 3.36 (t, 3J(H,H)=9.5 Hz,
1H; H5), 3.28 (d, 3J(H,H)=6.0 Hz, 1H; C1OH), 3.24 (dd, 3J(H6a’,H6b’)=
11.0 Hz, 3J(H6a’,H5’)=2.5 Hz, 1H; H6a’), 3.07 ppm (app.d, 3J(H,H)=
11.0 Hz, 1H; H6b’); 13C NMR (125 MHz, CDCl3, 258C, TMS): d=138.7,
138.6, 138.4, 138.2, 138.03, 137.99 (6C; Ph), 135.3 (CH=CH2), 128.6–127.4
(30”CH; Ph), 117.2 (CH=CH2), 98.5 (C1’), 82.1 (C4), 81.3 (C5), 81.0 (C3),
80.8, 80.5 (C6, C3’), 78.3 (C4’), 76.8 (C2), 75.9, 75.5, 75.3, 74.9 (4”CH2Ph),
ꢀ
ꢀ
ꢀ
3.91 (m, 6H; C1bH, C3bH2, H2, 2”H4), 3.87–3.68 (m, 10H; 2”H2’, 2”H3’,
2”H4’, 2”H5, 2”H3), 3.53–3.42 (m, 4H; 2”H6a’, 2”H6b’), 2.30–2.18 (m,
8H; 4”CH2CO), 1.61–1.50 (m, 8H; 4”CH2CH2CO), 1.38–1.20 (m, 80H;
40”CH2), 0.89 ppm (t, 3J(H,H)=6.6 Hz, 12H; 4”CH3); 13C NMR
(125 MHz, CDCl3, 258C, TMS): d=173.30, 173.25, 172.89, 172.83 (4”
CO), 139.17, 139.12, 138.9, 138.6, 138.5, 138.35, 138.33, 138.02, 137.96,
137.8, 135.75, 135.70 (16C; Ph), 128.8–127.1 (80”CH; Ph), 94.6, 94.9 (2”
C1’), 82.3 (2”C4), 80.9 (2”C5), 79.9 (2”C3), 77.7 (2”C3’), 76.5, 76.08, 75.3
(9”CH2Ph), 75.2, 74.8 (2”C2), 73.7, 73.2 (4”CH2Ph), 73.69 (2”C4’),
72.67 (2”C6), 72.4 (CH2Ph), 72.02, 71.96 (2”C1), 69.45, 69.39 (2”d, 3J-
(H,H)=7.6 Hz; 2”POCH2Ph), 69.6 (2”C5’), 69.93, 69.75 (2”d, 3J-
(H,H)=5.4 Hz; 2”C2Hglycerol), 69.1 (2”C6’), 65.9, 65.8 (2”d, 3J(H,H)=
ꢀ
74.0 (CH2 CH=CH2), 73.6 (C1), 73.5, 73.0 (2”CH2Ph), 71.0 (C5’), 67.7
(C6’), 64.3 ppm (C2’); FAB HRMS: m/z calcd for [C57H61N3O10+Na]+:
970.4255; found: 970.4276.
O-(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1!6)-1-O-
allyl-2,3,4,5-tetra-O-benzyl-d-myo-inositol (38): A mixture of compound
1524
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 1513 – 1528

Phospholipase Cleavage of d-and l-chiro-Glycosylphosphoinositides
FULL PAPER
23 (187 mg, 0.02 mmol) and NaH (66 mg, 1.67 mmol, 6 equiv) in dried
DMF (11 mL) was stirred for 10 min under Ar. After the mixture had
been cooled to ꢀ158C in an ice/salt bath, BnBr (265 mL, 2.23 mmol,
8 equiv) was added dropwise. The reaction mixture was stirred overnight,
NH4OH was added, and the mixture was then washed successively with a
saturated solution of NH4Cl, H2O, and brine. The organic layer was dried
(MgSO4) and concentrated, and the crude product was purified by flash
chromatography (n-hexane/AcOEt 10:1, AcOEt) to provide 38 as a
white foam (145 mg, 0.14 mmol, 68%). [a]2D0 =+51 (c=0.45 in CHCl3);
10.0 Hz, 3J(H3,H2)=2.0 Hz, 1H; H3), 3.47 (dd, 3J(H3,H4)=10.0 Hz, 3J-
(H3,H2)=2.0 Hz, 1H; H3), 3.42, 3.40 (2”t, 3J(H,H)=10.0 Hz, 2H; 2”
3
3
H5), 3.26–3.18 (m, 4H; 2”H2’, 2”H6a’), 3.11 (dt, J(H6b’,H6a’)=11.0 Hz, J-
(H6b’,H5’)=2.5 Hz, 2H; 2”H6b’), 2.30–2.21 (m, 8H; 4”CH2CO), 1.65–1.50
(m, 8H; 4”CH2CH2CO), 1.34–1.20 (m, 80H; 40”CH2), 0.89 ppm (t, 3J-
(H,H)=7.0 Hz, 12H; 4”CH3); 13C NMR (125 MHz, CDCl3, 258C, TMS):
d=173.3, 173.2, 173.0, 172.8 (4”CO), 139.0, 138.6, 138.2, 138.0 (16C;
Ph), 129.0–127.5 (80”CH; Ph), 98.0, 97.9 (2”C1’), 81.7 (2”C4), 80.8 (2”
C3, 2”C5), 80.7, 80.2 (2”C1, 2”C6), 79.9, 79.8 (2”C3’), 78.3 (2”C4’), 75.6
(2”C2), 76.0–72.9 (14”CH2Ph), 70.5 (2”C5’), 70.2, 69.8 (2”d, 3J(H,H)=
5.4 Hz, 2”C2Hglycerol,), 69.6, 69.4 (2”d, 3J(H,H)=7.8 Hz, 2”POCH2Ph),
67.7 (2”C6’), 66.1, 65.7 (2”d, 3J(H,H)=5.0 Hz; 2”C3H2glycerol), 63.2 (2”
C2’), 61.7, 61.6 (2”C1H2glycerol), 34.3, 34.2, 34.14, 34.09 (4”CH2CO), 32.1
(4”CH2CH2CH3), 29.8–29.2 (32”CH2), 25.0 (4”CH2CH2CO), 22.8 (4”
CH2CH2CH3), 14.3 ppm (4”CH3); 31P NMR (202 MHz, CDCl3, 258C,
TMS): d=ꢀ2.59, ꢀ2.77 ppm; elemental analysis calcd (%) for
C99H128N3O17·H2O: C 70.73, H 7.80, N 2.50; found: C 71.00, H 7.90, N
2.87; FAB HRMS: m/z calcd for [C99H128N3O17P+Na]+: 1684.8879;
found: 1684.8352.
1
Rf (n-hexane/AcOEt 3:1): 0.49; H NMR (300 MHz, CDCl3, 258C, TMS):
d=7.48–7.05 (m, 35H; Ph), 5.96 (ddd, 3J(H,H)=17.2 Hz, 3J(H,H)=
10.5 Hz, 3J(H,H)=5.1 Hz, 1H; CH=CH2), 5.75 (d, 3J(H,H)=3.6 Hz, 1H;
H1’), 5.30 (dd, 3J(H,H)=17.2, 3J(H,H)=1.2 Hz, 1H; CH=CH2), 5.22
(app.d, 3J(H,H)=10.5 Hz, CH=CH2), 5.05 (d, 3J(H,H)=10.8 Hz, 1H;
3
ꢀ
ꢀ
CH Ph), 5.00 (d, J(H,H)=10.5 Hz, 1H; CH Ph), 4.88 (2 AB, 4H; 2”
3
ꢀ
ꢀ
CH2Ph), 4.87 (m, 1H; CH Ph), 4.84 (d, J(H,H)=10.5 Hz, 1H; CH Ph),
3
ꢀ
ꢀ
4.74 (d, J(H,H)=11.1 Hz, 1H; CH Ph), 4.71–4.62 (m, 4H; CH Ph),
4.56 (d, J(H,H)=12.0 Hz, 1H; CH Ph), 4.40 (d, 3J(H,H)=10.8 Hz, 1H;
3
ꢀ
CH Ph), 4.26 (t, 3J(H,H)=9.6 Hz, 1H; H6), 4.24 (d, 3J(H,H)=12.0 Hz,
ꢀ
1H; CH Ph), 4.16 (t, 3J(H,H)=9.6 Hz, 1H; H4), 4.10–4.00 (m, 4H;
ꢀ
2-Amino-2-deoxy-d-glucopyranosyl-a-(1!6)-1-O-(1’,2’-di-O-myristoyl-
CH2 CH=CH2, H2, H5’) 3.97 (t, 3J(H,H)=9.8 Hz, 1H; H3’), 3.74 (t, 3J-
sn-glycero-3’-phosphatidyl)-d-myo-inositol (7):[23b]
A suspension of 21
ꢀ
(H,H)=9.8 Hz, 1H; H4’), 3.48–3.30 (m, 3H; H1, H3, H5), 3.33 (dd, 3J-
(H2’,H3’)=9.8 Hz, 3J(H2’,H1’)=3.6 Hz, 1H; H2’), 3.24 (app.d, 3J(H,H)=
9.8 Hz, 1H; H6a’), 3.12 ppm (dd, 3J(H6b’,H6a’)=9.8 Hz, 3J(H6b’,H5’)=
1.8 Hz, 1H; H6b’); 13C NMR (75 MHz, CDCl3, 258C, TMS): d=139.2,
139.0, 138.6 (7C; Ph), 134.7 (CH=CH2), 128.8–127.6 (35C; Ph), 117.3
(CH=CH2), 98.1 (C1’), 82.4 (C4), 82.3, 81.7, 81.2 (C1, C3, C5), 80.6 (C3’),
78.6 (C4’), 76.2, 76.0 (2”CH2Ph), 75.8 (C6), 75.7, 75.1, 74.5, 73.8, 73.3 (5”
(12 mg, 0.007 mmol) in an AcOEt/THF/EtOH/H2O mixture (2:1:1:0.1,
3.5 mL) was stirred for 16 h under H2 in the presence of Pd on charcoal
(10%, 22 mg, 0.021 mmol) as catalyst. The reaction mixture was filtered
over celite, washed with MeOH (10 mL), and carefully neutralized with a
solution of NaOH in MeOH (0.1%). The slurry was evaporated to dry-
ness and the residue was purified on Sephadex LH-20 in MeOH to give 7
(6 mg, 0.007 mmol, 98%) as a white dust. Rf (tBuOH/EtOH/aqueous
NH3 30%/H2O 4:2:0.5:1): 0.19. NMR experiments were performed at
pH 7.6; 1H NMR (500 MHz, [D4]MeOH, 258C, TMS): d=5.50 (d, 3J-
(H,H)=4.0 Hz, 1H; H1’), 5.25 (m, 1H; C2Hglycerol), 4.45 (dd, 3J(H,H)=
ꢀ
CH2Ph), 73.2, 71.3, 70.4 (C2, C5’, CH2 CH=CH2), 68.0 (C6’), 63.9 ppm
(C2’); FAB HRMS: m/z calcd for [C64H67N3O10+Na]+: 1060.4724; found:
1060.4791.
3
3
3
O-(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-a-d-glucopyranosyl)-(1!6)-
2,3,4,5-tetra-O-benzyl-d-myo-inositol (39):[34] Compound 39 (41 mg,
0.041 mmol, 72%) was obtained as a colorless syrup from 38 (59 mg,
0.057 mmol) by the same experimental procedure as that used for the
12.0 Hz, J(H,H)=3.0 Hz, 1H; C1Hglycerol), 4.20 (dd, J(H,H)=12.0 Hz, J-
(H,H)=6.5 Hz, 1H; C1Hglycerol), 4.17–4.11 (m, 2H; H1, H5’), 4.09 (t, 3J-
(H,H)=2.5 Hz, 1H; H2), 4.04 (m, 2H; C3H2glycerol), 3.97 (t, 3J(H,H)=
3
3
9.5 Hz, 1H; H6), 3.82 (dd, J(H6a’,H6b’)=11.5 Hz, J(H6a’,H5’)=2.5 Hz, 1H;
1
preparation of 27. H NMR (500 MHz, CDCl3, 258C, TMS): d=7.45–7.05
H6a’), 3.80 (t, 3J(H,H)=9.5 Hz, 1H; H3’), 3.71 (dd, 3J(H6b’,H6a’)=11.5 Hz,
(m, 35H; Ph), 5.45 (d, 3J(H,H)=3.0 Hz, 1H; H1’), 5.03 (d, 3J(H,H)=
3J(H6b’,H5’)=4.5 Hz, 1H; H6b’), 3.67 (t, J(H,H)=9.5 Hz, 1H; H4), 3.40 (t,
3
3
11.0 Hz, 1H; CH Ph), 4.99 (d, J(H,H)=11.5 Hz, 1H; CH Ph), 4.96 (d,
3J(H,H)=9.5 Hz, 1H; H4’), 3.37 (dd, 3J(H3,H4)=9.5 Hz, 3J(H3,H2)=
ꢀ
ꢀ
J(H,H)=10.5 Hz, 1H; CH Ph), 4.87 (AB, 2H; CH2Ph), 4.80 (d, 3J-
(H,H)=10.5 Hz, 1H; CH Ph), 4.77 (d, J(H,H)=11.5 Hz, 1H; CH Ph),
4.74 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.70 (s, 2H; CH2Ph), 4.64 (d, J-
2.5 Hz, 1H; H3), 3.28 (t, J(H,H)=9.5 Hz, 1H; H5), 3.08 (dd, J(H2’,H3’)=
3
3
3
ꢀ
3
9.5 Hz, 3J(H2’,H1’)=4.0 Hz, 1H; H2’), 2.34 (m, 4H; 2”CH2CO), 1.60 (m,
ꢀ
ꢀ
3
3
3
ꢀ
4H; 2”CH2CH2CO), 1.30 (app.s, 40H; 20”CH2), 0.90 ppm (t, J(H,H)=
3
(H,H)=11.0 Hz, 1H; CH Ph), 4.46 (d, J(H,H)=12.0 Hz, 1H; CH Ph),
7.0 Hz, 6H; 2”CH3); 13C NMR and HMQC (125 and 500 MHz,
[D4]MeOH, 258C, TMS): d=97.2 (C1’), 79.4 (C6), 78.4 (C1), 74.9 (C5),
74.2 (C4), 73.5 (C5’), 73.4 (C2), 72.5 (C3), 72.0 (C3’), 71.9 (C2Hglycerol), 71.5
(C4’), 65.0 (C3H2glycerol), 62.5 (C1H2glycerol), 62.1 (C6’), 56.2 (C2’), 35.1, 35.0
(2”CH2CO), 33.1 (2”CH2CH2CH3), 30.8–30.2 (16”CH2), 26.0 (2”
CH2CH2CO), 23.8 (2”CH2CH2CH3), 14.4 ppm (2”CH3); 31P NMR
(202 MHz, [D4]MeOD, 258C, TMS): d=ꢀ0.58 ppm.
ꢀ
ꢀ
4.41 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.14 (d, 3J(H,H)=12.0 Hz, 1H;
3
ꢀ
CH Ph), 4.10 (t, 3J(H,H)=9.1 Hz, 1H; H4), 4.01 (s, 1H; H2), 4.03–3.91
ꢀ
(m, 3H; H6, H3’, H5’), 3.73 (t, 3J(H,H)=9.5 Hz, 1H; H4’), 3.66 (m, 1H;
H1), 3.50 (m, 2H; H2’, H3), 3.38 (t, 1H; 3J(H,H)=9.1 Hz, 1H; H5), 3.22
3
(m, 2H; C1OH, H6a’), 3.06 ppm (app.d, J(H,H)=11.0 Hz, 1H; H6b’).
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-d-glucopyranosyl-a-(1!6)-1-O-(1’,2’-
di-O-myristoyl-sn-glycero-3’-(R,S)-benzylphosphatidyl)-1,4,5,6-tetra-O-
benzyl-d-myo-inositol (40): Compound 40 was obtained as a colorless
syrup and as a mixture of two diastereomers (1:1, 31 mg, 0.0188 mmol,
82%) from 39 (23 mg, 0.023 mmol) by the procedure described for the
preparation of 29. The crude product was fractionated on PLC plates (n-
hexane/AcOEt 3:1) previously treated with Et3N to give 40. [a]2D0 =+37
(c=0.31 in CHCl3); Rf (n-hexane/AcOEt 2:1): 0.54; 1H NMR (500 MHz,
CDCl3, 258C, TMS): d=7.43–7.01 (m, 80H; Ph), 5.45 (d, 3J(H,H)=
3.5 Hz, 1H; H1’), 5.42 (d, 3J(H,H)=3.5 Hz, 1H; H1’), 5.24 (m, 1H; C2aH-
2-Amino-2-deoxy-d-glucopyranosyl-a-(1!3)-1-O-(1’,2’-di-O-myristoyl-
sn-glycero-3’-phosphatidyl)-l-chiro-inositol (9): Compound
9 (12 mg,
0.0131 mmol, 98%) was obtained as
a white dust from 29 (23 mg,
0.0138 mmol) by the procedure described for the preparation of 7. Purifi-
cation was on Sephadex LH-20 in MeOH/CH2Cl2 (9:1). [a]2D0 =+13 (c=
0.19 in MeOH); Rf (tBuOH/EtOH/aqueous NH3 30%/H2O 4:2:0.5:1):
0.30. NMR experiments were performed at pH 7.6; 1H NMR (500 MHz,
[D4]MeOH, 258C, TMS): d=5.32 (d, 3J(H,H)=3.5 Hz, 1H; H1’), 5.24
3
3
(ddd, J(H,H)=6.5 Hz, J(H,H)=5.5 Hz, 3J(H,H)=3.5 Hz, 1H; C2Hglycerol),
4.46 (dd, 3J(H,H)=12.0 Hz, 3J(H,H)=3.5 Hz, 1H; C1Hglycerol), 4.42 (ddd,
3J(H1,HP)=8.5 Hz, 3J(H1,H2)=3.5 Hz, 3J(H1,H6)=2.5 Hz, 1H; H1), 4.20
(dd, 3J(H,H)=12.0 Hz, 3J(H,H)=6.5 Hz, 1H; C1Hglycerol), 4.06 (ddd, 3J-
(H,H)=11.0 Hz, 3J(H,H)=5.5 Hz, 3J(H,H)=5.5 Hz, 1H; C3Hglycerol), 4.03
(ddd, 3J(H5’,H4)=9.5 Hz, 3J(H5’,H6b’)=5.0 Hz, 3J(H5’,H6a’)=2.5 Hz, 1H;
ꢀ
glycerol), 5.22–5.15 (m, 2H; 2”CH Ph), 5.11–4.97 (m, 6H; C2bHglycerol, 5”
3
CH Ph), 4.94 (d, J(H,H)=10.5 Hz, 1H; CH Ph), 4.93 (d, 3J(H,H)=
ꢀ
ꢀ
ꢀ
10.5 Hz, 2H; 2”CH Ph), 4.87 (AB, 2H; CH2Ph), 4.86 (AB, 2H;
CH2Ph), 4.77 (app.d, J(H,H)=10.5 Hz, 3H; 3”CH Ph), 4.74–4.59 (m,
3
ꢀ
3
ꢀ
ꢀ
7H; 7”CH Ph), 4.51 (app.d, J(H,H)=12.0 Hz, 2H; 2”CH Ph), 4.47
(brt, 3J(H,H)=2.0 Hz, 1H; H2), 4.48 (brt, 3J(H,H)=2.0 Hz, 1H; H2),
H5’), 4.05 (m, 1H; H6), 4.00 (ddd, J(H,H)=11.0 Hz, J(H,H)=5.5 Hz, J-
3
3
3
3
4.45–4.41 (m, 1H; H1), 4.370 (d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.367
(H,H)=5.5 Hz, 1H; C3Hglycerol), 3.96 (dd, 3J(H2,H3)=9.0 Hz, 3J(H2,H1)=
ꢀ
3
3
3
ꢀ
(d, J(H,H)=11.0 Hz, 1H; CH Ph), 4.34–4.27 (m, 4H; H1, 2”H6, C1a-
3.5 Hz, 1H; H2), 3.82 (dd, J(H6a’,H6b’)=12.0 Hz, J(H6a’,H5’)=2.5 Hz, 1H;
H6a’), 3.78 (dd, 3J(H3’,H2’)=10.5 Hz, 3J(H3’,H4’)=9.5 Hz, 1H; H3’), 3.73
(dd, 3J(H6b’,H6a’)=12.0 Hz, 3J(H6b’,H5’)=5.0 Hz, 1H; H6b’), 3.72 (dd, 3J-
(H5,H4)=9.0 Hz, 3J(H5,H6)=4.0 Hz, 1H; H5), 3.67 (t, 3J(H,H)=9.0 Hz,
1H; H3), 3.59 (t, 3J(H,H)=9.0 Hz, 1H; H4), 3.41 (t, 3J(H,H)=9.5 Hz,
3
ꢀ
Hglycerol), 4.21 (app.d, J(H,H)=12.0 Hz, 2H; 2”CH Ph), 4.23–4.12 (m,
4H; C3aH2, C1aH, C1bHglycerol), 4.10 (t, 3J(H,H)=10.0 Hz, 2H; 2”H4),
ꢀ
4.06–4.00 (m, 5H; C3bH2glycerol, POCH Ph, 2”H5’), 3.98–3.92 (m, 4H;
3
ꢀ
2”H3’, POCH Ph, C1bHglycerol), 3.70 (m, 2H; 2”H4’), 3.52 (dd, J(H3,H4)=
Chem. Eur. J. 2006, 12, 1513 – 1528
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1525

M. Martín-Lomas et al.
1H; H4’), 3.07 (dd, 3J(H2’,H3’)=10.5 Hz, 3J(H2’,H1’)=3.5 Hz, 1H; H2’),
(5 mg, 0.0057 mmol, 94%) was obtained as a white dust from 22 (10 mg,
0.006 mmol) by the procedure described for compound 7. Compound 4
was purified on Sephadex LH-20 in MeOH. [a]2D0 =+18 (c=0.12 in
CHCl3); Rf (tBuOH/EtOH/aqueous NH3 (30%)/H2O 4:2:0.5:1): 0.25.
NMR experiments were performed at pH 6.5; 1H NMR ([D4]methanol,
500 MHz): d=5.47 (d, 3J(H,H)=4.0 Hz, 1H; H1’), 5.18 (m, 1H; C2Hgly-
cerol), 4.46 (td, 3J(H2,HP)=3J(H2,H3)=9.5 Hz, 3J(H2,H1)=3.0 Hz, 1H;
H2), 4.39 (dd, 3J(H,H)=12.0 Hz, 3J(H,H)=3.0 Hz, 1H; C1Hglycerol), 4.29
3
3
2.36 (t, J(H,H)=7.5 Hz, 4H; CH2CO), 2.33 (t, J(H,H)=7.5 Hz, 4H; 2”
CH2CO), 1.58 (m, 4H; 2”CH2CH2CO), 1.29 (app.s, 40H; 20”CH2),
0.90 ppm (t, 3J(H,H)=6.5 Hz, 6H; 2”CH3); 13C NMR and HMQC (125
and 500 MHz, [D4]MeOH, 258C, TMS): d=98.2 (C1’), 83.5 (C3), 76.8
(C1), 73.0 (C5’), 72.1 (C4), 71.7 (C5), 71.3 (C2, C6), 71.0 (C3’), 70.8 (C2Hglycerol),
70.4 (C4’), 63.7 (C3H2glycerol), 62.4 (C1H2glycerol), 60.6 (C6’), 55.5 (C2’), 36.0,
35.8 (2”CH2CO), 34.0 (2”CH2CH2CH3), 31.7–31.1 (16”CH2), 26.9 (2”
CH2CH2CO), 24.6 (2”CH2CH2CH3), 15.4 ppm (2”CH3); 31P NMR
(121 MHz, [D4]MeOD, 258C, TMS): d=ꢀ0.09 ppm; FAB HRMS: m/z
calcd for [C43H82NO17P+Na]+: 938.5218; found: 938.5239.
3
3
3
(app.t, J(H,H)=6.0 Hz, 1H; H5’), 4.13 (dd, J(H,H)=12.0 Hz, J(H,H)=
6.5 Hz, 1H; C1Hglycerol), 4.00 (t, 3J(H,H)=4.0 Hz, 1H; H1), 3.99, 4.06 (2”
t, 3J(H,H)=5.5 Hz, 2H; 2”C3Hglycerol), 3.92 (dd, 3J(H,H)=10.5 Hz, 3J-
3
(H,H)=3.0 Hz, 1H; H3’), 3.85 (m, 2H; H4’, H6), 3.84 (t, J(H,H)=9.5 Hz,
2-Amino-2-deoxy-d-galactopyranosyl-a-(1!3)-1-O-(1’,2’-di-O-myristoyl-
sn-glycero-3’-(R,S)-phosphatidyl)-l-chiro-inositol (10): Compound 10
(9 mg, 0.01 mmol, 98%) was obtained as a white dust from 30 (16 mg,
0.01 mmol) by the procedure described for compound 7. Compound 10
was purified on Sephadex LH-20 in MeOH. [a]2D0 =ꢀ32 (c=0.03 in
MeOH); Rf (nBuOH/EtOH/aqueous NH3 (30%)/H2O 2:2:1:3): 0.62.
NMR experiments were performed at pH 6.7; 1H NMR (500 MHz,
1H; H3), 3.72–3.57 (m, 4H; H4, H5, H6a’, H6b’), 3.35 (dd, 3J(H2’,H3’)=
11.0 Hz, 3J(H2’,H1’)=4.0 Hz, 1H; H2’), 2.28 (t, 3J(H,H)=7.0 Hz, 2H;
CH2CO), 2.24 (t, 3J(H,H)=7.5 Hz, 2H; CH2CO), 1.54 (m, 4H; 2”
CH2CH2CO), 1.23 (app.s, 40H; 20”CH2), 0.84 ppm (t, 3J(H,H)=6.5 Hz,
6H; 2”CH3); HMQC (500 MHz, [D4]MeOD, 258C, TMS): d=96.4 (C1’),
79.2 (C3), 77.4 (C2), 72.8 (C4), 72.38 (C6), 72.35 (C1), 71.7 (C5’), 71.6 (C5),
71.2 (C2Hglycerol), 69.3 (C4’), 68.1 (C3’), 64.3 (C1H2glycerol), 63.0 (C3H2glycerol),
61.7 (C6’), 51.9 (C2’), 34.3 (2”CH2CO), 32.8 (2”CH2CH2CH3), 29.1–31.4
(16”CH2), 25.4 (2”CH2CH2CO), 23.2 (2”CH2CH2CH3), 13.8 ppm (2”
CH3); 31P NMR (121 MHz, [D4]MeOD, 258C, TMS): d=ꢀ0.42 ppm;
FAB HRMS: m/z calcd for [C43H82NO17P+Na]+: 938.5218; found:
938.5164.
3
[D4]MeOD, 258C, TMS): d=5.28 (d, J(H,H)=3.5 Hz, 1H; H1’), 5.24 (m,
3
3
1H; C2Hglycerol), 4.46 (dd, J(H,H)=12.0 Hz, J(H,H)=3.5 Hz, 1H; C1Hgly-
cerol), 4.40 (ddd, 3J(H1,HP)=8.5 Hz, 3J(H1,H6)=3J(H1,H2)=4.5 Hz, 1H;
H1), 4.24 (t, 3J(H,H)=6.0 Hz, 1H; H5’), 4.20 (dd, 3J(H,H)=12.0 Hz, 3J-
(H,H)=6.0 Hz, 1H; C1Hglycerol), 4.07 (m, 1H; C3Hglycerol), 4.06 (t, 3J-
(H,H)=4.5 Hz, 1H; H6), 4.00 (dd, 3J(H,H)=11.0 Hz, 3J(H,H)=5.5 Hz,
1H; C3Hglycerol), 3.96 (app.d, 3J(H,H)=9.5 Hz, 1H; H2), 3.90 (d, 3J-
(H,H)=3.5 Hz, 1H; H4’), 3.82 (app.dd, 3J(H3’,H2’)=10.5 Hz, 3J(H3’,H4’)=
3.5 Hz, 1H; H3’), 3.77–3.70 (m, 3H; H5, H6a’, H6b’), 3.67 (t, 3J(H,H)=
9.5 Hz, 1H; H3), 3.59 (t, 3J(H,H)=9.5 Hz, 1H; H4), 3.26 (m, 1H; H2’),
2.35 (t, 3J(H,H)=7.5 Hz, 2H; CH2CO), 2.32 (t, 3J(H,H)=7.5 Hz, 2H;
CH2CO), 1.60 (m, 4H; 2”CH2CH2CO), 1.29 (app.s, 40H; 20”CH2),
0.90 ppm (t, 3J(H,H)=6.5 Hz, 6H; 2”CH3); HMQC (500 MHz,
[D4]MeOD, 258C, TMS): d=100.8 (C1’), 84.0 (C3), 77.7 (C1), 74.2 (C6),
73.2 (C4), 72.5 (C5’), 72.0 (C2, C5), 71.6 (C2Hglycerol), 70.5 (C3’), 70.0 (C4’),
64.9 (C3H2glycerol), 63.5 (C1H2glycerol), 62.5 (C6’), 49.0 (C2’), 34.8 (2”
CH2CO), 31.5–29.6 (18”CH2), 26.0 (2”CH2CH2CO), 23.2 (2”
CH2CH2CH3), 14.4 ppm (2”CH3); 31P NMR (202 MHz,[D4]MeOD, 258C,
TMS): d=ꢀ0.32 ppm; FAB HRMS: m/z calcd for [C43H82NO17P+Na]+:
938.5218; found: 938.5232.
2-Amino-2-deoxy-d-glucopyranosyl-a-(1!2)-1-O-(1’,2’-di-O-myristoyl-
sn-glycero-3’-(R,S)-phosphatidyl)-d-chiro-inositol (5): Compound
5
(7 mg, 0.0076 mmol, 42%) was obtained as a white dust from 33 (10 mg,
0.006 mmol) by the procedure described for compound 7. Compound 5
was purified on Sephadex LH-20 in MeOH. [a]2D0 =ꢀ64 (c=0.02 in
MeOH); Rf (tBuOH/EtOH/aqueous NH3 (30%)/H2O 4:2:0.5:1): 0.13.
NMR experiments were performed at pH 6.8; 1H NMR (500 MHz,
3
[D4]MeOD, 258C, TMS): d=5.33 (d, J(H,H)=4.0 Hz, 1H; H1’), 5.24 (m,
1H; C2Hglycerol), 4.57 (m, 1H; H1), 4.43 (dd, 3J(H,H)=11.9 Hz, 3J(H,H)=
3.2 Hz, 1H; C1Hglycerol), 4.18 (dd, 3J(H,H)=12.1 Hz, 3J(H,H)=6.9 Hz,
3
1H; C1Hglycerol), 4.05 (t, J(H,H)=3.7 Hz, 1H; H6), 4.04–3.93 (m, 4H; H2,
H5’, C3H2glycerol), 3.90 (t, 3J(H,H)=9.8 Hz, H3’), 3.82 (dd, 3J(H6a’,H6b’)=
12.1 Hz, 3J(H6a’,H5’)=2.3 Hz, 1H; H6a’), 3.75–3.69 (m, 2H; H5, H6b’), 3.67
(t, 3J(H,H)=9.5 Hz, 1H; H3), 3.60 (t, 3J(H,H)=9.5 Hz, 1H; H4), 3.40 (t,
3J(H,H)=9.8 Hz, 1H; H4’), 3.12 (dd, 3J(H2’,H3’)=9.8 Hz, 3J(H2’,H1’)=
4.0 Hz, 1H; H2’), 2.36 (t, 3J(H,H)=7.3 Hz, 2H; CH2CO), 2.33 (t, 3J-
(H,H)=7.3 Hz, 2H; CH2CO), 1.61 (m, 4H; 2”CH2CH2CO), 1.29 (m,
40H; 20”CH2), 0.90 ppm (t, 3J(H,H)=7.0 Hz, 6H; 2”CH3); HSQC
(500 MHz, [D4]MeOD, 258C, TMS): d=93.3 (C1’), 75.3 (C2), 74.4 (C4),
73.5 (C5’), 72.8 (C3), 72.3 (C1), 72.0 (C5), 71.9 (C6), 71.8 (C3’), 71.6 (C2Hgly-
cerol), 71.3 (C4’), 64.8 (C1H2glycerol), 63.3 (C3H2glycerol), 61.8 (C6’), 55.1 (C2’),
34.8 (2”CH2CO), 32.9 (2”CH2CH2CH3), 29.7–31.0 (16”CH2), 25.8 (2”
CH2CH2CO), 23.5 (2”CH2CH2CH3), 14.3 ppm (2”CH3); 31P NMR
(202 MHz, [D4]MeOD, 258C, TMS): d=ꢀ0.09 ppm; FAB HRMS: m/z
calcd for [C43H82NO17P+Na]+: 938.5218; found: 938.5159.
2-Amino-2-deoxy-d-glucopyranosyl-a-(1!3)-2-O-(1’,2’-di-O-myristoyl-
sn-glycero-3’-(R,S)-phosphatidyl)-l-chiro-inositol (3): Compound 10
(7 mg, 0.0079 mmol, 69%) was obtained as a white dust from 21 (19 mg,
0.0114 mmol) by the procedure described for compound 7. Compound 3
was purified on Sephadex LH-20 in MeOH. [a]2D0 =ꢀ21 (c=1.15 in
MeOH); Rf (tBuOH/EtOH/aqueous NH3 (30%)/H2O 4:2:0.5:1): 0.44.
NMR experiments were performed at pH 7.7; 1H NMR (500 MHz,
3
[D4]MeOD, 258C, TMS): d=5.51 (d, J(H,H)=3.2 Hz, 1H; H1’), 5.26 (m,
1H; C2Hglycerol), 4.55 (td, 3J(H2,HP)=3J(H2,H3)=9.0 Hz, 3J(H2,H1)=
2.5 Hz, 1H; H2), 4.47 (dd, 3J(H,H)=12.5 Hz, 3J(H,H)=3.5 Hz, 1H;
C1Hglycerol), 4.21 (dd, 3J(H,H)=12.0 Hz, 3J(H,H)=6.5 Hz, 1H; C1Hglycerol),
4.15 (m, 1H; H5’), 4.098 (t, 3J(H,H)=3.0 Hz, 1H; H1), 4.08, 4.06 (2”m,
2H; C3H2glycerol), 3.94 (t, 3J(H,H)=3.5 Hz, 1H; H6), 3.91 (t, 3J(H,H)=
9.0 Hz, 1H; H3), 3.85 (app.d, 3J(H,H)=11.5 Hz, 1H; H6a’), 3.82 (t, 3J-
(H,H)=10.5 Hz, 1H; H3’), 3.76 (dd, 3J(H5,H4)=9.5 Hz, 3J(H,H)=3.5 Hz,
1H; H5), 3.72 (dd, 3J(H6b’,H6a’)=11.5 Hz, 3J(H6b’,H5’)=4.5 Hz, 1H; H6b’),
3.67 (t, 3J(H,H)=9.0 Hz, 1H; H4), 3.41 (t, 3J(H,H)=9.5 Hz, 1H; H4’),
3.07 (dd, 3J(H2’,H3’)=10.5 Hz, 3J(H2’,H1’)=3.2 Hz, 1H; H2’), 2.37 (t, 3J-
2-Amino-2-deoxy-d-galactopyranosyl-a-(1!2)-1-O-(1’,2’-di-O-myristoyl-
sn-glycero-3’-(R,S)-phosphatidyl)-d-chiro-inositol (6)[23b]
: Compound 6
(8 mg, 0.0082 mmol, 37%) was obtained as a white dust from 31 (37 mg,
0.022 mmol) by the procedure described for compound 7. Compound 6
was purified on Sephadex LH-20 in MeOH. NMR experiments were per-
formed at pH 6.3; [a]2D0 =+35 (c=0.24 in MeOH); Rf (tBuOH/EtOH/
aqueous NH3 (30%)/H2O 4:2:0.5:1): 0.07; 1H NMR (500 MHz,
3
[D4]MeOD, 258C, TMS): d=5.36 (d, J(H,H)=4.0 Hz, 1H; H1’), 5.25 (m,
3
(H,H)=7.5 Hz, 2H; CH2CO), 2.34 (t, J(H,H)=7.5 Hz, 2H; 2”CH2CO),
1H; C2Hglycerol), 4.59 (m, 1H; H1), 4.45 (dd, 3J(H,H)=12.0 Hz, 3J(H,H)=
3.2 Hz, 1H; C1Hglycerol), 4.20 (m, 2H; H5’, C1Hglycerol), 4.08 (dd, 3J(H,H)=
10.6 Hz, 3J(H,H)=3.0 Hz, 1H; H3’), 4.05–3.95 (m, 4H; H6, H2, C3H2gly-
cerol), 3.93 (app.s, 1H; H4’), 3.80–3.70 (m, 3H; H6a’, H6b’, H5), 3.68 (t, 3J-
(H,H)=9.3 Hz, 1H; H3), 3.62 (t, 3J(H,H)=9.3 Hz, 1H; H4), 3.44 (dd, 3J-
(H2’,H3’)=10.6 Hz, 3J(H2’,H1’)=4.0 Hz, 1H; H2’), 2.37 (t, 3J(H,H)=
7.5 Hz, 2H; CH2CO), 2.34 (t, 3J(H,H)=7.5 Hz, 2H; CH2CO), 1.62 (m,
1.62 (m, 4H; 2”CH2CH2CO), 1.29 (app.s, 40H; 20”CH2), 0.92 ppm (t,
3J(H,H)=6.5 Hz, 6H; 2”CH3); HMQC (500 MHz, [D4]MeOD, 258C,
TMS): d=99.0 (C1’), 80.9 (C3), 78.0 (C2), 74.1 (C1), 73.7 (C5), 73.0 (C6),
72.9 (C5’), 72.3 (C3’), 71.84 (C2Hglycerol), 71.80 (C4’), 71.5 (C4), 65.0 (C1H2glycerol),
63.7 (C3H2glycerol), 62.4 (C6’), 56.5 (C2’), 34.9 (2”CH2CO), 33.0 (2”
CH2CH2CH3), 31.8–29.2 (16”CH2), 26.0 (2”CH2CH2CO), 23.6 (2”
CH2CH2CH3), 14.4 ppm (2”CH3); 31P NMR (121 MHz, [D4]MeOD,
258C, TMS): d=ꢀ0.74 ppm; FAB HRMS: m/z calcd for
[C43H82NO17P+Na]+: 938.5218; found: 938.5155.
3
4H; 2”CH2CH2CO), 1.31 (app.s, 40H; 20”CH2), 0.92 ppm (t, J(H,H)=
7.2 Hz, 6H; 2”CH3); HSQC (500 MHz, [D4]MeOD, 258C, TMS): d=
93.7 (C1’), 75.5 (C5), 74.5 (C3), 73.0 (C4), 72.5 (C6), 72.3 (C5’), 72.03 (C2),
71.95 (C1), 71.8 (C2Hglycerol), 69.8 (C4’), 68.7 (C3’), 64.8 (C1H2glycerol), 63.4
(C3H2glycerol), 62.3 (C6’), 52.0 (C2’), 34.9 (2”CH2CO), 33.1 (2”
2-Amino-2-deoxy-d-galactopyranosyl-a-(1!3)-2-O-(1’,2’-di-O-myristoyl-
sn-glycero-3’-(R,S)-phosphatidyl)-l-chiro-inositol (4): Compound
4
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Phospholipase Cleavage of d-and l-chiro-Glycosylphosphoinositides
FULL PAPER
CH2CH2CH3), 29.3–31.7 (16”CH2), 25.8 (2”CH2CH2CO), 24.2 (2”
CH2CH2CH3), 14.3 ppm (2”CH3); 31P NMR (202 MHz, [D4]MeOD,
258C, TMS): d=ꢀ0.10 ppm; FAB HRMS: m/z calcd for
[C43H82NO17P+Na]+: 938.5218; found: 938.5210.
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Received: July 18, 2005
Published online: November 28, 2005
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