New sugar-based permeant analogs of D-Myo-inositol 1,4,5-trisphosphate mimicking the effect of vasopressin: Synthesis and biologic evaluation

Article abstract of DOI:10.1081/CAR-200068070

On the basis of the xylose-inositol analogy, a series of permeant analogs of D-myo-inositol 1,4,5-trisphosphate (InsP₃) have been synthesized by various esterifications of the phosphate groups. Their ability to cross the cell membrane has been

Full text of DOI:10.1081/CAR-200068070

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New Sugar‐Based Permeant Analogs
of D‐Myo‐Inositol 1,4,5‐Trisphosphate
Mimicking the Effect of Vasopressin:
Synthesis and Biologic Evaluation
Françoise Chrétien a , Fabien Roussel a , Mauricette Hilly b
,
Jean‐Pierre Mauger b & Yves Chapleur a
a Groupe SUCRES , UMR 7565 CNRS, Université Henri Poincaré‐Nancy
I , Nancy Vandoeuvre, France
b U INSERM 442 , Université de Paris‐Sud , Orsay, France
Published online: 16 Aug 2006.
To cite this article: Françoise Chrétien , Fabien Roussel , Mauricette Hilly , Jean‐Pierre Mauger & Yves
Chapleur (2005) New Sugar‐Based Permeant Analogs of D‐Myo‐Inositol 1,4,5‐Trisphosphate Mimicking
the Effect of Vasopressin: Synthesis and Biologic Evaluation , Journal of Carbohydrate Chemistry,
24:4-6, 549-581
To link to this article: http://dx.doi.org/10.1081/CAR-200068070
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Journal of Carbohydrate Chemistry, 24:549–581, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1081/CAR-200068070
New Sugar-Based Permeant
Analogs of D-Myo-Inositol
1,4,5-Trisphosphate
Mimicking the Effect
of Vasopressin: Synthesis
and Biologic Evaluation
´
Franc¸ oise Chretien and Fabien Roussel
´
´
Groupe SUCRES, UMR 7565 CNRS, Universite Henri Poincare-Nancy I,
Nancy Vandoeuvre, France
Mauricette Hilly and Jean-Pierre Mauger
´
U INSERM 442, Universite de Paris-Sud, Orsay, France
Yves Chapleur
´
´
Groupe SUCRES, UMR 7565 CNRS, Universite Henri Poincare-Nancy I,
Nancy Vandoeuvre, France
On the basis of the xylose-inositol analogy, a series of permeant analogs of D-myo-inosi-
tol 1,4,5-trisphosphate (InsP3) have been synthesized by various esterifications of the
phosphate groups. Their ability to cross the cell membrane has been tested on vasopres-
sin cells. Very fast liberation of calcium occurs when active analogs are introduced in the
extracellular medium on intact cells. Membrane crossing as well as hydrolysis of the
phosphate is very rapid using acyloxymethyl esterification of all the phosphate
groups. The free compounds behave in the cell like InsP3. One of the analogs
Received February 22, 2005; accepted April 6, 2005.
Dedicated to the memory of Jacques H. van Boom and his outstanding contribution
to the field.
´
Address correspondence to Franc¸oise Chretien, Groupe SUCRES, UMR 7565 CNRS,
´
´
Universite Henri Poincare-Nancy I, BP 239, F-54506 Nancy Vandoeuvre, France.
E-mail: francoise.chretien@sucres.uhp-nancy.fr
549

´
F. Chretien et al.
550
prepared this way behaves like vasopressin for rat hepatocyte cells expressing
vasopressin receptor.
Keywords Inositol 1,4,5-trisphosphate, Permeant analogs, Carbohydrate, Vaso-
pressin, Cell, Calcium liberation
INTRODUCTION
The increasing knowledge of cellular mechanisms provides new opportunities
for the development of molecular tools for the studies of biologic processes.
Cellular response to an external stimulus is often the result of a cascade of
events. Most of primary messengers bind to a specific G protein-coupled
receptor (GPCR). It is then followed by a second series of signalling events
such as calcium liberation. The discovery of this calcium signalling pathway
has been a major event in cellular biology.[1–3] Even more interesting was
the identification of the second messengers derived from the phosphoinositide
pathway, in particular inositol 1,4,5-trisphosphate (InsP3) 1.[4–6] This small
molecule is formed by hydrolysis of a membrane-anchored phosphoinositide
(PIP3) by a phospholipase C, which is activated following ligand binding to a
GPCR. This hydrolysis produces diacylglycerol and the second messenger
inositol 1,4,5-triphosphate (InsP3). Binding of the latter to its specific receptor
(InsP3R), located on the endoplasmic reticulum, triggers calcium liberation
and cellular response via calmodulin and other calcium-sensitive proteins.
Complex cascades of events degrade InsP3 to inositol and then recycle inositol
to PIP3.
InsP3 is a ubiquitous second messenger, which prompted much interest in
the scientific community in the last 20 years. On the one hand, the possibility of
blocking its binding to InsP3R was explored, and the search for antagonists of
this receptor has been a long-standing interest.[7] Only a few antagonists of low
potency have been found. On the other hand, InsP3R agonists, that is, analogs
of InsP3, have been developed to study InsP3R, the signalling pathway, or to
trigger at will biologic events such as secretion, cell proliferation, or cell
contraction. This task was easier, and a number of synthetic analogs of InsP3
have been proposed.[8–10]
In our group, we studied heterocyclic structures designed by analogy with
InsP3. These sugar-based analogs of InsP3 are potent ligands of InsP3R.[11–15]
Moreover, while our studies were in progress, adenophostins 2, the most potent
agonists of InsP3R, have been isolated[16] and supported our idea of replacing
the cyclohexane ring of inositol by sugar templates. These molecules provide
good lead compounds for the design of potent agonists. The chemistry of adeno-
phostin[17] and congeners[18] has been thoroughly explored, in particular in
J. van Boom group. The biologic activity of all these analogs has been
explored on isolated receptor InsP3 or on permeabilized cells and confirmed

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
551
Figure 1: Sugars based analogs of InsP3.
the potency of these ligands to trigger calcium release from endoplasmic
reticulum stores.[19] We were also able to construct simplified analogs of adeno-
phostins, which we named xylophostins and which are very potent agonists of
InsP3R (Fig. 1).[15]
As above mentioned, one interest of these agonists is their possible use as
biochemical tools. Indeed, it would be of interest to trigger calcium release
using the second messenger itself without external stimulus by the normal
primary messenger and to trigger biologic events in intact cells. This would
be particularly useful on abnormal cells not expressing GPCR.
However, the intrinsic high polarity of these water-soluble polyphosphates
precludes their use on intact cells. Analogs of InsP3 that could be transported
across cell membranes and could undergo subsequent degradation to the free
phosphate derivatives, the so-called permeant derivatives, could provide a
solution to this problem. Various permeant inositol phosphates have already
been proposed,[20] and here we report the results of our investigations along
these lines using our xylose-based analogs. We also report the details of the
synthetic studies and some preliminary biologic results providing the clues
to cell external activation via InsP3 analogs.
RESULTS AND DISCUSSION
Among the InsP3 analogs synthesized in our group, compound 3 was found very
active[9] and would be a good candidate to test several strategies for making

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F. Chretien et al.
552
this compound permeant. Different approaches were tested, and six target
compounds depicted in Figure 2 were devised and prepared.
In the first series of experiments, three possibilities were explored. The
simplest way was the phosphorylation of the appropriate triol with a protected
alkylphosphate, assuming that the recovery of the phosphate group should be
carried out via a phospholipase hydrolysis of the alkylphosphate groups. The
synthesis of 5 bearing dialkylphosphate was envisioned. The chain length of
the alkyl group of the dialkylphosphate should play a crucial role either in
the transport or in the phosphate liberation inside the cell and would need to
be carefully chosen. We chose to limit our investigation to the medium-sized
octyl group. One drawback of this approach is the very low solubility in
water of this type of fully protected compound. To overcome this drawback, a
possibility was to decrease the number of protected phosphates Protection of
each of the three phosphates with single alkyl group as in 6 was thus
considered. Finally, the construction of 7 and 8, with only one of the three phos-
phates protected with two alkyl groups, was also envisioned. It is of note that
such monoprotected trisphosphates should act as a surfactant and interact
with cell membranes in several ways.
The synthesis of 5 and 6 started from the previously described triol 12
obtained in five steps from allyl-a-D-xylopyranoside via the 2-O-benzyl deri-
vative 17.[11] Reaction of 12 with the phosphoramidite 15 followed by in situ
oxidation with tBuOOH gave the protected derivative 13 in 42% yield. Hydro-
genolysis of this compound gave the free hydroxyl group of 5 in good yield.
The synthesis of 6 proceeded along the same lines by changing the phosphi-
tylating agent to 16. The protected trisphosphate 14 was obtained in 52% yield
Figure 2: Structure of target compounds.

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
553
Scheme 1: Reagents i: 15, 1H-tetrazole, CH2Cl2, rt, then 08C, tBuOOH; ii: Pd/C 10% H2, 20 bars,
MeOH.
for the two steps. Deprotection of the four benzyl groups by hydrogenolysis at
20 bars in methanol gave the free derivative 6 isolated as the sodium salt in
quantitative yield (Sch. 1).
It was known from the previous studies that the two vicinal phosphates at
position 4 and 5 of the inositol ring are crucial for recognition by the InsP3R.
Thus, it could be useful to modify only the phosphate of the aglycon that
mimics the phosphate at position 1 of InsP3. The synthetic strategy toward
the triol 12 allowed the construction of such unsymmetrical trisphosphates.
We started from the protected allyl xyloside 17 that was phosphorylated at
position 3 and 4 (carbohydrate numbering) using the phosphoramidite 23,[21]
followed by oxidation with tBuOOH to give the protected bisphosphate 18 in
70% yield. The double bond was cleaved using OsO4 and NaIO4 in a dioxane--
water mixture to yield the corresponding aldehyde 19 in 98% yield. Reduction
of the carbonyl group with sodium borohydride in MeOH for 3 min gave the
expected alcohol 20 in 57% yield. Phosphorylation of this alcohol by the phos-
phoramidite method using 15 gave the protected trisphosphate 21 in 42%
yield after oxidation. Deprotection of the cyano-ethyl groups of 21 gave the
semi-protected derivative 22. Deprotection of the cyano-ethyl groups and the
benzyl group of 21 was achieved by treatment with sodium in liquid
ammonia and gave quantitatively the free bisphosphate 7 (Sch. 2).
The same alcohol 20 was engaged in a phosphorylation reaction using the
H-phosphonate 25[22] in the presence of pivaloyl chloride[23] followed by
oxidation with iodine in a 98 : 2 pyridine/water mixture[24,25] to provide the
protected phosphate 24. Removal of the cyanoethyl and benzyl groups by treat-
ment with sodium in liquid ammonia afforded the free derivative 8 in 98% yield
(Sch. 3).
Preliminary studies of the properties of the four phosphates 5–8 were
rather disappointing. The biologic testings (vide infra) consisted of measuring
calcium liberation from intact cells under physiologic conditions. None of

´
F. Chretien et al.
554
Scheme 2: Reagents i: 23, 1H-tetrazole, CH2Cl2, rt, then 08C, tBuOOH; ii: OsO4, NaIO4,
dioxane/H2O, rt; iii: NaBH4, MeOH; iv: 15, 1H-tetrazole, CH2Cl2, rt, then 08C, tBuOOH; v: KOH,
MeOH 0.5 M, 408C, vi: from 21 Na, NH3 liq., dioxane.
these compounds showed significant activity, which means that either these
compounds do not cross the cell membrane or if they do, are not released signifi-
cantly to the free phosphates. Only compound 6 showed an ionophoric effect.
To improve the permeability and the hydrolysis to the free phosphate we
turned our attention to more labile phosphate protection.[26] Acyloxy deriva-
tives have been considered to produce prodrugs of charged or polar bioactive
compounds.[27] Acetoxymethyl groups have been introduced in the phosphoino-
sitol field to prepare permeant derivatives of InsP3.[20,28] These groups are
easily introduced on the free phosphates under smooth reaction conditions.
Three different acyloxymethyl groups, octyl, butyl, and ethyl, which differed
by the chain length, have been used in this study. The strategy consists of
Scheme 3: Reagents i: BF, pivaloyl chloride, pyridine, 2 208C, 2 hr then I2, pyridine/H2O
(98/2 v : v), 2 208C, then rt, 40 min; ii: Na, NH3 liq., dioxane.

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
555
the formation of the free xylose-derived trisphosphate 27 that could be
alkylated and then deprotected to give the expected permeant derivatives.
Phosphorylation of 12 using the phosphoramidite 23 followed by tBuOOH
oxidation gave the protected trisphosphate 26 in 58% yield. Removal of the
phosphate groups by treatment with potassium hydroxide in methanol gave
the free trisphosphate 27 in excellent yield (Sch. 4).
This key intermediate was then treated with iodomethyl octanoate
prepared by a three-step sequence from octanoic acid using modification of
literature procedures.[29] The fully protected derivative 30 was obtained in
modest yield. The same procedure was applied to 27 using iodomethylbutano-
ate prepared from the known bromoderivative[30] and bromomethyl acetate[31]
to provide the protected derivatives 29 and 28, respectively, in about 20% yield.
Final deprotection of the 2-O-benzyl group was cleanly achieved by hydrogeno-
lysis (20 bars H2) in ethylacetate in the presence of palladium on charcoal as
the catalyst to provide the permeant derivatives 9, 10, and 11.
Given the easy access to the enantiomerically pure xylose trisphosphate 3
and given its high potency, it seemed interesting to use it to construct more
complex molecular tools. Photoaffinity labelling is a key method to localize
receptors in cells for example. This has been used in the inositol field and devel-
oped recently by Prestwich.[32] In most of these studies a photoaffinity ligand is
carried by the 1-phosphate group of the inositol derivative. We have developed
xylose-based analogs of InsP3, which are equipped with an aglycon bearing the
phosphate group and an anchoring point. This has been used for linking the
adenine residue in the synthesis of xylophostin. We explored the same
approach to construct an affinity labelling ligand of InsP3R, in which the
three phosphates would be present in a protected form to make this
compound permeant. A derivative of cinnamic acid developed by Prestwich[33]
and acetoxy methyl groups were chosen as the photosensitive and protecting
groups, respectively (Sch. 5).
Scheme 4: Reagents i: KOH, MeOH 0.5 M, 408C; ii: bromomethyl acetate, DIEA, CH3CN, rt;
iii: iodomethylbutanoate, DIEA, CH3CN, rt; iv: iodomethyl octanoate, DIEA, CH3CN, rt; v: Pd/C
10%, H2, 20 bars, AcOEt.

´
F. Chretien et al.
556
Scheme 5: Reagents i: NaH, BnBr, DMF; ii: 1) NaN3, MeOCH2CH2OH, H2O, NH4Cl, 2) TFA/H2O;
(95/5 v : v), CH2Cl2; iii: Pd/C 10%, H2, MeOH; iv: 41, BOP, NEt3, DMF; v: 23, 1H-tetrazole,
CH2Cl2, rt, then 08C, tBuOOH; vi: KOH, MeOH 0.5 M, 408C; vii: bromomethyl acetate, DIEA,
CH3CN, rt; viii: Pd/C 10%, H2, 20 bars, AcOEt.
Benzylation of the 2-OH group of known epoxide 31[34] gave 32 in 91%
yield. Treatment of this compound with sodium azide in methoxyethanol in
the presence of ammonium chloride gave 33 in 80% yield. Attempts to carry
out the phosphorylation steps in the presence of the azide group did not lead
to the expected compounds in satisfactory yield and purity. Thus, the azide
function was reduced to the corresponding amine by hydrogenolysis using
10% Pd/C in methanol for 4 hr, providing 35 in 97% yield. Coupling of the
free amine with the E acid 41 in the presence of BOP reagent in DMF gave
the corresponding amide 36 in 72% yield. A set of two NMR signals was
observed for this compound, which is likely due to two conformational
isomers in the p-benzoylcinnamyl system. Phosphitylation of the free OH
groups of 36 with 23 and oxidation of the phosphites to the phosphates gave
37 in 42% overall yield. Removal of the phosphate-protecting groups in basic
medium gave the free trisphosphate 38 in excellent yield. After ion exchange
the diisopropylammonium salts were reacted with 10 equivalents of acetoxy-
methyl bromide in acetonitrile to provide the protected derivative 39 in 20%
yield. All these compounds were also mixtures of conformational isomers as
1
shown by H and 13C NMR. The last step was removal of 2-O-benzyl group
by hydrogenolysis, which was accompanied by reduction of the cinnamoyl
double bond. Treatment of 39 under 15 bars of hydrogen atmosphere for

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
557
10 hr in the presence of 10% Pd/C in ethyl acetate was needed to achieve the
benzyl group hydrogenolysis. The end product of the reaction 40 was also no
longer a mixture of conformers but was also no longer a benzophenone
system. Indeed, reduction of the carbonyl group occurred under such drastic
hydrogenolysis conditions. This type of reduction has some literature pre-
cedents.[35] Despite this unexpected loss of the carbonyl group, this
compound has an interest as a permeant analog of InsP3 bearing a lipophilic
group near the 10-O-phosphate. It could be used to test a hypothesis about
the potent activity of adenophostin, which should be due to lipophilic or
stacking interactions between adenosine and an appropriate pocket on
InsP3R.[36]
BIOLOGICAL ACTIVITIES
The binding efficiency of the compounds 3, 8, and 7 was assayed in competition
binding experiments of [3H]InsP3 to rat cerebellar membranes. The analogs
inhibited specific [3H]InsP3 binding in the following order of potency,
InsP3 . 3 . 8 . 7 (Fig. 3). This indicates that the protection of the 20-O-phos-
phate with one or two alkyl groups is not detrimental to binding to the
receptor, confirming the minor role of this phosphate group (corresponding to
the 1-O-phosphate in InsP3) in calcium liberation. It also indicates that the
phosphate esters are not cleaved under the experimental conditions to
produce 3.
Analog-evoked Ca21 Responses in Isolated Hepatocytes
Analogs 6, 9, or 40 were added to quin2-loaded rat hepatocyte suspension
and calcium responses were studied using spectrofluorimetry. All three analogs
induced an increase in quin2 fluorescence, indicating an increase of the
Figure 3: Inhibition of specific [3H]InsP3 binding to rat cerebellar cells InsP3R by 3, 7, and 8.

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F. Chretien et al.
558
cytosolic Ca2þ concentration ([Ca2þ]c). Maximal responses were obtained in the
presence of 100 mM of 6, 100 mM of 9, and 10 mM of 40. In the presence of extra-
cellular Ca2þ this increase of [Ca2þ]c is sustained, and the following addition of
10 nM of the Ca2þ mobilizing agonist vasopressin did not induce further
([Ca2þ
] increase, most likely because quin2 was saturated (Fig. 4). The
c
[Ca2þ]c increase developed within 1 to 2 min after the addition of 9 or 40.
This delay is most likely due to limiting steps like the entry of the analog
through the plasma membrane and to the hydrolysis of the ester function by
the cell esterases. The Ca2þ increase developed much more rapidly after the
addition of 6; this suggests that it induced a nonspecific Ca2þ entry through
the plasma membrane.
In the absence of extracellular Ca2þ, maximal concentrations of 6 (100 mM),
9 (100 mM), or 40 (10 mM) induced a transient [Ca2þ]c increase, which devel-
oped within 1 to 2 min after the addition of the analog (Fig. 5). Since there
was no Ca2þ entry from the extracellular medium, these increases are due to
the mobilization of Ca2þ accumulated in the intracellular Ca2þ store. The
addition of 10 nM vasopressin evoked a calcium release through the production
of InsP3.[37] This response was reduced when vasopressin was added after 6 or
suppressed when added after 9 or 40. This suggests that the analogs induced
the release of Ca2þ from the same intracellular store as the one, which is sen-
sitive to InsP3, namely, the endoplasmic reticulum. Accordingly, the previous
stimulation of the hepatocytes by vasopressin induced a transient increase of
[Ca2þ]c, which emptied the InsP3-sensitive Ca2þ store consequently, and the
following addition of the analogs did not induce further Ca2þ release.
We have studied in more detail the effects of the analog 40 on the Ca2þ
mobilization of quin2-loaded hepatocytes. Figure 5c indicates that the
addition of 0.1 mM of 40 to cells incubated in a Ca2þ-free medium did not
evoke Ca2þ release, and the following addition of vasopressin was not
precluded. The addition of 1 mM or 10 mM 40 induced progressive increases of
Figure 4: Calcium mobilization by 6, 9, and 40 in intact rat hepatocytes in the presence of
extracellular calcium (1.8 nM).

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
559
Figure 5: Calcium mobilization by 6, 9, and 40 in intact rat hepatocytes in the absence of
extracellular calcium.
[Ca2þ]c, which led to progressive decreases of the response to vasopressin. This
confirms that 40 and vasopressin mobilized Ca2þ from the same intracellular
Ca2þ store. The addition of 6 mM of thapsigargin, an inhibitor of the Ca2þ
pump that actively accumulates Ca2þ in the lumen of the endoplasmic reticu-
lum, evoked transient increases of [Ca2þ]c and precluded the effect of 40
(Fig. 6b). This confirms that 40 mobilized Ca2þ from the endoplasmic reticu-
lum. Accordingly, the addition of 40 before the addition of thapsigargin pre-
cluded the Ca2þ release by the Ca2þ pump inhibitor (Fig. 6a). These data
clearly indicate that 40 mimics the effect of InsP3 in the hepatocytes.
In the presence of extracellular Ca2þ, increasing concentrations of 40 from
0.1 mM to 10 mM induced progressive increase of the [Ca2þ]c (Fig. 4c). This
increase was maintained during the presence of the analog, and the further
addition of vasopressin induced a reduced increase of [Ca2þ]c. The sustained
Figure 6: Effect of thapsigargine on calcium mobilization by 40 on intact rat hepatocytes.

´
F. Chretien et al.
560
response observed in the presence of 40 suggests that the analog stimulated the
Ca2þ entry through the plasma membrane. This is in agreement with the capa-
citative hypothesis that assumes that the stimulation of Ca2þ influx directly
depends on the Ca2þ concentration in the lumen of the endoplasmic reticulum
of stimulated cells.[38]
CONCLUSION
Different protected analogs of our xylose-based full agonist of InsP3 3 have been
prepared and tested for their ability to bind the InsP3 R or to mobilize calcium
from its internal stores. Fully protected analogs of 3 on all phosphates did not
mobilize calcium in intact cells. Partially protected derivatives on 20-O-pho-
sphate do not cross cell membranes but bind to InsP3R, although less
potently than 3. The most efficient compound was the acetoxymethyl derivative
9, which crossed cell membranes and is able to mobilize calcium, other acyl oxy
derivatives being less interesting. Interestingly, compound 40, primarily
designed as a photoaffinity ligand, is also a good permeant agonist of InsP3R.
This should be in favor of the existence of a hydrophobic pocket on the
receptor that can accommodate a rather large aromatic group like adenosine
or the phenyl benzyl group of 40. All these analogs can serve as biologic tools
for further investigation of the calcium-signalling pathway.
EXPERIMENTAL SECTION
Optical rotations were determined at 208C with a Perkin Elmer model 141 auto-
matic polarimeter. NMR spectra were recorded at 258C with a Bruker AC250
spectrometer. Chemical shift (d) are given in ppm relative to the signal for
internal tetrametylsilane for 1H NMR and indirectly to the central line of
CDCl3, d 77.03 for 13C NMR spectra; those in deuterium oxide are reported
relative to external 2,3 dimethyl-2-silapentane-5 sulfate (DSS). Infrared
spectra were recorded on a Perkin-Elmer Spectrum 1000 FTIR spectrometer.
All reactions were monitored by thin layer chromatography on Kieselgel
60F254 (Merck) with detection by UV light and/or by charring with 15%
sulfuric acid in ethanol. Elemental analyses and mass spectra were performed
by the Service Central d’Analyses du CNRS at Vernaison (France).
General procedure for ozonolysis followed by a reductive cleavage
(Procedure A): A solution of allyl xyloside (1 mmol) in a dichloromethane-
methanol mixture (10 mL, 1 : 1 v/v) was cooled to 2708C and ozone was
bubbled through the solution (flow of 0.4 L/min) until the disappearance of
the starting material. The solution was degassed using a stream of argon for
15 min. Sodium borohydride (4 eq.) was added and the reaction mixture was
allowed to warm to rt. After completion of the reaction (TLC monitoring) the

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
561
solvents were evaporated under reduced pressure and the residue was diluted
with ethyl acetate. The organic layer was washed with 3N hydrochloric acid
and water, dried over magnesium sulfate, and concentrated to provide the cor-
responding 2-hydroxyethyl derivative.
General procedure for phosphorylation reactions (Procedure B): To a
solution of the alcohol derivative in dichloromethane (12 mL/mmol) were
added under argon 1H-tetrazole (4 eq./OH) and the required phosphoramidite
(2.5 eq./OH). The reaction mixture was stirred at rt until TLC and 31P NMR
spectroscopy analysis showed complete conversion of starting material into
the corresponding phosphite derivative. The solution was cooled to 08C, an
excess of tBuOOH was added, and the reaction mixture was warmed to rt.
After completion of the reaction (TLC monitoring), the medium was diluted
with dichloromethane; successively washed with water, saturated aqueous
NaHCO3 solution, and water; dried over magnesium sulfate; and
concentrated in vacuo. The residue was purified by column chromatography.
General procedure for hydrogenolysis reactions (Procedure C): To a
mixture of the phosphorylated derivative in methanol (40 mL/mmol) was
added the same amount of 10% Pd/C. The mixture was placed under
hydrogen atmosphere at 20 bars. After 14 hr, the catalyst was removed by
filtration on a pad of Celite and the solvent evaporated in vacuo. In the case
of the presence of benzylic phosphodiester groups, the residue was dissolved
in water and aqueous NH3 was added to reach pH 8.0, and the solution was
applied to a column of Bio-Rad Chelex 100 resin (Naþ form). The compound
was eluted with water; fractions containing the desired compound were
combined and freeze dried.
General procedure for debenzylation using sodium in liquid ammonia
(Procedure D): Liquid ammonia was condensed into a two-necked flask at
2788C and sodium was added. A solution of a benzylated compound in dry
dioxane was added and the mixture was stirred for 20 min. The reaction was
quenched with ethanol and the medium was warmed to rt. The solution was
degassed with nitrogen for 15 min, neutralized to pH 7.00 with DOWEX 50W
(Hþ) cation exchange resin, and filtered, and the resin was washed with deionized
water. After evaporation of the solvents, the residue was applied to a column of
Bio-Rad Chelex 100 resin (Naþ form). The compounds were eluted with water;
fractions containing the desired compound were combined and freeze dried.
General procedure for methanolic cleavage of cyanoethyl phosphate
(Procedure E): The protected compound was dissolved in a 0.5M potassium
hydroxide methanolic solution (2.3 OH equivalent/phosphate group). The
solution was heated at 358C until all the cyanoethyl groups were removed
(31P NMR monitoring) and then allowed to cool to rt. The solution was neutral-
ized to pH 7.00 with DOWEX 50W (Hþ) cation exchange resin and filtered, and

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F. Chretien et al.
562
the resin was washed with deionized water. After evaporation of the solvents
the residue was applied to a column of Bio-Rad Chelex 100 resin (Naþ form).
The column was eluted with water; fractions containing the desired
compound were combined and freeze dried.
(2-Hydroxyethyl) 2-O-benzyl-a-D-xylopyranoside (12). The allyl xyloside
17 (2.22 g, 7.92 mmol) was converted into 2-hydroxyethyl derivative according to
procedure A to provide 12 (1.84 g, 6.5 mmol, 82%) as a syrup. Rf 0.65 (CH2Cl2/
1
MeOH,9:1); [a]D þ 65.0 (c 2.8, CHCl3); IR 3374 (OH) cm21; H NMR (CDCl3) d
7.23–7.36 (m, 5H, Ar); 4.66 (AB, 1H, JAB 11.5 Hz, CH2Ph); 4.56 (d, 1H, J1-2
3.5 Hz, H-1); 4.52 (AB, 1H, CH2Ph); 4.49 (m, 1H, OH); 4.28 (m, 2H, OH); 3.83
(dd, 1H, J2-3 9.6, J3-4 8.5 Hz, H-3); 3.56–3.68 (m, 3H, H-10a, H-10b, H-5ax);
3.44–3.49 (m, 1H, H-20a); 3.38–3.42 (m, 1H, H-20b); 3.33–3.37 (m, 1H, H-4);
3.29 (dd, 1H, J4-5eq 4.5, J5eq-5ax 11.5 Hz, H-5eq); 3.23 (dd, 1H, H-2); 13C NMR
(CDCl3) d 137.8 (C ipso); 128.3; 127.9; 127.8; (5C, C Ar); 96.7 (C-1); 79.5 (C-3);
72.9 (C-2); 73.4 (CH2Ph); 69.9 (C-4); 69.5 (C-10); 61.3 (2C, C-5, C-20). Anal.
Calcd for C14H20O6: C, 59.10; H, 7.10. Found: C, 59.26; H, 7.31.
Bis octyloxy (diisopropylamino)phosphine (15). A solution of dichloro-
(N,N-diisopropylamino)-phosphine (28.08 g, 40mmol) in anhydrous diethyl
ether (6mL) was added dropwise under argon to a solution of octyl alcohol
(2eq., 12.59 mL, 80 mmol) and triethylamine (2eq., 11.15 mL, 80 mmol) in Et2O
(26 mL) at 08C. The mixture was stirred at rt until 31P NMR indicated
complete consumption of the dichlorophosphine (170 ppm) to give the corres-
ponding phosphoramidite (150ppm) (6hr). After filtration over Celite and
washing with Et2O (100 mL), the solvent was evaporated to afford 15 (95%,
1
14.79 g, 38 mmol) as an oil. H NMR (CDCl3) d 3.53–3.71 (m, 6H, 4CH(CH3)2,
H-1, H-10); 1.56–1.70 (m, 4H, H-2, H-20); 1.25–1.42 (m, 20H, 2(H-3, H-30, H-4,
H-40, H-5, H-50, H-6, H-60, H-7, H-70)); 1.19 (d, 12H, JH-H 6.8 Hz, 2 CH(CH3)2);
2
0.89 (t, 6H, 6H-8); 13C NMR (CDCl3) d 63.4 (2C, JC-P 16.7 Hz, 2C-1); 42.7 (2C,
3
2JC-P 10.8 Hz, 2CH(CH3)2); 31.8 (2C, 2C-6); 31.3 (2C, JC-P 6.9 Hz, 2C-2); 29.3
3
(4C, 2C-4, 2C-5); 26.0 (2C, 2C-3); 24.6 (4C, JC-P 6.9 Hz, 2CH(CH3)2); 22.6 (2C,
2C-7); 14.0 (2C, 2C-8); 31P NMR (CDCl3) d 150.0 (s).
[(2-Dioctylphosphonoxy)ethyl] 2-O-benzyl-3,4-bis(dioctylphosphate)-a-
D-xylopyranoside (13). The alcohol derivative 12 was phosphorylated
according to procedure B with the phosphoramidite 15 to give 13 (42%) as a
gum. Rf 0.5 (Hex/EtOAc, 3 : 7); [a]D þ 17.7 (c 1.66, CHCl3); IR 1232 (P55O);
1025 (P–O). cm21 1H NMR (400 MHz, CDCl3) d 7.22–7.41 (m, 5H, Ar);
;
4.65–4.75 (m, 3H, H-1, H-3, CH2Ph); 4.58 (AB, 1H, JAB 12.1 Hz, CH2Ph);
4.26 (m, 1H, H-4); 4.13–4.19 (m, 2H, H-20a, H-20b); 3.86–4.12 (m, 13H,
6H-100a, 6H-100b, H-5eq); 3.75 (m, 1H, H-10a); 3.54–3.66 (m, 2H, H-5ax,
H-10b); 3.44 (dd, 1H, J1-2 3.3, J2-3 9.3 Hz, H-2); 1.58–1.70 (m, 12H, 6H-200a,
6H-200b); 1.15-1.41 (m, 60H, 6(H-300a, H-300b, H-400a, H-400b, H-500a, H-500b,

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
563
H-600a, H-600b, H-700a, H-700b); 0.80–0.92 (m, 18H, 6CH3); 13C NMR (CDCl3) d:
2
137.5 (C ipso); 128.0; 127.5 (5C, C Ar); 96.6 (C-1); 77.4 (C-2); 77.1 (dd, JC-P
3
2
3
6.1, JC-P 7.3 Hz, C-3); 73.2 (dd, JC-P 3.8, JC-P 4.8 Hz, C-4); 72.4 (CH2Ph);
2
3
2
67.5–68.5 (6d, JC-P 6.2 Hz, 6C-100); 66.6 (d, JC-P 7.1 Hz, C-10); 65.5 (d, JC-P
5.7 Hz, C-20); 59.2 (C-5); 31.9 (6C-600); 29.9 (d, JC-P 6.7 Hz, 6C-200); 28.8
3
(6C-400, 6C-500); 25.1 (6C-300); 22.2 (6C-700); 13.7 (6C-800); 31P NMR (CDCl3) d:
0.55 (s, C-20OP); 20.27 (s, 2P, C-3OP, C-4OP). Anal. Calcd for C62H119O15P3:
C, 62.18; H, 10.02; P, 7.76. Found: C, 62.32; H, 10.21; P, 7.62.
[(2-Dioctylphosphonoxy)ethyl]-3,4-bis(dioctylphosphate)-a-D-xylopyr-
anoside (5). Tris(dioctyl)phosphate 13 (150 mg, 0.125 mmol) was hydrogen-
ated according to procedure C to yield 5 (99%, 137 mg, 0.124 mmol) as a gum.
Rf 0.59 (Hex/EtOAc,1 : 9); [a]D þ 24.0 (c 1.6, CHCl3); IR 1232 (P55O);
1
1026(P-O). cm21; H NMR (250 MHz, CDCl3) d 4.87 (d, 1H, J1-2 3.6 Hz, H-1);
3
4.51 (m, 1H, J2-3 9.4, J3-4 9.3, JH-P 8.1 Hz, H-3); 3.82–4.40 (m, 18H, H-4,
H-5eq, H-10a, H-10b, H-20a, H-20b, 6H-100a, 6H-100b); 3.54–3.78 (m, 2H, H-2,
H-5ax); 1.53–1.83 (m, 12H, 6H-200a, 6H-200b); 1.17–1.47 (m, 60H, 6(H-300a,
H-300b, H-400a, H-400b, H-500a, H-500b, H-600a, H-600b, H-700a, H-700b)); 0.77–0.98
2
3
(m, 18H, 6CH3); 13C NMR (CDCl3) d 99.0 (C-1); 79.0 (dd, JC-P 5.7, JC-P
2
3
7.6 Hz, C-3); 73.0 (dd, JC-P 5.2, JC-P 5.7 Hz, C-4); 71.4 (C-2); 67.5–68.5 (6d,
2
3
2
6C, JC-P 6.2 Hz, 6C-100); 67.5 (d, JC-P 6.2 Hz, C-10); 66.1 (d, 1C, JC-P 5.7 Hz,
3
C-20); 59.9 (C-5); 31.9 (s, 6C, 6C-600); 30.5 (d, JC-P 5.9 Hz, 6C-200); 29.3 (s, 12C,
6C-400, 6C-500); 25.6 (s, 6C, 6C-300); 22.8 (s, 6C, 6C-700); 14.2 (s, 6C, 6C-800); 31P
NMR (CDCl3) d: 1.09; 0.57; 20.21 (3s, 3P); ESI-MS (positive mode): calcd for
C55H113O15P3 m/z: 1108 [M þ H]þ; Anal. Calcd for C55H113O15P3: C, 59.64; H,
10.20; P, 8.38. Found: C, 59.44; H, 10.19; P, 8.36.
(Benzyloxy)(octyloxy)(diisopropylamino)phosphine
(16). Benzyloxy
(diisopropylamino)phosphine (17.34 g, 51.23 mmol) and 1H-tetrazole (1 eq.,
3.58 g, 51.23 mmol) were dissolved in dichloromethane (80 mL) under argon.
After stirring for 30 min, a solution of 1-octanol (0.95 eq., 7.66 mL,
51.2 mmol) in CH2Cl2 (55 mL) was added dropwise over 45 min. The mixture
was stirred for 2 hr, when 31P NMR indicated complete consumption of the
starting phosphine (126.7 ppm) to give a product with a signal at 147.5 ppm.
The medium was diluted with CH2Cl2 (600 mL) and washed with saturated
aqueous NaHCO3 solution (3 ꢀ 50 mL) and water. The organic layer was
dried over Na2SO4 and concentrated in vacuo, and the residue was purified
by column chromatography on silica gel deactivated with 5% of triethylamine
to afford 16 (61%, 11.48 g, 31.25 mmol) as a gum. 1H NMR (250 MHz, CDCl3) d
3
7.18–7.44 (m, 5H, H Ar); 4.58–4.95 (2dd, 2H, JAB 12.6, JH-P 8.8 Hz, CH2Ph);
3.54–3.90 (m, 4H, 2CH(CH3)2, H-1, H-10); 1.55–1.71 (m, 2H, H-2, H-20);
1.27–1.50 (m, 10H, H-3, H-30, H-4, H-40, H-5, H-50, H-6, H-60, H-7, H-70); 1.23
(d, 6H, JH-H 1.5 Hz, CH(CH3)2); 1.20 (d, 6H, JH-H 2.2 Hz, CH(CH3)2); 0.92
(t, 3H, H-8, H-80, H-800); 13CNMR (CDCl3) d 136.8 (Cipso); 127.8; 126.8; 126.6

´
F. Chretien et al.
564
2
2
(3s, 5C, CH Ar); 64.8 (d, JC-P 18.3 Hz, CH2Ph or C-1); 63.4 (d, JC-P 17.1 Hz,
2
3
CH2Ph or C-1); 42.6 (d, JC-P 13.4 Hz, CH(CH3)2); 31.5 (C-6); 31.0 (d, JC-P
3
7.3 Hz, C-2); 30.9 (2C, C-4, C-5); 25.6 (C-3); 24.2 (d, 4C, JC-P 6.1 Hz,
2CH(CH3)2); 22.3 (C-7); 13.8 (C-8); 31PNMR d 147.5 (s).
[(2-Benzyloctylphosphonoxy)ethyl]
2-O-benzyl-3,4-bis(benzyl-octyl-
phosphate) a-D-xylopyranoside (14). Compound 12 was phosphorylated
according to procedure B with the phosphoramidite 16 to give the correspond-
ing compound 14 (52%) as a gum after purification by column chromatography
(Hex/EtOAc 6 : 4 ! EtOAc). Rf 0.79 (Hex/EtOAc,2 : 8); [a]D þ 18.8 (c 0.85,
CHCl3); IR 1272 (P55O); 1019 (P-O) cm21 1H NMR (250 MHz, CDCl3) d
;
7.13–7.43 (m, 5H, H Ar); 4.88–5.19 (m, 6H, 3POCH2Ph); 4.47–4.87 (m, 4H,
H-1, H-3, CH2Ph); 4.33 (m, H-4); 4.06–4.21 (m, 2H, H-20a, H-20b); 3.81–4.05
(m, 7H, 6H-100, H-5a); 3.73 (ddd, 1H, H-10a); 3.50–3.67 (m, 2H, H-5b, H-10b);
3.44 (dd, 1H, J1-2 2.9, J2-3 9.5 Hz, H-2); 1.48–1.71 (m, 6H, 3(2H-200)); 0.85–
1.30 (m, 30H, 3(2H-300, 2H-400, 2H-500, 2H-600, 2H-700)); 0.79–0.99 (m, 9H,
3(2H-800)); 13CNMR (CDCl3) d 137.5 (Cipso); 135.7; 135.6 (2s, 3C, Cipso);
128.2; 128.1; 127.8; 127.6; 127.4; 127.2 (20C, CH Ar); 96.5 (C-1); 77.4 (C-2);
3
4
77.0 (dd, JC-P 6.1, JC-P 1.1 Hz, C-3); 73.4 (m, C-4); 72.5 (CH2Ph); 69.3 (d, 1C,
2
2JC-P 4.9 Hz, POCH2Ph); 68.6–69.0 (2d, 2C, 2POCH2Ph); 68.1 (d, 1C, JC-P
3
6.1 Hz, C-100); 67.7–68.0 (2d, 2C, 2C-100); 66.6 (d, 1C, JC-P 7.3 Hz, C-10); 65.6
2
3
(d, 1C, JC-P 6.1 Hz, C-20); 59.2 (C-5); 31.4 (C-600); 29.9 (d, JC-P 6.1 Hz, C-200);
28.8 (2C, C-400, C-500); 25.0 (C-300); 22.3 (C-700); 13.7 (C-800); 31PNMR (CDCl3) d
00
0.48 (s, 1P, C2 OP); 0.28 (s, 2P, C3OP, C4OP); FAB-MS (positive mode): calcd
for C59H89O15P3 m/z: 1132 [M þ H]þ. Anal. Calcd for C59H89O15P3: C, 62.68;
H, 7.87; P, 8.21. Found: C, 62.74; H, 7.84; P, 8.25.
[(2-Benzyloctylphosphonoxy)ethyl] 3,4-bis(benzyl-octylphosphate) a-D-
xylopyranoside (6). The title compound was obtained as a white powder in
99% yield by hydrogenolysis of 14 according to procedure C. [a]D þ12.0 (c 0.5,
1
H2O); IR 1230(P55O); 1105(P-O) cm21; H NMR (250 MHz, D20) d4.97 (d, 1H,
3
J1-2 3.0 Hz, H-1); 4.27 (dd, 1H, J2-3 9.5, J3-4 9.3, JH-P 8.0 Hz, H-3); 3.57–4.18
(m, 14H, H-2, H-4, H-5a, H-5b, H-10a, H-10b, H-20a, H-20b, 6H-100); 1.57–1.78
(m, 6H, 3(2H-200)); 1.23–1.39 (m, 30H, 3(2H-300, 2H-400, 2H-500, 2H-600, 2H-700));
0.83–1.06 (m, 9H, 3(3H-800)); 13CNMR (D2O) d 100.5 (C-1); 78.5 (dd, C-3); 73.8
3
2
(C-2); 73.5 (dd, C-4); 70.1 (d, 1C, JC-P 7.6 Hz, C-10); 69.1 (d, 1C, JC-P 4.8 Hz,
2
C-100); 68.6-68.7 (2d, 2C, 2C-100); 66.7 (d, 1C, JC-P 4.3 Hz, C-20); 63.2 (C-5); 34.4
(3C, C-600); 33.0 (d, 3C, 3JC-P 6.0 Hz, C-200); 31.9 (6C, C-400, C-500); 28.1 (3C, C-300);
25.1 (3C, C-700); 16.3 (3C, C-800); 31PNMR (D2O) d 4.34; 4.29; 3.79 (3s, 3P).
ESI-MS (positive mode): Calcd for C31H62O15P3Na3 m/z: 859 [M þ Na]þ; 837
[M þ H]þ; 815 [M-Na þ 2H]þ; 793 [(M-2Na þ 3H]þ.
Allyl 2-O-benzyl-3,4-bis[(2-cyanoethyl) phosphate] a-D-xylopyranoside
(18). Compound 17 was phosphorylated with 23 according to the procedure B

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
565
to afford 18 (70%) as a gum. Rf 0.38 (EtOAc/MeOH,97:3); [a]D þ 31.1 (c 0.8,
1
CH2Cl2); IR 2254 (CN); 1256 (P55O); 1040 (P-O) cm21; H NMR (400 MHz,
0
CDCl3) d 7.30–7.45 (m, 5H, H Ar); 5.93 (m, 1H, H-7); 5.38 (dd, 1H, J7-8 17.1,
J8-8 1.5 Hz, H-80); 5.27 (dd, 1H, J7-8 11.5 Hz, H-8); 4.92 (d, 1H, J1-2 3.5 Hz,
0
3
H-1); 4.78 (m, 1H, J3-4 9.3, JP-H 8.8 Hz, H-3); 4.64 (AB, 2H, JAB 11.4 Hz,
CH2Ph); 4.03–4.50 (m, 10H, H-4, H-6, 4CH2CH2CN); 3.92–4.02 (m, 2H, H-5,
H-60); 3.72 (dd, 1H, J4-5 10.1, J5-5 10.8 Hz, H-50); 3.59 (dd, 1H, J2-3 9.8 Hz,
0
H-2); 2.79–2.88 (m, 4H, CH2CH2CN); 2.70–2.77 (m, 2H, CH2CH2CN); 2.47–
2.57 (m, 1H, CH2CH2CN); 2.33–2.42 (m, 1H, CH2CH2CN); 13CNMR (CDCl3)
d 137.2 (s, Cipso); 133.1 (C-7); 128.6; 128.3; 128.2 (3s, 5C, CH Ar); 118.6
(C-8); 117.0; 116.8; 116.7 (3s, CN); 94.8 (C-1); 78.2 (m, C-3); 77.5 (C-2); 74.4
2
(m, C-4); 72.5 (CH2Ph); 68.6 (C-6); 62.1–63.3 (4d, 4C, JC-P 4.9 Hz,
4CH2CH2CN); 59.0 (C-5); 19.3–19.5 (4d, 4C, 4CH2CH2CN); 31PNMR (CDCl3)
d 21.86; 21.94 (2s, 2P). Anal. Calcd for C27H34O11N4P2: C, 49.72; H, 5.21; N,
8.58; P, 9.49. Found: C, 49.79; H, 5.24; N, 8.43; P, 9.54.
(Oxoethanyl) 2-O-benzyl-3,4-bis[(2-cyanoethyl) phosphate] a-D-xylo-
pyranoside (19). Compound 18 (1.86 g, 2.85 mmol) was dissolved in dioxane
(25 mL) and water was added (25 mL). To this solution were added dropwise
2.65 mL of osmium tetroxide (2.5% in tert-butanol) and sodium periodate
(5 eq., 3.05 g, 14.2 mmol). After stirring for 2 hr 30 min, TLC analysis showed
complete consumption of the starting material and the solution was filtered
over Celite. The mixture was diluted with CH2Cl2 (300 mL) and the organic
layer was washed twice with water (50 mL), dried over MgSO4, and concen-
trated in vacuo to afford 19 (99%, 1.86 g, 2.84 mmol) as a yellow gum. Rf 0.45
(EtOAc/MeOH,9 : 1); [a]D þ 32.2 (c 1.3, CHCl3); IR 2255 (CN); 1734 (C55O);
1280(P55O); 1039(P-O) cm21 1H NMR (250 MHz, CDCl3) d 9.70 (s, 1H,
;
COH); 7.22–7.48 (m, 5H, H Ar); 4.92 (d, 1H, J1-2 3.9 Hz, H-1); 4.60–4.85
(m, 2H, CH2Ph, H-3); 4.64 (AB, 1H, JAB 10.7 Hz, CH2Ph); 3.95–4.50 (m, 11H,
H-4, H-5a, H-10a, 4CH2CH2CN); 3.45–3.84 (m, 3H, H-2, H-5b, H-10b); 2.31–
2.89 (m, 8H, 4CH2CH2CN), 13C NMR (CDCl3) d: 198.3 (C55O); 137.2 (Cipso);
128.7; 128.5 (2s, 5C, CH Ar); 116.9 (s, 4C, CN); 96.2 (C-1); 77.6 (m, C-3); 76.6
2
3
(C-2); 74.1 (dd, JC-P 5.7, JC-P 2.8 Hz, C-4); 72.9 (CH2Ph); 72.3 (C-10); 62.3–
2
63.4 (4d, 4C, JC-P 5.2 Hz, 4CH2CH2CN); 59.4 (C-5); 19.5–19.8 (4d, 4C,
4CH2CH2CN); 31P NMR (CDCl3)
d 21.96 (s, 2P). Anal. Calcd for
C26H32O12N4P2: C, 47.74; H, 4.89; N, 8.56; P, 9.46. Found: C, 47.70; H, 4.94;
N, 8.53; P, 9.47.
(2-Hydroxyethyl) 2-O-benzyl-3,4-bis[(2-cyanoethyl) phosphate] a-D-
xylopyranoside (20). To a solution of 19 (1.86 g, 2.79 mmol) in methanol
(88 mL) was added NaBH4 (2 eq., 224 mg, 5.58 mmol). After stirring for 3 min,
the solution was neutralized with 3 N HCl, the solvent was evaporated, and
the residue was diluted in CH2Cl2 (30 mL). The organic layer was washed
with water (2 ꢀ 40 mL), dried over MgSO4, and concentrated in vacuo. The

´
F. Chretien et al.
566
crude mixture was purified by preparative HPLC (EtOAc/MeOH 96.5 : 3.5) to
give 20 (57%, 1.07 g, 1.60 mmol) as a gum. Rf 0.36 (EtOAc/MeOH,9 : 1);
1
[a]D þ 30.1 (c 1.8, CHCl3); IR 2255 (CN); 1276 (P55O); 1037 (P-O) cm21; H
NMR (400 MHz, CDCl3) d 7.32–7.47 (m, 5H, H Ar); 4.80–4.89 (m, 2H, H-1,
H-3); 4.68 (s, 2H, CH2Ph); 4.14–4.50 (m, 9H, H-4, 4CH2CH2CN); 4.00 (dd,
1H, J4-5eq 6.0, J5eq-5ax 11.1 Hz, H-5eq); 3.73–3.86 (m, 4H, H-5ax, H-10a, H-20a,
H-20b); 3.59 (dd, 1H, J1-2 3.3, J2-3 9.8 Hz, H-2); 3.49 (m, 1H, H-10b); 2.78–2.91
(m, 4H, 2CH2CH2CN); 2.67–2.74 (m, 2H, CH2CH2CN); 2.48–2.62 (m, 2H,
CH2CH2CN); 1.90 (s, 1H, OH), 13C NMR (CDCl3) d: 137.1 (s, Cipso); 128.6;
128.5; 128.3 (3s, 5C, CH Ar); 116.8 (s, 4C, CN); 96.3 (C-1); 78.1 (m, C-3); 77.7
2
3
(C-2); 74.4 (dd, JC-P 5.7, JC-P 2.8 Hz, C-4); 72.8 (CH2Ph); 70.1 (C-10);
2
62.2–63.3 (4d, 4C, JC-P 5.2 Hz, 4CH2CH2CN); 61.2 (C-20); 59.0 (C-5); 19.3–
19.5 (4d, 4C, 4CH2CH2CN)); 31P NMR (CDCl3) d 23.31; 23.09 (2s, 2P). Anal.
Calcd for C26H34O12N4P2: C, 47.59; H, 5.18; N, 8.53; P, 9.44. Found: C, 47.52;
H, 5.24; N, 8.62; P, 9.45.
[(2-Dioctylphosphonoxy)ethyl] 2-O-benzyl-3,4-bis[(2-cyanoethyl)phos-
phate] a-D-xylopyranoside (21). The alcohol 20 was phosphorylated with
the phosphoramidite 15 according to the procedure B. The residue was
purified by column chromatography (EtOAc/MeOH 98 : 2 ! 96 : 4) to give 21
(60%) as a gum. Rf 0.5 (EtOAc/MeOH,95 : 5); [a]D þ 29.8 (c 1.56, CH2Cl2); IR
1
2255 (CN); 1277(P55O); 1037 (P-O) cm21; H NMR (400 MHz, CDCl3) d 7.22–
7.42 (m, 5H, H Ar); 4.92 (d, 1H, J1-2 3.6 Hz, H-1); 4.63–4.80 (m, 2H, CH2Ph,
H-3); 4.56 (AB, 1H, JAB 11 Hz, CH2Ph); 3.89–4.48 (m, 16H, 4H-100, H-4,
4CH2CH2CN, H-5a, H-20a, H-20b); 3.82 (m, 1H, H-10a); 3.61–3.76 (m, 4H,
H-5ax, H-10b); 3.55 (dd, 1H, J2-3 9.8 Hz, H-2); 2.64–2.86 (m, 6H, CH2CH2CN);
2.24–2.55 (m, 2H, CH2CH2CN); 1.51–1.72 (m, 4H, 2H-200a, 2H-200b); 1.12–
1.42 (m, 20H, 2(H-300a, H-300b, H-400a, H-400b, H-500a, H-500b, H-600a, H-600b,
H-700a, H-700b)); 0.77–0.93 (m, 6H, 6H-800); 13C NMR (CDCl3) d 137.4 (Cipso);
128.8; 128.4; (2s, 5C, CH Ar); 116.9; 116.8 (2s, 4C, CN); 96.4 (C-1); 78.1
(m, C-3); 77.7 (C-2); 74.4 (m, C-4); 72.5 (CH2Ph); 68.2 (m, 2C, 2C-100); 67.2
3
2
2
(d, JC-P 7.3 Hz, C-10); 66.2 (d, 1C, JC-P 5.7 Hz, C-20); 63.2 (d, 1C, JC-P 6.2 Hz,
2
CH2CH2CN); 63.0 (m, 2C, 2CH2CH2CN); 62.5 (d, 1C, JC-P 4.9 Hz,
CH2CH2CN); 59.3 (C-5); 31.9 (s, 2C, 2C-600); 30.5 (d, 2C, JC-P 7.3 Hz, 2C-200);
3
30.4; 29.3 (2s, 4C, 2C-400, 2C-500); 25.6 (s, 2C-300); 22.8 (s, 2C-700); 19.4-19.9 (4d,
4C, 4CH2CH2CN); 14.3 (s, 6C-800); 31P NMR (CDCl3) d: 0.49; 21.92; 21.95
(3s, 3P). Anal. Calcd for C42H67O15N4P3: C, 52.49; H, 6.97; N, 5.82; P, 9.66.
Found: C, 52.20; H, 7.07; N, 5.77; P, 9.63.
[(2-Dioctylphosphonoxy)ethyl] 3,4-bis[(2-cyanoethyl)phosphate] a-D-
xylopyranoside (22). Compound 21 (280 mg, 0.291 mmol) was dissolved in
a solution of KOH (0.5 M in methanol, 4.85 mL) and heated at 408C for 3 hr.
The reaction mixture was then cooled to rt and neutralized to pH 7 with
Dowex 50W (Hþ) cation-exchange resin. The resin was filtered off and

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
567
washed twice with methanol (10 mL). The filtrate was concentrated to dryness;
the residue was dissolved in deionized water (3 mL) and applied to a column of
Bio-Rad Chelex 100 resin (Naþ form). The compound was eluted with deionized
water; fractions containing compound 22 were combined and freeze dried to
give 22 (99%, 241 mg, 0.288 mmol) as a white powder. [a]D þ 4.5 (c 1.60,
1
H2O); IR (KBr) 1263 (P55O); 1037 (P-O) cm21; H NMR (D2O; 250 MHz) d
7.17–7.53 (m, 5H, H Ar); 4.92 (AB, 1H, JAB 12 Hz, CH2Ph); 4.73 (d, 1H, J1-2
3
3.4 Hz, H-1); 4.67 (AB, 1H, CH2Ph); 4.42 (ddd, 1H, J2-3 9.5, J3-4 9.6, JH-P
7.3 Hz, H-3); 3.75–4.33 (m, 9H, 4H-100, H-4, H-5a, H-10a, H-20a, H-20b); 3.44–
3.71 (m, 3H, H-2, H-5b, H-10b); 1.50–1.73 (m, 4H, 2H-200a, 2H-200b); 1.19–1.47
(m, 20H, 2(H-300a, H-300b, H-400a, H-400b, H-500a, H-500b, H-600a, H-600b, H-700a,
H-700b)); 0.81–1.06 (m, 6H, 6H-800); 13C NMR (D2O) d 139.3 (Cipso); 128.9;
128.8 (2s, 5C, CH Ar); 99.9 (C-1); 80.7 (C-2); 78.4 (dd, C-3); 75.8 (CH2Ph);
2
2
74.1 (dd, C-4); 70.3–71.3 (2d, 2C, JC-P 6.7 Hz, 2C-100); 69.1 (d, 1C, JC-P
3
5.7 Hz, C-20); 68.8 (d, JC-P 7.6 Hz, C-10); 63.4 (C-5); 34.3 (s, 2C, 2C-600); 32.2–
32.6 (m, 2C, 2C-200); 31.8; 31.6 (2s, 4C, 2C-400, 2C-500); 28.0 (s, 2C-300); 25.1
(s, 2C-700); 16.2 (s, 6C-800); 31P NMR (D2O) d 6.58; 4.13 (2s, 2P, 2OPO3); 23.36
(s, 1P, C20OP). ESI-MS (negative mode): Calcd for C30H55O15P3 m/z: 747 [M-
H]2; 769 [M-2H þ Na]2.
[(2-Dioctylphosphonoxy)ethyl] 3,4-bis(dihydrogen-phosphate) a-D-xylo-
pyranoside tetrasodium salt (7). Compound 7 was obtained in 99% yield
as a white powder by treatment of 21 according to procedure D. [a]D þ 3.3
1
(c 0.45, H2O); H NMR (400 MHz, D20) d4.84 (d, 1H, J1-2 3.5 Hz, H-1); 4.15–
4.24 (m, 2H, H-20b, H-20a); 4.01–4.14 (m, 5H, H-3, 4H-100); 3.84–3.95 (m, 3H,
H-4, H-5 eq, H-10a); 3.62–3.78 (m, 3H, H-2, H-5ax, H-10b); 1.53–1.71 (m, 4H,
2H-200a, 2H-200b); 1.07–1.43 (m, 20H, 2(H-300a, H-300b, H-400a, H-400b, H-500a,
H-500b, H-600a, H-600b, H-700a, H-700b)); 0.71–0.89 (m, 6H, 6H-800); 13CNMR
2
(D20) d 101.3 (C-1); 76.0 (m, C-3); 73.8 (C-2); 74.1 (m, C-4); 71.3 (d, 2C, JC-P
6.2 Hz, 2C-100); 68.9–69.5 (m, 2C, C-10, C-20); 64.6 (C-5); 34.4 (s, 2C, 2C-600);
3
32.7 (d, 2C, JC-P 6.7 Hz, 2C-200); 31.8; 31.7 (4C, 2C-400, 2C-500); 28.1 (2C,
2C-300); 25.3 (2C, 2C-700); 16.6 (2C, 2C-800); 31PNMR (D20) d 7.96; 7.31 (2s, 2P,
2OPO3Na2); -3.18 (s, 1P, C20OP); ESI-MS (negative mode): Calcd for
C23H49O15P3 m/z: 657 [M-H]2; 679 [M-2H þ Na]2; 701 [M-3H þ 2Na]2.
Octyl phosphonate triethylamonium salt (25). To a solution of 1-octanol
(651 mg, 5 mmol) and NEt3 (1.39 mL, 10 mmol) in dioxane (7 mL) was added
under argon a dioxane solution (5 mL) of 2-chloro-1,3,2-benzodioxaphosphori-
n-4-one (1.01 g, 5 mmol). After 30 min at rt, 31P NMR indicated complete con-
sumption of the starting chlorophosphite (149.6 ppm) to give a product with a
signal at 125.6 ppm; water (1 mL) was then added to the medium. The
solvents were evaporated to dryness and the residue was coevaporated twice
with toluene. Compound 25 (812 mg, 2.73 mmol) was as a white solid in 55%
yield after purification by column chromatography (AcOEt ! AcOEt/MeOH

´
F. Chretien et al.
568
85 : 15). 1H NMR (250 MHz, CDCl3): d 6.78 (d, 1H, JP-H 628 Hz, P-H); 3.85
(m, 2H, 2H-1); 3.03 (q, 6H, J 7.1 Hz, N(CH2CH3)3); 1.53–1.67 (m, 2H, 2H-2);
1.13–1.40 (m, 19H, 2(H-3, H-30, H-4, H-40, H-5, H-50, H-6, H-60, H-7, H-70),
N(CH2CH3)3)); 0.83 (t, 3H, JH-H 5.8 Hz, 3H-8). 13C NMR (CDCl3): d 63.8
2
3
(d, JC-P 409 Hz, C-1); 4504 (s, 3C, N(CH2CH3)3; 3104 (s, C-6); 3003 (d, JC-P
703 Hz, C-2); 2808 (s, 2C, C-4, C-5); 25.4 (s, C-3); 22.2 (s, C-7); 13.8 (s, C-8);
8.2 (s, 3C, N(CH2CH3)3); 31P NMR (CDCl3): d 3.51 (s, 1P).
[(2-Octylphosphonoxy)ethyl] 2-O-benzyl-3,4-bis[(2-cyanoethyl)phosphate]
a-D-xylopyranoside (24). To a solution of xyloside 20 (250 mg, 0.37 mmol)
and H-phosphonate monoester 25 (1 eq., 110 mg, 0.37 mmol) in pyridine
(5 mL) was added dropwise a solution of pivaloyl chloride (3 eq., 0.13 mL,
1.11 mmol) in pyridine (1 mL) at 2208C for 25 min under argon. After being
stirred for 2 hr, a solution of iodine (1.25 eq., 118 mg, 0.46 mmol) in a
pyridine/water mixture (0.9 mL, 98 : 2, v/v) was added. The medium was
warmed to rt and after 45 min, a solution of sodium sulfite in water (1%) was
added until complete discoloration of the medium. The solvents were evapor-
ated in vacuo, the residue was coevaporated three times with toluene and the
crude product was purified by column chromatography on silicic acid (EtOAc/
MeOH 98 : 2 ! 80 : 20) and then on LH20 Sephadex gel (CHCl3/MeOH 1 : 1) to
give 24 (40%, 140 mg, 0.15 mmol) as a gum. [a]D þ 23.0 (c 0.98, CHCl3); IR 2251
(CN); 1280 (P55O); 1038 (P-O) cm21; 1H NMR (250 MHz, CDCl3/MeOD, 1 : 1) d
7.07–7.29 (m, 5H, H Ar); 4.73 (d, 1H, J1-2 2.9 Hz, H-1); 4.37–4.60 (m, 3H, H-3,
CH2Ph); 3.47–4.34 (m, 17H, H-4, H-5, H-50, H-10a, H-10b, H-20a, H-20b, 2H-100,
4CH2CH2CN); 3.41 (dd, 1H, J2-3 9.5 Hz, H-2); 3.14 (q, 6H, J 7.1 Hz,
N(CH2CH3)3); 2.50–2.75 (m, 6H, 3CH2CH2CN); 2.17–2.49 (m, 2H,
CH2CH2CN); 1.32–1.61 (m, 2H, 2H-200); 0.85–1.30 (m, 19H, 2(H-300a, H-300b,
H-400a, H-400b, H-500a, H-500b, H-600a, H-600b, H-700a, H-700b), N(CH2CH3)3));
0.56–0.77 (t, 3H, JH-H 5.8 Hz, 3H-800); 13CNMR(CDCl3/MeOD, 1 : 1) d 136.8
(Cipso); 128.0; 127.9; 127.6 (5C, CH Ar); 117.6 (s, CN); 95.8 (C-1); 77.9
(m, C-3); 77.1 (C-2); 74.1 (m, C-4); 72.1 (CH2Ph); 67.2 (m, C-10); 62.2–63.3
(m, 6C, C-100, C-20, 4CH2CH2CN); 58.6 (C-5); 45.8 (s, 3C, N(CH2CH3)3); 31.4
3
(C-600); 30.0 (d, JC-P 7.2 Hz, C-200); 29.2; 28.9 (2C, C-400, C-500); 25.4 (C-300); 22.2
(C-700); 13.4 (C-800); 8.0 (3C, N(CH2CH3)3); 31PNMR (CDCl3/MeOD, 1 : 1)d
22.43; 21.78 (2s, 2OPO(OCH2CH2CN)2); 0.40 (s, OPORO22 ); ESI-MS
(negative mode): Calcd for C34H51O15N4P3 m/z: 847 [M-H]2.
[(2-Octylphosphonoxy)ethyl] 3,4-bis(dihydrogen-phosphate) a-D- xylo-
pyranoside pentatriethylamonium salt (8). The title compound was
obtained as a white powder from xyloside 24 in 98% yield according to pro-
cedure D. [a]D þ 16.1 (c 1.05, H2O); IR 1232 (P55O); 1100 (P-O) cm21
;
1H
NMR (250 MHz, D2O) d 4.96 (d, 1H, J1-2 3 Hz, H-1); 4.23 (ddd, 1H, J2-3 9.4,
3
J3-4 9.5, JH-P 8.0 Hz, H-3); 3.62–4.14 (m, 10H, H-2, H-4, H-5a, H-5b, H-10a,
H-10b, H-20a, H-20b, 2H-100); 1.52–1.75 (m, 2H, 2H-200); 1.18–1.48 (m, 10H,

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
569
2(H-300a, H-300b, H-400a, H-400b, H-500a, H-500b, H-600a, H-600b, H-700a, H-700b); 0.80–
0.98 (t, 3H, JH-H 5.8 Hz, 3H-800); 13CNMR (D2O) d 99.2 (C-1); 75.4 (m, C-3); 72.5
2
3
2
(C-2); 71.5 (dd, JC-P 5.4 Hz, C-4); 68.5 (d, JC-P 7.1 Hz, C-10); 67.6 (d, JC-P
2
3
6.2 Hz, C-100); 65.6 (d, JC-P 5.7 Hz, C-20); 63.0 (C-5); 32.1 (C-600); 30.9 (d, JC-P
7.2 Hz, C-200); 29.4 (2C, C-400, C-500); 26.1 (C-300); 23.1 (C-700); 14.5 (C-800);
31PNMR (D20) d 7.80; 7.20 (2s, 2OPO32-); 4.60 (s, OPORO22 ); ESI-MS
(negative mode): Calcd for C15H33O15P3 m/z: 545 [M-H]2.
[(2-Dicyanoethylphosphonoxy)ethyl] 2-O-benzyl-3,4-bis[(2-cyanoethyl)-
phosphate] a-D-xylopyranoside (26). Compound 12 was phosphorylated
according to procedure B with the phosphoramidite 23 to give the compound
26 in 58% as a gum after purification by column chromatography (AcOEt/
MeOH 98 : 2 ! 88 : 12). Rf 0.36 (AcOEt/MeOH,9 : 1); [a]D þ 26.1 (c 0.72,
CHCl3); IR 2255 (CN); 1279 (P55O); 1036 (P-O) cm21 1H NMR (250 MHz
.
CDCl3): d 7.28–7.43 (m, 5H, H Ar); 4.83 (d, 1H, J1-2 3.6 Hz, H-1); 4.76 (ddd,
3
1H, J2-3 9.5, J3-4 9.2, JH-P 7.9 Hz, H-3); 4.66 (s, 2H, CH2Ph); 4.22–4.52
(m, 15H, H-4, H-20a, H-20b, 6 CH2CH2CN); 4.01 (dd, 1H, J4-5eq 6.0, J5eq-5ax
11.7 Hz, H-5eq); 3.89 (m, 1H, H-10a); 3.75 (dd, 1H, J4-5ax 9.7 Hz, H-5ax); 3.53–
3.68 (m, 2H, H-2, H-10b); 2.33–2.92 (m, 12H, 6 CH2CH2CN); 13C NMR
(CDCl3): d 137.0 (s, Cipso); 128.3; 128.0; 127.8; (3s, 5C, CH Ar); 116.8; 116.7;
2
3
116.6 (3s, 3CN); 95.8 (s, C-1); 77.6 (dd, JC-P 7.8, JC-P 5.9 Hz, C-3); 77.2
2
3
2
(s, C-2); 73.7 (dd, JC-P 5.2, JC-P 3.3 Hz, C-4); 72.3 (s, CH2Ph); 66.7 (d, JC-P
3
2
5.7 Hz, C-20); 66.7 (d, JC-P 6.7 Hz, C-10); 62.3-63.3 (6d, 6C, JC-P 5.7 Hz, 6
CH2CH2CN); 58.7 (s, C-5); 19.0-19.3 (6d, 6C, 6 CH2CH2CN); 31P NMR (CDCl3):
d 21.38; 21.48 (2s); 21.92 (s, C-20OP). Anal. Calcd for C32H41O15N6P3: C,
45.61; H, 4.90; N, 9.97; P, 11.03. Found: C, 45.37; H, 4.85; N, 10.15; P, 10.92.
[2-(Phosphonoxy)ethyl] 2-O-benzyl-3,4-bis(dihydrogen-phosphate) a-D-
xylopyranoside hexasodium salt (27). Compound 27 was obtained as a
white solid by treatment of 26 according to procedure E. [a]D þ 26.0 (c 1.5,
1
H2O); IR 1232 (P55O), 1100 (P-O) cm21. H NMR (250 MHz D2O): d 7.37–
7.62 (m, 5H, H Ar); 4.95 (AB, 1H, JAB 11.9 Hz, CH2Ph); 4.70–4.87 (m, 2H,
3
H-1, CH2Ph); 4.46 (ddd, 1H, J2-3 9.2, J3-4 9.0, JH-P 7.9 Hz, H-3); 4.09 (dddd,
1H, H-4); 3.90–4.02 (m, 2H, H-20a, H-20b); 3.74–3.89 (m, 2H, H-5eq, H-10a);
3.57–3.73 (m, 3H, H-2, H-5ax, H-10b); 13C NMR (D2O): d 138.2 (s, Cipso);
3
129.4; 129.0 (2s, 5C, CH Ar); 99.4 (s, C-1); 79.9 (d, JC-P 3.8 Hz, C-2); 79.1
2
3
2
(dd, JC-P 5.9, JC-P 4.7 Hz, C-3); 76.0 (s, CH2Ph); 74.5 (dd, JC-P 4.8 Hz, C-4);
70.0 (d, JC-P 7.6 Hz, C-10); 65.9 (d, JC-P 4.8 Hz, C-20); 59.1 (s, C-5). 31P NMR
(D2O): d 7.66; 6.84 (2s, 2OPO23-); 4.25 (s, C-20OP); ESI-MS (Negative mode):
Calcd for C14H23O15P3 m/z: 611 [M-5H þ 4Na]2; 589 [M-4H þ 3Na]2; 567 [M-
3H þ 2Na]2; 545 [M-2H þ Na]2; 523 [M-H]2.
3
2
[(2-(Diacetoxymethylphosphonoxy)ethyl] 2-O-benzyl-3,4 bis(diacethoxy-
methyl)phosphate a-D-xylopyranoside (28). Compound 27 (139 mg,

´
F. Chretien et al.
570
0.21 mmol) was dissolved in water (2 mL) and applied to column of DOWEX
50W Hþ resin. The column was eluted with deionized water; fractions contain-
ing the free phosphate compound (114 mg, 0.21 mmol) were combined and
freeze dried. The resulting powder was suspended in acetonitrile (1.45 mL) con-
taining N,N-diisopropylethylamine (0.3 mL). The suspension was sonicated
and concentrated. This operation was repeated twice or more to get an oily
residue, which was dissolved in acetonitrile (1.45 mL), and then DIEA
(0.73 mL) and acetoxymethyl bromide (1.95 g, 12.78 mmol) were added. The
solution was stirred 28 hr (31P RMN monitoring) at rt and the solvent was con-
centrated. Compound 28 (80 mg, 0.08 mmol, 39%) was obtained as a gum after
purification by column chromatography (AcOEt). Rf 0.24 (AcOEt/MeOH, 98:
1
2); [a]D þ 22.8 (c 0.7, CHCl3); IR: 1766 (C55O); 1216 (P55O) cm21; H NMR
(250 MHz CDCl3): d 7.21–7.39 (m, 5H, H Ar); 5.42–5.73 (m, 12H, 6O-CH2-O);
4.62–4.78 (m, 3H, H-1, H-3, CH2Ph); 4.55 (AB, 1H, JAB 11.7 Hz, CH2Ph);
4.37 (dddd, 1H, H-4); 4.18–4.30 (m, 2H, H-20a, H-20b); 3.88 (ddd, 1H, J4-5eq
4
5.8, J5eq-5ax 11. JH-P 2,9 Hz, H-5eq); 3.76 (dddd, 1H, H-10a); 3.51–3.70
(m, 1H, H-5ax, H-10b); 3.44 (dd, 1H, J1-2 3.6, J2-3 9.5 Hz, H-2); 2.12; 2.10; 2.07
(3s, 18H, 6CH3C55O); 13C NMR (CDCl3): d 168.9 (s, 6C, 6C55O); 137.1
(s, Cipso); 128.2; 127.8 (2s, 5C, CH Ar); 96.4 (s, C-1); 81.5–82.5 (6d, 6C,
2
2
6OCH2C55O); 77.7 (dd, JC-P 7.3 Hz, C-3); 77.0 (s, C-2); 73.6 (dd, JC-P 4.9 Hz,
C-4); 72.8 (s, CH2Ph); 66.5 (m, 2C, C-10, C-20); 58.7 (s, C-5); 20.3 (s, 6C,
6CH3C55O); 31P NMR (CDCl3; 100 MHz): d 22.98; 23.31 (2s); 23.95
(s, C-20OP). ESI-MS (positive mode): Calcd for C32H47O27P3 m/z: 956 [M]þ.
[(2-(Dibutyryloxymethylphosphonoxy)ethyl] 2-O-benzyl-3,4 bis(dibuty-
ryloxymethyl phosphate) a-D-xylopyranoside (29). Compound 29 was
obtained as a gum in 22% yield after purification by column chromatography
(AcOEt/Hex 65 : 35 ! 75 : 25) by treatment of 27 with butyryloxymethyl iodide
using the procedure described for the preparation of 28. Rf 0.3 (Hex/AcOEt,
1
3 : 7), [a]D þ 20.0 (c 0.6, CHCl3); IR 1766 (C55O); 1218 (P55O) cm21; H NMR
(250 MHz CDCl3): d 7.22–7.42 (m, 5H, H aromatics); 5.46–5.77 (m, 12H,
6O-CH2-O); 4.65–4.82 (m, 3H, H-1, H-3, CH2Ph); 4.55 (AB, 1H, JAB 11.7Hz,
CH2Ph); 4.36 (dddd, 1H, H-4); 4.17–4.32 (m, 2H, H-20a, H-20b); 3.89 (ddd, 1H,
4
J4-5eq 5.8, J5eq-5ax 11. JH-P 2.9 Hz, H-5eq); 3.77 (dddd, 1H, H-10a); 3.50–3.69
(m, 2H, H-5ax, H-10b); 3.44 (dd, 1H, J1-2 2.9, J2-3 9.5 Hz, H-2); 2.23–2.43
(m, 12H, 6H-100a, 6H-100b); 1.56–1.75 (m, 12H, 6H-200a, 6H-200b); 0.88–1.04
(m, 18H, 6CH3); 13C NMR (CDCl3): d 171.5 (s, 6C, 6C55O); 137.1 (s, Cipso);
128.2; 127.8 (2s, 5C, CH Ar); 96.4 (s, C-1); 82.0–82.8 (6d, 6C, 6OCH2C55O);
2
3
77.7 (dd, JC-P 6.1, JC-P 7.3 Hz, C-3); 77.0 (s, C-2); 73.6 (dd, JC-P 4.8 Hz, C-4);
72.7 (s, CH2Ph); 66.4 (2d, 2C, C-10, C-20); 58.7 (s, C-5); 35.3 (s, 6C, 6C-100); 17.6
(s, 6C, 6C-200); 13.2 (s, 6C, 6CH3); 31P NMR (CDCl3; 100 MHz): d 23.07; 23.37
(2s); 23.99 (s, C-20OP); Anal. Calcd for C44H71O27P3; C, 46.98; H, 6.36; P, 8.26.
Found: C, 46.87; H, 6.48; P, 8.13.

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
571
[(2-(Dioctanoyloxymethylphosphonoxy)ethyl] 2-O-benzyl-3,4-bis(dioc-
tanoyloxymethylphosphate) a-D-xylopyranoside (30). Compound 30
was obtained as a gum in 35% yield after purification by column chromato-
graphy (AcOEt/Hex 3 : 7 ! 45 : 55) by treatment of 27 with octanoyloxymethyl
iodide using the procedure described for the preparation of 28. Rf 0.43 (Hex/
AcOEt, 5 : 5); [a]D þ 12.7 (c 1.14, CHCl3); IR 1766 (C55O); 1284 (P55O) cm21
;
1H NMR (250 MHz CDCl3): d 7.27–7.42 (m, 5H, H Ar); 5.45–5.75 (m, 12H,
6O-CH2-O); 4.65–4.82 (m, 3H, H-1, H-3, CH2Ph); 4.57 (AB, 1H, JAB 11.7 Hz,
CH2Ph); 4.38 (dddd, 1H, H-4); 4.17–4.32 (m, 2H, H-20a, H-20b); 3.89 (ddd,
4
1H, J4-5eq 5.8, J5eq-5ax 11. JH-P 2.9 Hz, H-5eq); 3.78 (dddd, 1H, H-10a); 3.51–
3.70 (m, 2H, H-5ax, H-10b); 3.45 (dd, 1H, J1-2 3.6, J2-3 9.5 Hz, H-2); 2.27–2.45
(m, 12H, 6H-100a, 6H-100b); 1.50–1.73 (m, 12H, 6H-200a, 6H-200b); 1.18–1.43
(m, 48H, 6(H-300a, H-300b, H-400a, H-400b, H-500a, H-500b, H-600a, H-600b); 0.79–
0.99 (m, 18H, 6CH3); 13C NMR (CDCl3): d 171.6; 171.5 (2s, 6C, 6C55O); 137.1
(s, Cipso); 128.1; 127.7 (2s. 5C, CH Ar); 96.3 (s, C-1); 82.0–82.7 (6d, 6C,
2
3
6OCH2C55O); 77.7 (dd, JC-P 6.1, JC-P 7.3 Hz, C-3); 77.0 (s, C-2); 73.6 (dd,
2
JC-P 4.8 Hz, C-4); 72.7 (s, CH2Ph); 66.5 (d, JC-P 4.9 Hz, C-20); 66.3 (d, 1C,
3JC-P 7.3 Hz, C-10); 58.7 (s, C-5); 33.4 (s, 6C, C); 31.2 (s, 6C, C) 28.5 (s, 6C, C);
24.4 (s, 6C, C); 24.0 (s, 6C, C); 22.5 (s, 6C, C); 13.6 (s, 6C, 6C700); 31P NMR
(CDCl3; 100 MHz): d 23.18; 23.46 (2s); 24.12 (s, C-20OP). Anal. Calcd for
C68H119O27P3: C, 55.88; H, 8.21; P, 6.36. Found: C, 55.99; H, 8.14; P, 6.68.
[(2-(Diacetoxymethylphosphonoxy)ethyl] 3,4-bis(diacethoxymethyl)-
phosphate a-D-xylopyranoside (9). To a solution of 28 (20 mg, 0.020 mmol)
in ethyl acetate (10 mL) was added Pd/C 10% (20 mg). The medium was hydro-
genated under 20 bars. After 12 hr, the catalyst was removed on a pad of Celite
and the solvent evaporated in vacuo to give compound 9 (17.8 mg, 0.020 mmol) as
a gum. Rf ¼ 0.37 (AcOEt/MeOH, 93 : 7); [a]D ¼ þ33.0 (c 1.54, CHCl3); IR 1767
1
(C55O); 1216 (P55O) cm21; H NMR (400 MHz CDCl3): d 5.58–5.78 (m, 12H,
3
6O-CH2-O); 4.92 (d, 1H, J1-2 3.7 Hz, H-1); 4.63 (ddd, 1H, J2-3 9.3, J3-4 9.3, JH-P
8.0 Hz, H-3); 4.41 (dddd, 1H, H-4); 4.19–4.36 (m, 2H, H-20a, H-20b); 3.89–4.01
(m, 2H, H-5eq, H-10a); 3.59–3.75 (m, 3H, H-10b, H-5ax, H-2); 2.14; 2.11; 2.09
(3s, 18H, 6CH3C55O); 13C NMR (CDCl3): d 168.9 (s, 6C, 6C55O); 98.5 (s, C-1);
2
2
82.4–82.8 (6d, 6C, 6OCH2C55O); 79.6 (dd, JC-P 6.1 Hz, C-3); 73.3 (dd, JC-P
3
2
4.9 Hz, C-4); 70.7 (s, C-2); 67.1 (d, JC-P 6.1 Hz, C-10); 66.8 (d, JC-P 3.7 Hz,
C-20); 59.1 (s, C-5); 20.3 (s, 6C, 6CH3C55O); 31P NMR (CDCl3): d 22.90; 23.08
(2s); 23.83 (s, C-20OP); LSIMS-MS (positive mode): Calcd for C25H41O27P3
m/z: 867 [M þ H]þ.
[(2-(Dibutyryloxymethylphosphonoxy)ethyl] 3,4-bis(dibutyryloxymethyl)
phosphate a-D-xylopyranoside (10). Compound 10 was quantitatively
obtained as a gum by treatment of 29 using the procedure described for the prep-
aration of 9. Rf 0.55(Hex/AcOEt, 4 : 6); [a]D þ 35.2 (c 0.68, CHCl3); IR 1767 (C55O);
1
1211 (P55O) cm21; H NMR (250 MHz CDCl3): d 5.53–5.79 (m, 12H, 6O-CH2-O);

´
F. Chretien et al.
572
4.89 (d, 1H, J1-2 3.6 Hz, H-1); 4.60 (ddd, 1H, J2-3 9.3, J3-4 9.3, 3JH-P 8.0 Hz, H-3); 4.37
(dddd, 1H, H-4); 4.21–4.33 (m, 2H, H-20a, H-20b); 3.84–4.00 (m, 2H, H-5eq, H-10a);
3.53–3.76 (m, 3H, H-10b, H-5ax, H-2); 2.28–2.43 (m 12H, 6H-100a, 6H-100b); 1.55–
1.77 (m, 12H, 6H-200a, 6H-200b); 0.88–1.07 (m, 18H, 6CH3); 13C NMR (CDCl3): d
172.2; 172.1 (2s, 6C, 6C55O); 99.2 (s, C-1); 82.7–83.3 (6d, 6C, 6OCH2C55O); 80.2
(dd, 2JC-P 6.1, 3JC-P 7.3 Hz, C-3); 74.0 (dd, JC-P 6.0 Hz, C-4); 71.4 (s, C-2); 67.5 (2d,
2C, C-10, C-20); 59.8 (s, C-5); 36.0 (s, 6C, 6C-100); 18.3 (s, 6C, 6C-200); 13.8 (s, 6C,
31
6CH3); P NMR (CDCl3; 100 MHz): 22.83; 23.11 (2s); 23.90 (s, C-20OP); ESI-
MS (positive mode): Calcd for C37H65O27P3 m/z: 1057 [M-H þ Na]þ; 1035 [M]þ.
[(2-(Dioctanoylphosphonoxy)ethyl] 3,4-bis(dioctanoyloxymethyl)phos-
phate a-D-xylopyranoside (11). Compound 11 was quantitatively obtained
as a gum by treatment of 29 using the procedure described for the preparation
of 9. Rf 0.55 (Hex/AcOEt, 4 : 6); [a]D þ 23.3 (c 1.24, CHCl3), IR 1770 (C55O);
;
1277 (P55O) cm21 1H NMR (250 MHz CDCl3): d 5.52–5.77 (m, 12H,
6O-CH2-O); 4.88 (d, 1H, J1-2 3.7 Hz, H-1); 4.60 (ddd, 1H, J2-3 9.3, J3-4 9.3,
3JH-P 8.1 Hz, H-3); 4.37 (dddd, 1H, H-4); 4.17–4.33 (m, 2H, H-20a, H-20b);
3.82–3.99 (m, 2H, H-5eq, H-10a); 3.53–3.75 (m, 3H, H-10b, H-5ax, H-2);
2.24–2.47 (6s, 12H, 6H-100a, 6H-100b); 1.50–1.73 (m, 12H, 6H-200a, 6H-200b);
1.18–1.43 (m, 48H, 6(H-300a, H-300b, H-400a, H-400b, H-500a, H-500b, H-600a,
H-600b); 0.78–0.98 (m, 18H, 6CH3); 13C NMR (CDCl3): d 178.1; 171.5 (2s, 6C,
6C55O); 98.5 (s, C-1); 82.0–82.8 (6d, 6C, 6OCH2C55O); 79.5 (dd, JC-P 6.1 Hz,
2
3
C-3); 73.3 (dd, JC-P 4.9 Hz, C-4); 70.7 (s, C-2); 66.8 (d, JC-P 6.1 Hz, C-10); 66.7
2
(d, JC-P 3.7 Hz, C-20); 59.0 (s, C-5); 33.4 (s, 6C, C); 31.2 (s, 6C, C); 28.5 (s, 6C,
C); 24.4 (s, 6C, C); 24.0 (s, 6C, C); 22.2 (s, 6C, C); 13.6 (s, 6C, 6C-700); 31P
NMR (CDCl3; 100 MHz): d 22.90; 23.29 (2s); 24.05 (s, C-20OP); Anal. Calcd
for C61H113O27P3 C, 53.40; H, 8.31; P, 6.78. Found: C, 53.72; H, 8.36; P, 6.65;
ESI-MS (positive mode): Calcd for C61H113O27P3 m/z: 1371 [M]þ.
(2(S)-Hydroxy-3-azidopropyl) 2-O-benzyl-3,4-O-[(2S,3S) (2,3-dimethoxy-
butane-2,3-diyl)]-a-D-xylopyranoside (33). To a solution of 32 (1.53 g,
3.73 mmol) in 2-methoxyethanol (40 mL) were added NH4Cl (490 mg,
9.32 mmol), water (13 mL), and NaN3 (970 mg, 14.9 mmol). The mixture was
heated under reflux for 1 hr and concentrated under reduced pressure, and
the residue was diluted in water (20 mL). The aqueous layer was extracted
with ethyl acetate. The organic layers were combined and dried over MgSO4
and concentrated in vacuo. Compound 33 (1.350 g, 2.98 mmol, 80%) was
obtained as a gum after purification by chromatography (Hex/AcOEt,1 : 1). Rf
0.26 (Hex/AcOEt,7 : 3); [a]D þ 157.3 (c 0.96, CHCl3); IR 3445 (OH); 2101 (N3)
cm21. 1H NMR (250 MHz CDCl3): d 7.22–7.40 (m, 5H, H Ar); 4.90 (AB, 1H,
JAB 11.7 Hz, CH2Ph); 4.73 (d, 1H, J1-2 3.7 Hz, H-1); 4,64 (AB, 1H, CH2Ph);
0
0
0
0
4.09 (dd, 1H, J2-3 9.6, J3-4 9.4 Hz, H-3); 3.87 (m, 1H, J2 -3 a 4.4, J2 -3 b 4.9 Hz,
H-20); 3.68–3.78 (m, 3H, H-4, H-5a, H-10a); 3.46–3.62 (m, 3H, H-2, H-5b,
H-10b); 3.21–3.41 (m, 8H, 2OCH3, H-30a, H-30b); 1.82 (s, 1H, OH); 1.35; 1.31

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
573
(2s, 6H, 2CH3). 13C NMR (CDCl3): d 137.8 (s, Cipso); 128.0; 127.4 (2s, 5C, CH Ar);
99.3; 99.1; (2s, 2CH3COCH3); 98.6 (s, C-1); 76.3 (s, C-2); 73.4 (s, CH2Ph); 70.0
(s, 1C, C-3); 69.8 (s, C-10); 69.0 (s, C-20); 66.0 (s, 1C, C-4); 59.3 (s, C-5); 53.0
(s, C-30); 47.5 (s, 2C, 2OCH3); 17.5; 17.2 (2s, 2CH3); Anal. Calcd for
C21H31N3O8: C, 55.60; H, 6.89; N, 9.27. Found: C, 55.45; H, 6.72; N, 9.14.
(2(S)-Hydroxy-3-azidopropyl) 2-O-benzyl-a-D-xylopyranoside (34). To a
solution of 33 (1.017 g, 3 mmol) in CH2Cl2 (20 mL) was added 95% aqueous
TFA solution (15 mL). The mixture was stirred at rt for 10 min and concen-
trated under reduced pressure. The residue was diluted with ethyl acetate
and the solution was neutralized by adding solid NaHCO3. Compound 34
(814 mg, 2.49 mmol) was obtained as a gum in 83% yield after purification by
column chromatography (Hex/AcOEt,1 : 9). Rf 0.46 (AcOEt); [a]D. þ 74.0 (c
1
0.36, CHCl3), IR 3390 (OH); 2101 (N3) cm21; H NMR (250 MHz CDCl3): d
7.23–7.40 (m, 5H, H Ar); 4.72 (AB, 1H, JAB 11.6 Hz, CH2Ph); 4.65 (d, 1H, J1-2
3.6 Hz, H-1); 4.60 (AB, 1H, CH2Ph); 3.77–4.08 (m, 2H, H-3, H-20); 3.43–3.71
(m, 4H, H-4, H-5a, H-5b, H-10a); 3.18–3.42 (m, 4H, H-2, H-10b, H-30a, H-30b);
1.72 (s, 3H, 3OH); 13C NMR (CDCl3): d 137.0 (s, Cipso); 128.4; 128.1 (2s, 5C,
CH Ar); 96.8 (s, C-1); 79.3 (s, C-2); 73.3 (s, CH2Ph); 72.8 (s, C-3); 69.5 (s, C-4);
69.4 (s, C-10); 69.1 (s, C-20); 61.2 (s, C-5); 52.9 (s, C-30); Anal. Calcd for
C15H21N3O6: C, 53.09; H, 6.24; N, 12.38. Found: C, 53.25; H, 6.17; N, 13.54.
(2(S)-Hydroxy-3-aminopropyl) 2-O-benzyl-a-D-xylopyranoside (35). To a
solution of 34 (228 mg, 0.67 mmol) in methanol (4.4 mL) was added Pd/C 10%
(34 mg) and the solution hydrogenated for 4 hr. The catalyst was removed by
filtration through a pad of Celite and the solvent was concentrated to afford
compound 35 (203 mg, 0.065 mmol; 97%) as a white solid. Rf 0.10 (CH2Cl2/
MeOH/H2O (65 : 25 : 4)); [a]D þ 72.7 (c 1.02, MeOH); IR 3368 (OH, NH2)
cm21 1H NMR (250 MHz CD3OD): d 7.13–7.43 (m, 5H, H Ar); 4.66–4.76
;
(m, 2H, CH2Ph, H-1); 4.58 (AB, 1H, JAB 11.7 Hz, CH2Ph); 3.54–3.80 (m, 3H,
H-3, H-20, H-10a); 3.35–3.53 (m, 3H, H-4, H-5a, H-5b); 3.17–3.34 (m, 2H,
H-2, H-10b); 2.52–2.83 (m, 2H, H-30a, H-30b); 13C NMR (CD3OD): d 140.2
(s, Cipso); 130.1; 130.0 (2s, 5C, CH Ar); 99.3 (s, C-1); 81.7 (s, 2C, C-2, C-20);
75.0 (s, C-3); 74.9 (s, CH2Ph); 72.1 (s, 2C, C-4, C-10); 63.5 (s, C-5); 45.9
(s, C-30), Anal. Calcd for C15H23NO6: C, 57.48; H, 7.40; N, 4.47. Found: C,
57.64; H, 7.49; N, 4.35.
[3-(p-Benzoylcinnamido)-2(S)-hydroxy-propyl] 2-O-benzyl-a-D-xylopyra-
noside (36). To a solution of 41 (257 mg, 1 mmol) in DMF (3 mL) were added
NEt3 (0.15 mL, 1.12 mmol) and BOP (495 mg, 1.12 mmol). The mixture was
stirred at rt for 15 min and then a solution of 35 (319 mg, 1 mmol) in DMF
(7 mL) was added. After 40 min, the solvent was removed in vacuo and the
residue was diluted with ethyl acetate. The organic layer was washed with
3N HCl and water, dried over Na2SO4, and concentrated. Compound 36

´
F. Chretien et al.
574
(398 mg, 0.73 mmol) was obtained as a gum in 72% yield after purification by
column chromatography (CH2Cl2/MeOH 97 : 3 ! 94 : 6). Rf 0.50 (CH2Cl2/
MeOH 9 : 1); [a]D þ 33.4 (c 1.0, CHCl3); IR 3351 (OH, NH); 1658; 1651; 1645
1
(PhC(O)Ph-, OC(O)N, -Ph-C55C-) cm21; H NMR (250 MHz CDCl3): d 7.65–
7.78 (m, 2H, H Ar); 7.39–7.63 (m, 8H, -C55CHPh, H Ar); 7.19–7.35 (m, 5H,
H Ar); 7.11 (t, 1H, J, NH); 6.56 (d, 1H, Jtrans 15.4 Hz, -C55CHC(O)); 4.48–
4.77 (m, 3H, CH2Ph, H-1); 3.82–4.00 (m, 3H, H-3, H-20, OH); 3.23–3.70
(m, 8H, H-4, H-5a, H-5b, H-2, H-10a, H-10b, H-30a, H-30b); 2.93 (s, 2H, OH);
13C NMR (CDCl3): 196.1 (C55O); 166.6 (C(O)NH); 139.3 (C55C-Ph); 138.3;
137.7; 137.1; 136.8 (4s, 4C, Cipso); 132.7; 130.2; 129.6; 128.3; 128.0; 127.3 (6s,
14C, CH Ar); 122.8 (C55C-C(O)); 96.8 (s, C-1); 79.4 (s, C-2); 73.3 (s, CH2Ph);
72.8 (s, C-3 or C-20); 69.9 (s, C-3 or C-20); 69.8 (s, C-10); 68.9 (s, C-4); 61.3
(s, C-5); 42.7 (s, C-30); Anal. Calcd for C31H33NO8: C, 67.99; H, 6.07; N, 2.56.
Found: C, 68.12; H, 6.19; N, 2.45.
[3-(p-Benzoylcinnamido)-2(S)-(2-dicyanoethylphosphonoxy)-propyl] 2-O-
benzyl-3,4-bis[(2-dicyanoethyl) phosphate]-a-D-xylopyranoside (37). The
triol derivative 36 was phosphorylated with the phosphoramidite 23 according
to procedure B. The residue was purified by column chromatography (CH2Cl2/
MeOH 98 : 2 ! 9 : 1) to give 37 (42%) as a gum. Rf 0.12 (CH2Cl2/MeOH 9 : 1);
[a]D þ 24.6 (c 0.8, CHCl3); IR 3320 (NH); 2251 (CN); 1658; 1651; 1646
(PhC(O)Ph-, OC(O)N, –Ph-C55C–; 1279 (P55O); 1040 (P-O) cm21 1H NMR
;
(250 MHz CDCl3): d 7.66–7.78 (m, 2H, H Ar); 7.38–7.64 (m, 8H, –C55CHPh, H
Ar); 7.22–7.37 (m, 5H, H Ar); 6.90 (t, 1H, J, NH); 6.62 (d, 1H, Jtrans 15.2 Hz,
–C55CHC(O)); 4.92 (d, 1H, J1-2 2.9 Hz, H-1); 4.58–4.84 (m, 4H, CH2Ph, H-3,
H-20); 3.94–4.52 (m, 16H, H-4, H-5eq, H-10a, H-10b, 6CH2CH2CN); 3.88 (dd, 1H,
J4-5ax 6.6 Hz, H-5ax); 3.50–3.79 (m, 3H, H-2, H-30a, H-30b); 13C NMR (CDCl3): d
195.7 (C55O); 165.8 (C(O)NH); 139.7 (C55C-Ph); 138.3; 137.9; 136.9 (3s, 4C,
Cipso); 132.4; 130.2; 129.6; 128.4; 127.8; 127.5 (6s, 14C, CH Ar); 122.5
(C55C–C(O)); 116.9; 116.7; 116.6 (3s, 3CN); 96.1 (s, C-1); 77.6 (m, C-3); 77.1
(s, C-2); 76.6 (m, C-20); 73.8 (s, C-4); 72.4 (s, CH2Ph); 67.7 (m, C-10); 58.8 (s, C-5);
40.6 (s, C-30); 21.2 (s, 6C, 6CH2CH2CN); 31P NMR (CDCl3; 100 MHz): d 21.47;
21.61; 21.98 (s, 3P); Anal. Calcd for C49H54N7O17P3: C, 53.22; H, 4.92; N, 8.87;
P, 8.40. Found: C, 53.05; H, 4.75; N, 8.98; P, 8.23.
[3-(p-Benzoylcinnamido)-2(S)-(dihydrogen phosphate)-propyl] 2-O-
benzyl-3,4-bis-dihydrogen phosphate a-D-xylopyranoside hexasodium
salt (38). Compound 38 was obtained as a white solid by treatment of 37
according to procedure E. [a]D þ 23.8 (c 1.2, H2O), IR 3317 (NH); 1658; 1652;
1646 (PhC(O)Ph-, OC(O)N, -Ph-C55C-C(O)); 1278(P55O) cm21
;
1H NMR
(250 MHz D2O): 7.03–7.65 (m, 15H, H Ar, –C55CHPh); 6.66 (d, 1H, Jtrans
15.0 Hz, –C55CHC(O)); 4.59–4.94 (m, 3H, H-1, CH2Ph); 4.40–4.57 (m, 2H,
H-3, H-20); 4.28 (m, 1H, H-4); 3.85–4.11 (m, 4H, H-5ax, H-5eq, H-10a, H-10b);
3.79 (m, 1H, H-2); 3.42–3.65 (m, 2H, H-30a, H-30b); 13C NMR (D2O): d 199.4

Permeant Analogs of D-Myo-Inositol 1,4,5-Trisphosphate
575
(C55O); 168.0 (C(O)NH); 139.4 (C55C-Ph); 138.0; 137.1; 136.4 (3s, 4C, Cipso);
133.6; 130.2; 128.1; 127.9; 127.7 (5s, 14C, CH Ar); 123.2 (C55C-C(O)); 99.6
(s, C-1); 76.4 (s, C-2); 73.1 (s, CH2Ph); 70.5 (s, 2C, C-3 or C-4 or C-20); 70.0
(m, C-10); 69.4 (m, 1C, C-3 or C-4 or C-20); 65.8 (s, C-5); 42.6 (s, C-30); 31P NMR
(D2O; 100 MHz): d 7.65; 7.50; 7.26 (3s, 3P); Anal. Calcd for C31H30N1O17P3Na6:
C, 40.50; H, 3.29; N, 1.52; P, 10.11. Found: C, 40.26; H, 3.51; N, 1.36; P, 9.97.
[3-(p-Benzoylcinnamido)-2(S)-(diacetoxymethylphosphonoxy) propyl]
2-O-benzyl-3,4-bis (diacetoxymethyl)phosphate-a-D-xylopyranoside
(39). Compound 39 was obtained as a gum in 20% yield after purification by
column chromatography (AcOEt/MeOH 98 : 2 ! 95 : 5) by treatment of 38
using the procedure described for the preparation of 28. [a]D þ 27.5 (c 0.5,
CHCl3); IR 3315 (NH); 1769 (OC(O)CH3); 1662; 1660; 1656 (PhC(O)Ph-,
;
OC(O)N, -Ph-C55C-C(O)); 1217 (P55O) cm21 1H NMR (250 MHz CDCl3): d
7.72–7.83 (m, 2H, H Ar); 7.53–7.70 (m, 2H, -C55CHPh, H Ar); 7.21–7.52
0
0
(m, 13H, H Ar); 6.91 (t, 1H, JNH-H3 a ¼ JNH-H3 b 5.8 Hz, NH); 6.62 (d, 1H,
Jtrans 15.3 Hz, -C55CHC(O)); 5.44–5.75 (m, 12H, O-CH2-O); 4.57–4.83
(m, 5H, H-1, CH2Ph, H-3, H-20); 4.36 (m, 1H, H-4); 3.53–3.99 (m, 6H, H-5eq,
H-5ax, H-10a, H-10b, H-30a, H-30b); 3.50 (dd, 1H, J1-2 3.5, J2-3 9.5 Hz, H-2). 13C
NMR (CDCl3): d 196.0 (C55O); 169.2 (s, 6C, 6OC55OCH3); 165.9 (C(O)NH);
139.7 (C55C-Ph); 138.4; 136.8 (2s, 4C, Cipso); 132.2; 130.2; 129.6; 128.3; 127.9
(5s, 14C, CH Ar); 122.5 (C55C-C(O)); 96.7 (s, C-1); 81.5–82.55 (6d, 6C,
6OCH2C55O); 77.7 (m, 2C, C-2 or C-3 or C-20); 77.0 (m, 1C, C-2 or C-3 or
C-20); 73.5 (m, C-4); 72.8 (s, CH2Ph); 68.0 (m, C-10); 58.8 (s, C-5); 40.9
(m, C-30); 20.4 (s, 6C, 6CH3C55O). 31P NMR (CDCl3; 100 MHz): d 23.37;
23.61; 24.00 (3s, 3P); Anal. Calcd for C49H60N1O29P3: C, 48.24; H, 4.96; N,
1.15; P, 7.62. Found: C, 48.09; H, 5.12; N, 1.02; P, 7.43.
[3-Biphenyldihydrocinnamido)2(S)
(diacetoxymethylphosphonoxy)
propyl] 3,4-bis(diacetoxymethyl)phosphate a-D-xylopyranoside (40).
Compound 40 was quantitatively obtained as a gum by treatment of 39
using the procedure described for the preparation of 9. [a]D þ 27.0 (c 0.5,
CHCl3); IR 3315 (NH); 1768 (OC(O)CH3); 1668 (OC(O)N); 1216(P55O);
1151(P-O) cm21 1H NMR (250 MHz CDCl3): d 7.23–7.33 (m, 2H, H Ar);
;
0
0
7.12–7.21 (m, 7H, H Ar); 6.48 (t, 1H, JNH-H3 a ¼ JNH-H3 b 5.9 Hz, NH); 5.50–
5.76 (m, 12H, O-CH2-O); 4.81 (d, 1H, J1-2 3.6 Hz, H-1); 4.53–4.68 (m, 2H,
H-3, H-20); 4.37 (m, 1H, H-4); 3.87–3.99 (m, 3H, H-5a, Ph-CH2-Ph-); 3.74 (dd,
1H, J1 a-1 b 11.0, J1 a-2 6.6 Hz, H-10a); 3.40–3.68 (m, 5H, H-2, H-5b, H-10b,
H-30a, H-30b); 2.91 (m, 2H, CH2Ph-); 2.51 (m, 2H, CH2C(O)); 13C NMR
(CDCl3): d 172.6 (C(O)NH); 169.1; 168.9 (2s, 6C, 6OC55OCH3); 140.8; 138.8;
138.1 (3s, 3C, Cipso); 128.7; 128.5; 128.1 (3s, 5C, CH Ar); 98.5 (s, C-1); 81.6–
0
0
0
0
0
2
3
82.8 (6d, 6C, 6OCH2C55O); 79.6 (dd, JC-P 5.2, JC-P 7.1 Hz, C-3); 77.0 (m, 1C,
2
3
3
C-20); 73.4 (dd, JC-P 4.8, JC-P 5.2 Hz, C-4); 70.7 (s, C-2); 67.1 (d, JC-P 4.3 Hz,
C-10); 59.1 (s, C-5); 41.2 (s, -PhCH2Ph); 39.9 (m, C-30); 37.6 (s, CH2C(O)); 30.7

´
F. Chretien et al.
576
(s, CH2Ph-); 20.3 (s, 6C, 6CH3C55O); 31P NMR (CDCl3; 100 MHz): d 23.36;
23.83; 23.91 (3s, 3P); Anal. Calcd for C42H58N1O28P3: C, 45.13; H, 5.23; N,
1.25; P, 8.31. Found: C, 45.31; H, 5.11; N, 1.07; P, 8.19.
Biological Evaluation
The preparation of rat cerebellar microsomes was achieved according to
ref. 15.
Rat hepatocytes were isolated by the two-step collagenase perfusion
technique,[39] modified as described in ref. 40. Cell viability, as assessed by
trypan blue exclusion, was .96%. After isolation, cells were maintained in
suspension in Eagle’s medium supplemented with 15 g/L gelatin and gassed
with O2/CO2 (19 : 1) at rt, during the following 8 hr, during which [Ca2þ]c
determination was performed.
Equilibrium [3H]InsP3-binding studies were achieved according to the
procedure described in ref 15.
Spectrofluorimetry studies: Before experiment, cells were loaded with the
Ca2þ-sensitive indicator quin2-acetoxymethyl (AM) ester. Briefly, an aliquot
corresponding to 106 cells was incubated at 378C in 0.5 mL incubation
medium containing 50 mM quin2-AM for 180 seconds, centrifuged, washed,
and resuspended in 2 mL of Dulbecco’s modified Eagle medium. The cell sus-
pension was then transferred to the oxygenated spectrofluorimeter cuvette
(under O2/CO2 (19 : 1)) and stirred magnetically at 378C (JY3D fluorometer,
Jobin & Yvon). Calcium movements were measured from the observed
changes in the fluorescence of quin2 (excitation wavelength, 340 nm;
emission wavelength, 492 nm). At the end of each experiment, the fluorescence
signal was calibrated by making the cell membrane permeable with 5 mg/mL
digitonin (quin2 saturation by external Ca2þ gave maximal fluorescence) and
then by adding 20 mM EGTA to the suspension of lyzed cells to obtain the
minimal fluorescence.
Experiments in Ca2þ-free medium were performed to evaluate the mobiliz-
ation of intracellular Ca2þ during hormone or analog stimulation. The Ca2þ
influx was suppressed by adding EGTA (4 mM) to the incubation medium.
The hormone or analogs were then added to the medium before the addition
of the hormone or analog.
REFERENCES AND NOTES
[1] Rasmussen, H. Cell communication, calcium ion, and cyclic adenosine monophos-
phate. Science 1970, 170, 404–412.
[2] Putney, J.W.J. Inositol Phosphate and Calcium Signalling; Raven Press: New York,
1992.