2-Deoxy derivative is a partial agonist of the intracellular messenger inositol 3,4,5,6-tetrakisphosphate in the epithelial cell line T84

Article abstract of DOI:10.1021/jm970781n

We have synthesized the first deoxy analogues of myo-inositol 3,4,5,6- tetrakisphosphate (1) [Ins(3,4,5,6)P₄], rac-2-deoxy-myo-inositol 3,4,5,6- tetrakisphosphate (rac-2), 2-deoxy-myo-inositol 1,4,5,6-tetrakisphosphate (ent-2), and rac-1-deoxy-

Full text of DOI:10.1021/jm970781n

J . Med. Chem. 1998, 41, 3635-3644
3635
2
3
-Deoxy Der iva tive Is a P a r tia l Agon ist of th e In tr a cellu la r Messen ger In ositol
,4,5,6-Tetr a k isp h osp h a te in th e Ep ith elia l Cell Lin e T84
Marco T. Rudolf, Wen-hong Li,†,# Nora Wolfson, Alexis E. Traynor-Kaplan, and Carsten Schultz*
§
§,‡
Institut f u¨ r Organische Chemie, Abt. Bioorganische Chemie, Universit a¨ t Bremen UFT, Leobener Strasse,
2
8359 Bremen, Germany, and Departments of Medicine and of Chemistry and Biochemistry, University of California,
San Diego, La J olla, California 92093
Received November 17, 1997
We have synthesized the first deoxy analogues of myo-inositol 3,4,5,6-tetrakisphosphate (1)
[
1
Ins(3,4,5,6)P
4
], rac-2-deoxy-myo-inositol 3,4,5,6-tetrakisphosphate (rac-2), 2-deoxy-myo-inositol
,4,5,6-tetrakisphosphate (ent-2), and rac-1-deoxy-myo-inositol 3,4,5,6-tetrakisphosphate (rac-
). In order to evaluate the binding properties of the three derivatives to the yet unidentified
, the analogues were converted to membrane-
3
intracellular binding sites for Ins(3,4,5,6)P
4
permeant derivatives. Starting with common inositol precursors, various forms of Barton-
McCombie deoxygenation and classical protection/deprotection procedures yielded the desired
precursors rac-1-O-butyryl-2-deoxy-myo-inositol (rac-12), ent-3-O-butyryl-2-deoxy-myo-inositol
(ent-12), and rac-2-O-butyryl-1-deoxy-myo-inositol (rac-19), respectively. Phosphorylation and
subsequent deprotection yielded rac-2, ent-2, and rac-3. Alternatively, phosphorylation followed
by alkylation with acetoxymethyl bromide gave the membrane-permeant derivatives 1-O-
butyryl-2-deoxy-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (rac-5), 3-O-
butyryl-2-deoxy-myo-inositol 1,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (ent-5), and
2
6
-O-butyryl-1-deoxy-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (rac-
), respectively. We examined the potency of the membrane-permeant deoxy derivatives in
inhibition of calcium-mediated chloride secretion (CaMCS) in intact T84 cells. Compared to
the 1,2-di-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (4),
the membrane-permeant derivative of Ins(3,4,5,6)P
a slightly weaker inhibitory effect, while the enantiomerically pure 2-deoxy-Ins(1,4,5,6)P
) and the 1-deoxy derivative (rac-6) were inactive. As expected, the effect was stereoselective.
Thus, the 1-hydroxyl group is apparently essential for binding and the inhibitory effect of Ins-
3,4,5,6)P on chloride secretion, whereas the 2-hydroxyl group plays a less important role.
4
(1), the 2-deoxy derivative (rac-5) exhibited
4
(ent-
5
(
4
2
0,21
In tr od u ction
General recognition of the fundamental cellular role
interest because chloride secretion
water flux across mucosal epithelia
and resulting
are pathologi-
2
2,23
1
,2
cally altered in cystic fibrosis and secretory diarrhea.
of several inositol-containing compounds
in signal
3
,4
5,6
transduction has led to proliferation of biological
and chemical7 studies aimed at unraveling the details
of the complex actions of inositol polyphosphates. The
ubiquitous second messenger myo-inositol 1,4,5-tris-
phosphate [Ins(1,4,5)P3] couples agonist stimulation of
a variety of cell surface receptors to intracellular calcium
Although intracellular Ins(3,4,5,6)P4 levels were shown
to be increased following receptor stimulation,
ably due to an inhibitory effect of Ins(1,3,4)P3 on the
Ins(1,3,4,5,6)P5 1-phosphatase/Ins(3,4,5,6)P4 1-kinase
the membrane-permeant, bioactivat-
able Ins(3,4,5,6)P4 derivative 1,2-O-Bt2-Ins(3,4,5,6)P4/
AM (4) manifested the intracellular messenger function
The treatment of T84 cells with 4
inhibited CaMCS without altering intracellular calcium
-9
18,19
prob-
1
7,24
equilibrium,
1
0
mobilization. Recently, particular attention has been
focused on the more highly phosphorylated inositol
1
9
of Ins(3,4,5,6)P4.
1
1-14
polyphosphates.
While early studies were largely
1
4
19
confined to describing their complex metabolism,
levels.
Hence, the elevation of Ins(3,4,5,6)P4 levels
recent investigations have concentrated on elucidating
their physiological functions.12 Inositol tetrakisphos-
phates derived from InsP5 have been a focal point,
whether it was through muscarinic stimulation or
addition of 4 resulted in an uncoupling of Cl secretion
-
1
5-17
from intracellular calcium levels. At this point, we can
only speculate as to the mechanism by which Ins-
(3,4,5,6)P4 inhibits CaMCS. However, recent studies
using the whole cell patch clamp technique with intra-
cellular perfusion of inositol tetrakisphosphates indi-
cated that Ins(3,4,5,6)P4 directly modulated an apically
fueled by the discovery that Ins(3,4,5,6)P4 is responsible
for the inhibition of calcium-mediated chloride secretion
1
8,19
(CaMCS) in the intestinal epithelial cell line T84.
The
identification of this negative regulator was of special
2
+
-
*
Author for correspondence. Tel: +49-421-218-7665. Fax: +49-421-
located Ca /calmodulin kinase-regulated Cl channel
2
18-7643. E-mail: schultz@slater.chemie.uni-bremen.de.
25
2+
-
in T84 cells and a Ca -stimulated Cl current of
CFPAC-1 cells, the latter being a cell line carrying a
defect in the cystic fibrosis conductance regulator
(CFTR). Furthermore, Ca -dependent Cl channels
reconstituted in planar lipid bilayers were also regu-
†
Department of Chemistry and Biochemistry.
Department of Medicine.
Current address: Division of Biology 139-74, Beckman Institute,
§
#
California Institute of Technology, Pasadena, CA 91125.
2
6
2+
-
‡
Current address: Inologic Inc., 562 First Ave. S., Suite 601, Seattle,
WA 98104.
S0022-2623(97)00781-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/21/1998

3
636 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Rudolf et al.
complex signals for H-2ax and H-2eq in the 1.6-2.4 ppm
region (Experimental Section). Deprotection of rac-9
3
4
with DDQ gave the alcohol rac-10. Esterification of
the free hydroxyl group with butyric anhydride yielded
rac-11, and subsequent removal of the benzyl groups
by catalytic hydrogenolysis gave the desired 1-O-bu-
tyryl-2-deoxy-myo-inositol (rac-12) in 34% yield from
rac-7.
Phosphorylation was accomplished using dibenzyl
3
7
N,N-diisopropylphosphoramidite, followed by oxida-
tion with peracetic acid to give the fully protected
tetrakisphosphate derivative rac-13 in 71% yield after
purification by preparative HPLC (50 × 250 mm, RP-
1
8, 10 µm) with 92% methanol as the eluent. Depro-
tection by catalytic hydrogenolysis yielded rac-1-O-
butyryl-2-deoxy-Ins(3,4,5,6)P4 (rac-14) as the free acid.
The butyrate was removed by treatment with 1 M KOH
(pH 12.8) to yield rac-2-deoxy-Ins(3,4,5,6)P4 (rac-2).
Alternatively, acid rac-14 was alkylated with acetoxy-
methyl bromide (AMBr) in the presence of diisopropy-
lethylamine (DIEA) to give the uncharged, bioactivat-
able octakis(acetoxymethyl) ester rac-5. The same
synthetic sequence was repeated, starting from 1,4,5,6-
tetra-O-benzyl-3-O-(4-methoxybenzyl)-myo-inositol (ent-
F igu r e 1. Structures of myo-inositol 3,4,5,6-tetrakisphosphate
1), 2-deoxy-Ins(3,4,5,6)P (rac-2), 1-deoxy-Ins(3,4,5,6)P (rac-
), and the membrane-permeant Ins(3,4,5,6)P acetoxymethyl
ester derivatives 1,2-O-Bt -Ins(3,4,5,6)P /AM (4), 1-O-Bt-2-
deoxy-Ins(3,4,5,6)P /AM (rac-5), and 2-O-Bt-1-deoxy-Ins(3,4,5,6)-
/AM (rac-6). Numbering of myo-inositol derivatives refers
(
3
4
4
4
2
4
4
P
4
7
1
) which was prepared from enantiomerically pure
,4,5,6-tetra-O-benzyl-myo-inositol. The absolute con-
to the D-configuration throughout this paper, exclusively. In
case of racemic compounds only D-enantiomers are shown.
3
4
figuration was confirmed by comparing the optical
rotation value of the intermediate 1,4,5,6-tetra-O-ben-
zyl-2-deoxy-myo-inositol (ent-10) to the published data.38
lated by low levels of Ins(3,4,5,6)P4, suggesting that the
binding site is closely associated with the chloride
2
7
Syn th esis of 1-Deoxy-In s(3,4,5,6)P Der iva tives.
channel.
4
The synthesis of 1-deoxy compounds required a slightly
Extending the use of membrane-permeant inositol
phosphate derivatives, we are now aiming to use this
methodology to characterize the unidentified Ins(3,4,5,6)-
P4-binding site(s). Deoxy derivatives proved to be useful
analogues to study ligand/protein interaction in vitro
different approach since dehalogenation of an axial iodo
group in the 1 position failed. Therefore, the 1-hydroxyl
group of rac-3,4,5,6-tetra-O-benzyl-myo-inositol (rac-15)
was regioselectively esterified with equimolar amounts
of O-phenyl chlorothionoformate in dry pyridine in the
presence of DMAP to give rac-16 (Scheme 2). Higher
amounts of O-phenyl chlorothionoformate or longer
reaction times resulted in the formation of a 1,2-cyclic
thiocarbonate. The structure of the latter was verified
through the reaction of rac-3,4,5,6-tetra-O-benzyl-myo-
in the past.2
8-33
Therefore, we synthesized three mem-
brane-permeant deoxy derivatives of Ins(3,4,5,6)P4 (1):
-O-butyryl-2-deoxy-myo-inositol 3,4,5,6-tetrakisphos-
1
phate octakis(acetoxymethyl) ester (rac-5) [1-O-Bt-2-
deoxy-Ins(3,4,5,6)P4/AM], as a negative control, the
enantiomerically pure Ins(1,4,5,6)P4 derivative 3-O-
butyryl-2-deoxy-myo-inositol 1,4,5,6-tetrakisphosphate
octakis(acetoxymethyl) ester (ent-5) [3-O-Bt-2-deoxy-Ins-
3
9
inositol (rac-15) with thiophosgen in pyridine/DMAP
which gave the identical compound.
(
3
(
1,4,5,6)P4/AM], and 2-O-butyryl-1-deoxy-myo-inositol
,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester
rac-6) [2-O-Bt-1-deoxy-Ins(3,4,5,6)P4/AM] (Figure 1).
The remaining 2-OH group of rac-16 was esterified
with butyric anhydride, and subsequently the reduction
of the fully protected phenoxythiocarbonyl compound
rac-17 with tributyltin hydride utilizing Barton’s meth-
odology yielded the 1-deoxy derivative rac-18. The
benzyl groups were removed by catalytic hydrogenolysis
to form the tetrol rac-19. Phosphorylation to the fully
protected tetrakisphosphate rac-20 proceeded as de-
scribed above. Hydrogenolysis afforded compound rac-
Here we present the chemical synthesis and the effects
of the compounds on CaMCS.
3
6
Resu lts
Syn th esis of 2-Deoxy-In s(3,4,5,6)P 4 Der iva tives.
The Ins(3,4,5,6)P4 analogue rac-2-deoxy-myo-inositol
3
permeant derivative rac-1-O-butyryl-2-deoxy-myo-inosi-
tol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) es-
ter (rac-5) were prepared from the common precursor
rac-3,4,5,6-tetra-O-benzyl-1-O-(4-methoxybenzyl)-myo-
inositol (rac-7).
al., the free hydroxyl group at the 2 position of the
starting material was easily substituted by an iodo
2
1, which was treated with KOH (pH 12.8), followed by
ion-exchange chromatography to give rac-2-O-butyryl-
-deoxy-Ins(3,4,5,6)P4 (rac-3) as the free acid. Com-
,4,5,6-tetrakisphosphate (rac-2) and its membrane-
1
pound rac-21 was alkylated with AMBr, as described
above, to give the octakis(acetoxymethyl) ester rac-6.
3
4
Following the method of Garegg et
Bioch em istr y. We have previously shown that
exposure of T84 cells to the racemic mixture of 400 µM
1,2-di-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate
octakis(acetoxymethyl) ester [1,2-O-Bt2-Ins(3,4,5,6,)P4/
AM] extracellularly corresponded to an intracellular
concentration of 4 µM Ins(3,4,5,6)P4 which reduced the
3
5
group with inversion at the reaction center (Scheme 1).
Free-radical dehalogenation36 of rac-8 at C-2 with AIBN/
n-Bu3SnH gave compound rac-9. Evidence of the de-
halogenation was provided by the appearance of two
-
thapsigargin-induced Cl secretion by approximately

2
-Deoxy Derivative as Partial Agonist of Ins(3,4,5,6)P
4
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3637
Sch em e 1a
a
Reagents and conditions: (i) I2, imidazole, (Ph)3P, toluene; (ii) n-Bu3SnH, AIBN, toluene; (iii) DDQ, CH2Cl2-H2O; (iv) Bt2O, DMAP,
i
pyridine; (v) H2, Pd/C (10%), AcOH; (vi) a. (BnO)2PNPr 2, 1H-tetrazole, MeCN, b. - 40 °C, AcOOH; (vii) 1 M KOH, pH 12.8; (viii) AMBr,
DIEA, MeCN.
Sch em e 2a
4
0%. In contrast, 200 µM enantiomerically pure 2,3-
1
9
O-Bt2-Ins(1,4,5,6)P4/AM was without effect.
Here we demonstrate for the first time the ability of
Ins(3,4,5,6)P4 to inhibit carbachol-induced CaMCS by
incubation with enantiomerically pure 1,2-O-Bt2-Ins-
(
3,4,5,6,)P4/AM (4).34 Cells were grown to confluence on
Snap-wells and mounted in modified Ussing chambers
as described before.40 For these experiments, T84 cells
were pretreated with 200 µM 1,2-O-Bt2-Ins(3,4,5,6)P4/
AM (4) for 30 min at 37 °C. CaMCS was stimulated by
carbachol, a muscarinic receptor agonist which mobi-
lizes intracellular calcium through a phospholipase
C-dependent pathway. Preincubation of the monolayers
with 1,2-O-Bt2-Ins(3,4,5,6)P4/AM (4) inhibited CaMCS
by up to 50% (Figures 2 and 3).
To investigate the properties of the two deoxy Ins-
(
3,4,5,6)P4 derivatives, rac-1-O-Bt-2-deoxy-Ins(3,4,5,6)-
P4/AM (rac-5) and rac-2-O-Bt-1-deoxy-Ins(3,4,5,6)P4/AM
rac-6), on carbachol-mediated chloride secretion in T84
(
cells, short circuit current (Isc) was measured after
preincubation with these derivatives. As shown in
Figure 2, only pretreatment with rac-1-O-Bt-2-deoxy-
Ins(3,4,5,6)P4/AM (rac-5) inhibited CaMCS, resulting in
a 28% reduction of Isc in response to carbachol, whereas
pretreatment with rac-2-O-Bt-1-deoxy-Ins(3,4,5,6)P4/AM
(rac-6) was without effect. Inhibition was maximal at
a concentration of 400 µM rac-5, while 200 µM was
ineffective (n ) 3, data not shown) and 800 µM (n ) 3,
data not shown) showed no increased potency. Because
the 2-deoxy-Ins(3,4,5,6)P4 derivative (rac-5) consists of
a racemic mixture of Ins(3,4,5,6)P4/Ins(1,4,5,6)P4 deriva-
tives and to confirm that the inhibition was enantiose-
a
Reagents and conditions: (i) PhOC(S)Cl, DMAP, pyridine; (ii)
Bt2O, DMAP, pyridine; (iii) n-Bu3SnH, AIBN, toluene; (iv) H2, Pd/C
(
i
10%), AcOH; (v) a. (BnO)2PNPr 2, 1H-tetrazole, MeCN, b. -40 °C,
AcOOH; (vi) 1 M KOH, pH 12.8; (vii) AMBr, DIEA, MeCN.

3
638 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Rudolf et al.
F igu r e 3. Inhibition of CaMCS by membrane-permeant deoxy
InsP derivatives compared to 1,2-O-Bt -Ins(3,4,5,6)P /AM (4).
4 2 4
Monolayers were preincubated with the indicated membrane-
permeant tetrakisphosphate acetoxymethyl ester for 30 min
prior to mounting in the Ussing chamber. CaMCS was
-
4
stimulated with carbachol (10 M). Data are mean peak Isc
SEM expressed as percent control for 4-6 experiments.
Control values represent the response to carbachol in coincu-
bated monolayers. Significant differences are denoted by
probability values derived by Student’s 2-tailed t-test (*p <
0
1
.02; **p < 0.04). Concentrations of the InsP
,2-O-Bt -Ins(3,4,5,6)P /AM (200 µM) (4), 1-O-Bt-2-deoxy-Ins-
/AM (400 µM) (rac-5), 3-O-Bt-2-deoxy-Ins(1,4,5,6)-
/AM (200 µM) (ent-5), and 2-O-Bt-1-deoxy-Ins(3,4,5,6)P /AM
4
derivatives were
2
4
(3,4,5,6)P
4
P
4
4
(400 µM) (rac-6), respectively.
The lack of a biological effect of rac-2-O-Bt-1-deoxy-
Ins(3,4,5,6)P4/AM (rac-6) does not necessarily reflect the
potential binding properties to the binding protein(s).
Therefore we investigated whether rac-6 would compete
with naturally produced Ins(3,4,5,6)P4 after carbachol
stimulation. To analyze this potential antagonistic
effect, rac-6 was assayed for the reversal of the reported
-
Ins(3,4,5,6)P4-induced inhibition of Cl secretion by
1
9
thapsigargin. Cells were preincubated with rac-2-O-
Bt-1-deoxy-Ins(3,4,5,6)P4/AM (rac-6) for 30 min, mounted
in Ussing chambers, and then treated with carbachol
-
which induces transient Cl secretion followed by
1
9
prolonged elevation of Ins(3,4,5,6)P4 levels. After 30
min thapsigargin was added to stimulate a second cycle
2
+
of [Ca ]i elevation and subsequent chloride secretion.
No significant differences in thapsigargin-induced Isc
were observed in rac-6-pretreated monolayers compared
to controls (n ) 4, data not shown) indicating that the
1
-deoxy compound rac-6 did not reverse the inhibition
of CaMCS by physiologically mediated elevation of Ins-
3,4,5,6)P4 levels. Therefore, rac-6 does not inhibit
(
CaMCS and thus is unable to bind to the Ins(3,4,5,6)-
P4-binding protein(s).
Finally, neither compound had any effect on [Ca2+]i
levels or on thapsigargin-induced elevation of Ca
levels in T84 cells, as was verified by continuous Fura-2
calcium ratio imaging for more than 30 min after
incubation with the membrane-permeant inositol poly-
phosphate derivatives (data not shown).
F igu r e 2. Time course of CaMCS inhibition by membrane-
permeant Ins(3,4,5,6)P derivatives. T84 monolayers were
incubated for 30 min with (A) 4, (B) rac-5, or (C) rac-6 or
4
2+
vehicle (DMSO/5% Pluronic) prior to mounting into Ussing
-
chambers. Cl secretion was measured as short circuit current
-
4
(Isc). Carbachol (10 M) was added to the basolateral side to
induce CaMCS. Data reflect monitoring every 4 s and are the
average of 6 experiments.
Discu ssion
lective, we synthesized the enantiomerically pure de-
rivative 3-O-Bt-2-deoxy-Ins(1,4,5,6)P4/AM (ent-5). As
expected, extracellular application of 200 µM ent-5 did
not inhibit CaMCS (Figure 3), showing that the effect
of rac-5 is due to the Ins(3,4,5,6)P4 enantiomer.
Recent confirmation that Ins(3,4,5,6)P4 is an impor-
tant intracellular negative regulator of CaMCS in
epithelial cells
1
9,21,25-27
points out the need to character-
ize the intracellular target proteins. The latter should

2
-Deoxy Derivative as Partial Agonist of Ins(3,4,5,6)P
4
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3639
be interesting targets for drug design, especially with
respect to diseases where Cl secretion is pathological,
In summary, the extracellular application of mem-
brane-permeant derivatives of inositol 3,4,5,6-tetrak-
isphosphate provides a technique for in vivo mapping
of Ins(3,4,5,6)P4-binding proteins in T84 cells. Using this
methodology we established that the role of the 2-hy-
droxyl group in ligand recognition is less important but
that the role of the 1-hydroxyl group is essential. This
information may aid in the search for drug candidates
for the treatment of cystic fibrosis or secretory diarrhea.
-
e.g., in cystic fibrosis or secretory diarrhea. Agonists
which mimic the Ins(3,4,5,6)P4-mediated inhibition of
-
CaMCS would result in a decrease in Cl secretion,
while antagonists of Ins(3,4,5,6)P4 would be expected
to augment CaMCS. Unfortunately, no Ins(3,4,5,6)P4-
binding protein has yet been identified. However, using
CaMCS as a readout, the availability of membrane-
permeant derivatives of inositol polyphosphates now
enables us to characterize the unidentified binding sites
in their natural environment.
Exp er im en ta l Section
All chemical reagents were obtained in the highest purity
available. Where necessary, solvents were dried and/or dis-
tilled before use. Acetonitrile was distilled from phosphorus-
In principle, Ins(3,4,5,6)P4 offers two target groups
for systematic modification of its structure and therefore
its molecular interaction potential: (i) the hydroxyl
groups and (ii) the phosphates. Because previous
experiments showed that proper stereochemistry and
(V) oxide and stored over molecular sieves (3 Å), as was
dimethylformamide (DMF). Pyridine and toluene were stored
over molecular sieves (4 Å). Diisopropylethylamine (DIEA)
was dried over sodium wire. Palladium on charcoal (10%),
orientation of the phosphates were essential for the
3
iodine, and tri-n-butyltin hydride (n-Bu SnH) were from Acros
physiological function,2
5-27
we modified the interaction
Chemie. Dibenzyl N,N-diisopropylphosphoramidite, tetrazole,
peracetic acid (32% v/w), dibutyltin oxide, R,R′-azoisobutyroni-
trile (AIBN), and acetoxymethyl bromide were from Aldrich.
Butyric anhydride and DIEA were from Merck. 4-(Dimethy-
lamino)pyridine (DMAP), cesium fluoride (CsF), 2,3-dichloro-
potential of the hydroxyl groups by synthesizing deoxy
derivatives. Usually hydroxyl groups serve as either
hydrogen-bonding acceptors or donors or both. Replac-
ing the hydroxyl groups with hydrogen atoms results
in complete loss of the hydrogen bond interaction
properties. From the biological data in this study, we
can conclude that the hydroxyl group at the 2 position
exhibits a relatively minor role in the recognition of Ins-
5
,6-dicyano-1,4-benzoquinone (DDQ), and O-phenyl chlorothion-
oformate were from Fluka. The ion-exchange resin Dowex 50
+
WX 8, H -form, was from Serva, Heidelberg.
Thapsigargin was purchased from Alexis Biochemicals, La
J olla, CA. Carbachol was obtained from ICN, Irvine, CA. Fura
2
-acetoxymethyl ester (Fura-2/AM) was purchased from Cal-
(3,4,5,6)P4 by its unidentified target protein in T84 cells.
biochem, La J olla, CA. Pluronic 127 was a kind gift of BASF,
Germany. Cell culture membrane inserts (Snapwell, 0.4 µm
pore size polycarbonate) were obtained from Corning Costar
Corp. (Cambridge, MA).
On the other hand, the lack of an effect of the 1-deoxy
compound rac-6 indicates that the hydroxyl group at
position 1 is critical. Thus, the 1-hydroxyl group of Ins-
(
3,4,5,6)P4 may be engaged in essential hydrogen-
HPLC was performed on a LDC/Milton Roy Constametric
III pump with a LDC/Milton Roy UV monitor D (254 nm) or a
Knauer refractive index detector. The analytical column was
a Merck Hibar steel tube (250 mm × 4 mm) filled with RP 18
material (Merck, LiChrosorb; 10 µm). Preparative HPLC was
performed using a Shimadzu LC 8A pump with a preparative
LDC UV III monitor (254 nm) or a Knauer refractive index
detector and a Merck Prepbar steel column (250 mm × 50 mm)
filled with RP 18 material (Merck, LiChrospher 100; 10 µm).
The eluents were methanol-water mixtures; compositions are
given in % methanol (MeOH).
bonding interactions with the appropriate amino acid
residues of its target, while the 2-hydroxyl group ap-
pears to be less involved. Nevertheless, the biological
activity of rac-5 was 50% of that of the membrane-
permeant Ins(3,4,5,6)P4 derivative 4, and the effect
could not be increased by raising the concentration.
Therefore the 2-deoxy compound rac-5 represents only
a partial agonist, perhaps due to discrimination between
2
5
the different binding sites postulated by Xie et al.
1H and 31P NMR spectra were recorded on a Brucker AM
The method described here for mapping proteins in
living cells may be a promising approach for future
investigations of inositol phosphate/protein interactions.
Classical mapping approaches have been carried out in
cell-free systems relying on highly purified proteins.
This has been especially problematic when working with
membrane-bound proteins or proteins which are modifi-
able by a number of solute cofactors present in cells.
Although a higher number of membrane-permeant
structurally modified Ins(3,4,5,6)P4 derivatives would
be desirable, the method used in the present study
provides an alternative approach which enables us to
elucidate the binding character of both identified and
unidentified proteins in their natural environment. A
prerequisite for this approach is the conversion of the
membrane-impermeant inositol phosphates or one of its
derivatives into the membrane-permeant, bioactivatable
AM ester. These and previous studies demonstrated
that the acyloxymethyl ester method is effective for
delivering inositol phosphates to the cytosol of intact
3
60 spectrometer. Chemical shifts were measured in ppm
1
relative to tetramethylsilane for H NMR spectra and external
85% H PO for 31P NMR spectra. J values are given in Hz.
3
4
Mass spectra were recorded using a Finnigan Mat 8222 mass
spectrometer with fast atom bombardment (FAB) ionization.
High-resolution masses were determined relative to known
compounds with a mass not differing more than 10%. Optical
rotations were measured at the sodium D-line in a 10-cm cell
with a Perkin-Elmer 1231 polarimeter. Meltings points
(uncorrected) were determined using a B u¨ chi B-540 apparatus.
Ultrafiltration of the palladium/charcoal catalyst was per-
formed with a Sartorius filtration apparatus SM 162 01 using
filters from regenerated cellulose (Sartorius, SM 116 04).
Elemental analyses were performed by Mikroanalytisches
Labor Beller, G o¨ ttingen, Germany.
rac-3,4,5,6-Tetra-O-benzyl-myo-inositol (rac-15), rac-3,4,5,6-
tetra-O-benzyl-1-O-(4-methoxybenzyl)-myo-inositol (rac-7), and
enantiomerically pure 1,4,5,6-tetra-O-benzyl-myo-inositol (ent-
1
3,34
15) were synthesized as described before.
ent-15 had an
2
4
43
optical rotation value of [R]
D
+19 (c ) 1.05 in CHCl
)]. The synthesis of enantio-
merically pure 2-deoxy-myo-inositol 1,4,5,6-tetrakisphosphate
ent-2) and 3-O-butyryl-2-deoxy-myo-inositol 1,4,5,6-tetrak-
3
) [lit.
2
4
[R]
D
+23.4 (c ) 4.5 in CHCl
3
(
1
9,41,42
cells without perturbing the plasma membrane
and with minimal interference of other intracellular
pathways.
isphosphate octakis(acetoxymethyl) ester (ent-5) followed the
pathway for the racemic compounds. All analytical data were
in accordance with those obtained for the racemic compounds.

3
640 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Cell Cu ltu r e. All studies were performed using monolayers
Rudolf et al.
the mixture was stirred for 10 min. Iodine was added in
portions until the toluene phase remained iodine-colored and
was stirred for an additional 15 min. Excess of iodine was
removed by the addition of aqueous sodium dithionite solution.
The organic and the aqueous phase were separated, and the
organic phase was washed with brine twice, dried over Na2-
SO , and filtered. Evaporation of the solvent and crystalliza-
tion from methanol gave pure rac-8 (1.01 g, 73%). Mp: 131.5-
132.4 °C (from methanol). H NMR (CDCl , 360 MHz): 3.49
of the T84 cell line and cells from passage numbers 20-29 only.
Methods for the maintenance of T84 cells for use in transep-
thelial electrolyte transport studies have been described
1
8
previously.
In brief, T84 cells were grown in Dulbecco’s
modified Eagle’s/F12 media (J RH, Lenexa, KS) supplemented
with 5% newborn calf serum (Hyclone, Logan, UT) and 50
U/mL each of penicillin/streptomycin (Core Cell Culture Facil-
ity, UCSD). Medium was replaced every third day. Cells were
passaged by trypsinization. For the measurement of chloride
4
1
3
(1 H, dd, J ) 9.27, 9.27 Hz, H-4), 3.50 (1 H, dd, J ) 9.27, 9.27
Hz, H-6), 3.61 (1H, dd, J ) 9.50, 9.50 Hz, H-5), 3.64-3.67 (2
H, m, H-1, H-3), 3.80 (3 H, s, OMe), 4.06 (1 H, dd, J ) 11.08,
11.08 Hz, H-2), 4.82-4.92 (10 H, m, 4 × CH Ph and CH in
6
secretion, 10 cells were seeded onto microporous filter inserts
(
see above) and maintained for 8-12 days prior to experiments
in order to develop confluent monolayers with stable transepi-
thelial resistance.
2
2
PMB), 6.87 (2 H, d, PMB ArH), 7.23-7.37 (22 H, m, 4 × CH Ph
2
Ch lor id e Secr etion . Chloride secretion was measured as
short circuit current (Isc) across monolayers of T84 cells,
mounted in modified Ussing chambers (Physiologic Instru-
ments, San Diego, CA), bathed with Ringer solution warmed
to 37 °C and gassed continuously with 95% O /5% CO at a
2 2
rate of 30-35 mL/min. The spontaneous potential difference
across the monolayer was short-circuited with a voltage clamp
and PMB ArH). MS: m/z (+ve ion FAB) 924 [(M + NBA +
+
+
H) , 1], 121 [PMB , 100]. Anal. (C42
43 6
H O I) C: calcd, 65.45;
found, 65.89. H: calcd, 5.62; found, 5.72.
D-1,4,5,6-Tet r a -O-b en zyl-2-d eoxy-2-iod o-3-O-(4-m et h -
oxyben zyl)-scyllo-in ositol (en t-8). Compound ent-7 (1.18
g, 1.8 mmol) was halogenated in the same way as described
for rac-8 to give the title compound ent-8 (949 mg, 68%) as a
(
model VCC MC6, Physiologic Instruments, San Diego, CA).
Short circuit current (Isc) and conductance were recorded at
-s intervals using Acquire and Analyze Software 1.2 (Physi-
24
white solid. Mp: 132-133 °C (from methanol). [R]
D
-14.7
1
(
c ) 0.92 in CHCl
with those obtained for rac-8.
3
). H NMR and MS data were in accordance
4
ologic Instruments, San Diego, CA). Increased Isc values
stimulated through cholinergic pathways or through calcium-
mobilizing agonists in T84 cells are wholly reflective of Cl
secretion.
5
0
r a c-3,4,5,6-Tetr a -O-ben zyl-2-d eoxy-1-O-(4-m eth oxyben -
zyl)-m yo-in ositol (r a c-9). Compound rac-8 was dissolved in
dry toluene (150 mL), and AIBN (63 mg, 355 mol) and n-Bu -
3
SnH (1.63 mL, 6.3 mmol) were added. The solution was heated
under an argon atmosphere for 2 h. The reaction mixture was
cooled and then washed once with phosphate buffer (30 mL)
and once with brine (30 mL). The organic layer was dried over
-
4
0,44
+
Ringer’s solution contained (in mM) 140 Na ,
+
2+
2+
-
-
-
4
.2 K , 1.2 Ca , 0.8 Mg , 119.8 Cl , 25 HCO
3
, 2.4 H
2
PO
,
-
4
, and 10 glucose.
.4 HPO
P r im in g of T84 Cells w ith Mem br a n e-P er m ea n t In sP
4
Der iva tives. Confluent T84 cells on Snap-well inserts were
rinsed with 1 mL of Ringer’s solution on each side of the Snap-
well; 100 µL of Ringer’s solution containing inositol tetrak-
isphosphate octakis(acetoxymethyl) ester and 2 µL of DMSO/
Na
lization from methanol gave rac-9 (692 mg, 76%). Mp: 76.2-
6.8 °C (from methanol). 1H NMR (CDCl
, 360 MHz): 1.64 (1
2 4
SO and filtered. Evaporation of the solvent and crystal-
7
3
H, ddd, J ) 12.73, 12.73, 12.73 Hz, H-2 (ax)), 2.39 (1 H, ddd,
J ) 12.73, 4.39, 4.39 Hz, H-2 (eq)), 3.39-3.46 (2 H, m, H-1,
H-3), 3.46 (1 H, dd, J ) 9.22, 8.87 Hz, H-5), 3.55 (1 H, dd, J )
5
% Pluronic 127 was added to the basolateral reservoir. The
cells were incubated at 37 °C for 30 min. The incubation
mixture was discarded, and cells were washed with Ringer’s
solution (1 mL) and were ready for mounting. Cells for control
experiments were incubated only with 2 µL of DMSO/5%
Pluronic 127.
D-1,4,5,6-Tet r a -O-b en zyl-3-O-(4-m et h oxyb en zyl)-m yo-
in ositol (en t-7). Dry ent-15 (2.7 g, 5 mmol) and dry dibutyl
tin oxide (1.26 g, 5.06 mmol) were heated to reflux in dry
toluene (150 mL) in a Soxhlet apparatus filled with activated
molecular sieves (3 Å) for 4 h. The reaction mixture was cooled
to room temperature and evaporated to dryness under dimin-
ished pressure. CsF (1.5 g, 9.9 mmol) was added to the
residual oil, and the mixture was kept under high vacuum for
9
3
.22, 9.22 Hz, H-4), 3.56 (1 H, dd, J ) 9.22, 9.22 Hz, H-6),
.81 (3 H, s, OMe), 4.60-4.94 (10 H, m, 4 × CH Ph and CH
2
2
in PMB), 6.86 (2 H, d, PMB ArH), 7.24-7.33 (22 H, m, 4 ×
CH Ph and PMB ArH). MS: m/z (-ve ion FAB) 643 [(M -
2
-
+
+ -
H ) , 1], 523 [(M - PMB ) , 100]. Anal. (C42
8.23; found, 77.78. H: calcd, 6.88; found, 6.78.
D-1,4,5,6-Tetr a-O-ben zyl-2-deoxy-3-O-(4-m eth oxyben zyl)-
44 6
H O ) C: calcd,
7
m yo-in ositol (en t-9). Compound ent-8 (837 mg, 1.11 mmol)
was dehalogenated as decribed for rac-9 to give the title
compound ent-9 (571 mg, 80%) as a white solid. Mp: 75.8-
1
7
6.3 °C (from methanol). [R]24
D
-3.5 (c ) 0.88 in CHCl
3
).
H
NMR and MS data were in accordance with those obtained
2
h. The residual syrup was dissolved in dry DMF (20 mL)
for rac-9.
under argon, and 4-methoxybenzyl chloride (6 mL, 40 mmol)
was added. After the solution stirred for 5 h at 60 °C, HPLC
analysis (90% MeOH; 1.5 mL/min; t ) 5.30 min) showed no
further reaction. Excess of 4-methoxybenzyl chloride and DMF
were removed in high vacuum. The crude product was
chromatographed by preparative HPLC (90% MeOH; 37.5 mL/
r a c-3,4,5,6-Tetr a -O-ben zyl-2-d eoxy-m yo-in ositol (r a c-
0). DDQ (367 mg, 1.62 mmol) was added to a solution of rac-9
600 mg, 931 µmol) in CH Cl (10 mL) containing small
amounts of water (5%). After the suspension was stirred at
room temperature for 28 min, HPLC analysis (90% MeOH;
.5 mL/min; t ) 4.27 min) showed the reaction to be complete.
R
The solvent was evaporated under reduced pressure, and the
reaction mixture was purified by preparative HPLC (90%
R
20.00 min) to give rac-10 (314 mg, 65%)
as a solid. Mp: 126.7 °C (from methanol) [lit. mp 121-122
°C]. H NMR (CDCl , 360 MHz): 1.64 (1 H, ddd, J ) 12.73,
3
12.73, 12.73 Hz, H-2 (ax)), 2.39 (1 H, ddd, J ) 12.73, 4.39,
4.39 Hz, H-2 (eq)), 3.39-3.46 (2 H, m, H-1, H-3), 3.46 (1 H,
dd, J ) 9.22, 8.87 Hz, H-5), 3.55 (1 H, dd, J ) 9.22, 9.22 Hz,
H-4), 3.56 (1 H, dd, J ) 9.22, 9.22 Hz, H-6), 3.81 (3 H, s, OMe),
1
(
R
2
2
1
min; t ) 35.00 min) to give compound ent-7 (2.6 g, 78%) as a
solid. Mp: 127 °C (from methanol) (lit. mp 128-129 °C).
R
34
2
4
1
[
3
2
R]
D
3 3
+1.7 (c ) 1.2 in CHCl ). H NMR (CDCl , 360 MHz):
.37 (1 H, dd, J ) 9.54, 2.73, H-1), 3.40 (1 H, dd, J ) 9.50,
.73 Hz, H-3), 3.48 (1 H, dd, J ) 9.52, 9.52 Hz, H-5), 3.83 (3
MeOH; 40 mL/min; t
3
2
1
H, s, OMe), 3.99 (1 H, dd, J ) 9.54, 9.52 Hz, H-6), 4.03 (1 H,
dd, J ) 9.52, 9.50 Hz, H-4), 4.22 (1H, dd, J ) 2.73, 2.73 Hz,
H-2), 4.68 (2H, s, CH
2
in PMB), 4.77-4.98 (8 H, m, 4 × CH
2
-
Ph
Ph), 6.69 (2 H, d, PMB ArH), 7.28-7.43 (22 H, m, 4 × CH
2
+
-
and PMB ArH). MS: m/z (-ve ion FAB) 659 [(M - H ) , 100],
+
5
69 [M - Bn , 15].
4.60-4.94 (10 H, m, 4 × CH
2
Ph and CH
2
in PMB), 6.86 (2 H,
2
Ph and PMB ArH).
d, PMB ArH), 7.24-7.33 (22 H, m, 4 × CH
r a c-3,4,5,6-Tetr a -O-ben zyl-2-d eoxy-2-iod o-1-O-(4-m eth -
+
+
MS: m/z (+ve ion FAB) 525 [(M + H) , 1], 91 [Bn , 100].
oxyben zyl)-scyllo-in ositol (r a c-8). A mixture of dry rac-7
1.32 g, 2 mmol), triphenylphosphine (2.15 g, 8.2 mmol),
(
D-1,4,5,6-Tetr a -O-ben zyl-2-d eoxy-m yo-in ositol (en t-10).
The 4-methoxybenzyl group of ent-9 (540 mg, 873 µmol) was
removed as described for compound rac-10 to give ent-10 (315
imidazole (549 mg, 8.2 mmol), and iodine (1.52 g, 6 mmol) was
stirred under reflux in dry toluene (100 mL) for 41 h. The
reaction mixture was cooled to room temperature, saturated
aqueous sodium hydrogen carbonate (100 mL) was added, and
3
2
mg, 69%) as a solid. Mp: 126 °C (from methanol) [lit. mp
121-122 °C]. [R]24
-20.1 (c ) 0.68 in CHCl
) [lit. [R]
38
24
-24
D
3
D

2
-Deoxy Derivative as Partial Agonist of Ins(3,4,5,6)P
4
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3641
-1.67 (1 P, s), -1.33 (1 P, s), -0.85 (1 P, s), -0.64 (1 P, s).
(
c ) 2 in CHCl
3
)]. 1H NMR and MS data were in accordance
+
+
with those obtained for rac-10.
MS: m/z (+ve ion FAB) 1275 [(M + H) , 4], 91 [Bn , 100].
+
-
-
MS: m/z (-ve ion FAB)1183 [(M -Bn ) , 16], 277 [OPO(OBn) ,
r a c-3,4,5,6-Tet r a -O-b en zyl-1-O-b u t yr yl-2-d eoxy-m yo-
in ositol (r a c-11). A solution of rac-10 (186 mg, 355 µmol),
butyric anhydride (70 µL, 426 mol), and DMAP (12 mg, 10
µmol) in dry pyridine (5 mL) was stirred at room temperature
for 18 h. The solvent was evaporated under high vacuum to
give an oil. Residual pyridine was removed by evaporating
three times with octane. The residue was dissolved in tert-
butyl methyl ether (30 mL) and was washed with phosphate
buffer (20 mL), with sodium hydrogen carbonate (20 mL), with
sodium hydrogen sulfate (20 mL), again with phosphate buffer
+
1
1
71 18 4
00]. MS: m/z 1275.366 (M + H) (calcd for C66H O P ,
275.359).
D-3-O-Bu t yr yl-2-d eoxy-m yo-in osit ol 1,4,5,6-Tet r a k is-
(d iben zyl)p h osp h a t e (en t-13). Tetrol ent-12 (49 mg, 209
µmol) was phosphorylated as described for compound rac-13
to give the fully protected phosphate ent-13 (197 mg, 74%) as
2
4
a clear oil. [R]
D
+1.6 (c ) 0.97 in CHCl
3
). MS: m/z 1275,
+
1
31
350 (M + H) (calcd for C66
NMR data were in accordance with those obtained for rac-13.
H
71
O
18
P
4
, 1275.359). H and
P
(
20 mL), and finally with brine (20 mL). The organic layer
was dried over Na SO and filtered. Evaporation of the solvent
and crystallization from methanol gave pure rac-11 (196 mg,
r a c-1-O-Bu t yr yl-2-d eoxy-m yo-in osit ol 3,4,5,6-Tet r a k -
isp h osp h a te (r a c-14). Compound rac-13 (165 mg, 130 µmol)
was hydrogenated with palladium (10%) on charcoal as
described for compound rac-12 to give the title compound rac-
2
4
1
9
4%) as needles. Mp: 110.1-110.3 °C (from methanol).
H
1
NMR (CHCl , 360 MHz): 0.93 (3 H, t, J ) 7.37 Hz, CH ), 1.45
(
H, m, -CH
1
3
3
14 (65 mg, 90%) as a hygroscopic solid after freeze-drying. H
1 H, ddd, J ) 12.25, 12.25, 12.25 Hz, H-2 (ax)), 1.56-1.67 (2
NMR (D O, 360 MHz): 0.67 (3 H, t, J ) 7.41 Hz, CH ), 1.33-
2
3
2
), 2.12-2.28 (2 H, m, -CH
2
), 2.42 (1 H, dd, J )
1.43 (2 H, m, -CH ), 1.62 (1 H, ddd, J ) 11.72, 11.71, 11.38
2
2.25, 4.35, 4.35 Hz, H-2 (eq)), 3.50-3.61 (4 H, m, H-3, H-4,
Hz, H-2 (ax)), 2.17 (2 H, m, -CH ), 2.25 (1 H, ddd, J ) 11.38,
2
H-5, H-6), 4.61-4.95 (9H, m, CH
m, CH Ph). MS: m/z (+ve ion FAB) 595 [(M + H) , 2], 91
Bn , 100]. Anal. (C38 ) C: calcd, 76.74; found, 76.87.
H: calcd, 7.12; found, 7.31.
2
Ph, H-1), 7.23-7.37 (20 H,
2.24, 2.24 Hz, H-2 (eq)), 4.07-4.23 (4 H, m, H-3, H-4, H-5, H-6),
+
31
1
2
4.85 (1 H, m, H-1). P NMR (D O, H decoupled, 145.8 MHz):
2
+
[
H
42
O
6
-0.39 (2 P, s), -0.13 (1 P, s), 0.41 (1 P, s). MS: m/z (+ve ion
+
+
+ +
FAB) 555 [(M + H) , 100], 485 [(M - Bt + 2 H ) ], 40]. MS:
+
-
D-1,4,5,6-Tetr a -O-ben zyl-3-O-bu tyr yl-2-d eoxy-m yo-in os-
itol (en t-11). Compound ent-10 (169 mg, 322 µmol) was
butyrylated as described for rac-11 to give the fully protected
inositol ent-11 (183 mg, 96%) as a solid. Mp: 110.4-111.3 °C
m/z (-ve ion FAB) 553 [(M - H ) , 100]. MS: m/z 552.963
(M - H ) (calcd for C10
+
-
21 18 4
H O P , 552.967).
D-3-O-Bu tyr yl-2-deoxy-myo-in ositol 1,4,5,6-Tetr akisph o-
sp h a te (en t-14). Compound ent-13 (160 mg, 125 µmol) was
hydrogenated as described for rac-14 to give the free acid ent-
2
4
Å
(
from methanol). [R]
and MS data were in accordance with those obtained for rac-
1.
r a c-1-O-Bu tyr yl-2-d eoxy-m yo-in ositol (r a c-12). Com-
pound rac-11 (177 mg, 297 mol) was suspended in acetic acid
D
-14.0 (c ) 1.18 in CHCl
3
). H NMR
2
4
14 (66.4 mg, 96%) as a hygroscopic solid. [R] -2.4 (c ) 1.15
D
+
-
1
in H O). MS: m/z 552.972 (M - H ) (calcd for C H O P ,
2
10 21 18
4
552.967). 1H and P NMR data were in accordance with those
31
obtained for rac-14.
(
(
4 mL) and hydrogenated with palladium (10%) on charcoal
178 mg, 1.78 mmol) under a hydrogen atmosphere in a self-
r a c-1-O-Bu t yr yl-2-d eoxy-m yo-in osit ol 3,4,5,6-Tet r a k -
isph osph ate Octakis(acetoxym eth yl) Ester (r a c-5). DIEA
(201 µL, 1.19 mmol) and acetoxymethyl bromide (119 µL, 1.19
mmol) were added to a suspension of compound rac-14 (37 mg,
66 µmol) in dry acetonitrile (2 mL) under an argon atmosphere.
After the reaction mixture stirred in the dark for 4 days, all
volatile compounds were removed under reduced pressure and
the crude residue was purified by preparative HPLC (69%
MeOH; 40.00 mL/min; tR ) 12.10) to give compound rac-5 (31
built hydrogenation apparatus for 6 h. The catalyst was
removed by ultrafiltration, and the filtrate was freeze-dried
to give tetrol rac-12 (68 mg, 98%) as a solid. Mp: 174.9-175.9
°
t, J ) 7.25 Hz, CH
Hz, H-2 (ax)), 1.48-1.58 (2 H, m, -CH
1
2
4
1
C (from ethanol). H NMR (DMSO-d
6
, 360 MHz): 0.87 (3 H,
3
), 1.24 (1 H, ddd, J ) 12.37, 12.37, 12.37
), 1.90 (1 H, ddd, J )
),
2
2.37, 4.21, 4.21 Hz, H-2 (eq)), 2.26 (1 H, t, J ) 7.25, -CH
2
1
.93-3.02 (2 H, m, H-3, H-5), 3.17-3.30 (2 H, m, H-4, H-6),
.52 (1 H, ddd, J ) 12.37, 9.59, 4.21 Hz, H-1), 4.79 (1 H, d, J
4.01 Hz, OH), 4.81 (1 H, d, J ) 3.90 Hz, OH), 4.86 (1 H, d,
mg, 50%) as a clear syrup. H NMR (toluene-d , 360 MHz):
8
0.96 (3 H, t, J ) 7.48 Hz, CH ), 1.54 (1 H, ddd, J ) 12.70,
3
)
12.70, 12.31 Hz, H-2 (ax)), 1.63-1.74 (2 H, m, -CH ), 1.81-
2
J ) 3.90 Hz, OH), 4.94 (1 H, d, J ) 4.01 Hz, OH). MS: m/z
1.87 (24 H, 8 s, 8 × OAc), 2.30 (1 H, dt, J ) 16.28, 7.68 Hz,
+
(+ve ion FAB) 235 [(M + H) , 100]. MS: m/z (-ve ion FAB)
-CH ), 2.52 (1 H, dt, J ) 16.28, 7.88 Hz, -CH ), 2.64 (1 H,
2
2
+
-
-
2
5
33 [(M - H ) , 28], 87 [BtO , 100]. Anal. (C10
1.27; found, 51.09. H: calcd, 7.75; found, 7.57.
D-3-O-Bu tyr yl-2-d eoxy-m yo-in ositol (en t-12). Compound
H
18
O
6
) C: calcd,
ddd, J ) 12.31, 5.31, 5.31 Hz, H-2 (eq)), 4.40-4.59 (4 H, m,
H-3, H-4, H-5, H-6), 5.03 (1 H, ddd, J ) 12.20, 9.53, 5.31 Hz,
3
1
H-1), 5.65-5.95 (16 H, m, 8 × CH
2
OAc). P NMR (toluene-
1
ent-11 (170 mg, 286 µmol) was hydrogenated as described for
d
8
, H decoupled, 145.8 MHz): -4.68 (1 P, s), -4.32 (1 P, s),
rac-12 to give tetrol ent-12 (65 mg, 97%) as a solid. Mp: 174-
-3.77 (1 P, s), -3.65 (1 P, s). MS: m/z (+ve ion FAB) 1131
74.8 °C (from ethanol). [R]24
+25.8 (c ) 1.39 in MeOH). H
1
+
+
+ +
1
D
[(M + H) , 12], 987 [(M - 2 CH
2
-
OAc + 3 H ) , 100]. MS:
+
-
2
,
NMR and MS data were in accordance with those obtained
for rac-12.
m/z (-ve ion FAB) 1059 [(M - Bt ) , 16], 241 [OP(OCH
2
OAc)
+
+
100]. MS: m/z 1059.130 (M - CH
, 1059.131).
D-3-O-Bu tyr yl-2-deoxy-myo-in ositol 1,4,5,6-Tetr akisph o-
2
OAc + 2 H) (calcd for
31 51 32 4
C H O P
r a c-1-O-Bu tyr yl-2-d eoxy-m yo-in ositol 3,4,5,6-Tetr a k is-
d iben zyl)p h osp h a te (r a c-13). A solution of tetrol rac-12
(
(
44 mg, 190 µmol) and tetrazole (160 mg, 2.28 mmol) in
sp h a t e Oct a k is(a cet oxym et h yl) E st er (en t-5). The free
acid ent-14 (40 mg, 72 µmol) was alkylated as described for
compound rac-5 to give the acetoxymethyl ester ent-5 (45 mg,
acetonitrile (2 mL) was treated with dibenzyl N,N-diisopro-
pylphosphoramidite (767 µL, 2.28 mmol) for 36 h and subse-
quently oxidized with peracetic acid (153 µL, 2.28 mmol) at
2
4
56%) as a clear syrup. [R]
D
+
-1.0 (c ) 1 in toluene). MS:
+
-
40 °C. After the mixture warmed to room temperature, the
2
51 32 4
m/z 1059.138 (M - CH OAc + 2 H) (calcd for C31H O P ,
1
31
solvent was removed under reduced pressure and the residual
oil was purified by preparative HPLC (92% MeOH; 40 mL/
1059.131). H and P NMR data were in accordance with
those obtained for rac-5.
min; t
3 (172 mg, 71%) as an oil. H NMR (CDCl
3 H, t, J ) 7.57 Hz, CH ), 1.45 (2 H, tq, J ) 7.57, 7.57 Hz,
CH ), 1.46 (1 H, ddd, J ) 12.92, 11.14, 11.14 Hz, H-2 (ax)),
.08 (2 H, t, J ) 7.57 Hz, -CH ), 2.58 (1 H, ddd, J ) 12.92,
.90, 4.90 Hz, H-2 (eq)), 4.28-4.37 (1 H, m, H-3), 4.44 (1 H,
R
) 21.55 min) to give the fully protected compound rac-
r a c-2-Deoxy-m yo-in osit ol 3,4,5,6-Tet r a k isp h osp h a t e
(r a c-2). Compound rac-14 (17 mg, 30 µmol) was treated with
1 M KOH (260 µL) to adjust the pH value to 12.8. The solution
was stirred at room temperature for 2 days. The reaction
mixture was directly poured onto an ion-exchange column
1
1
(
-
2
4
3
, 360 MHz): 0.79
3
2
2
+
(Dowex 50 WX 8, H ) for purification. Lyophilization gave
dd, J ) 9.24, 9.24, 9.24 Hz, H-5), 4.53 (1 H, ddd, J ) 9.24,
.02, 9.02 Hz, H-4), 4.56 (1 H, ddd, J ) 9.24, 9.24, 9.24 Hz,
H-6), 4.83-5.08 (17 H, m, 8 × CH Ph, H-1), 7.12-7.32 (40 H,
compound rac-2 (14 mg, 95%). 1H NMR (D
2
O, 360 MHz): 1.55
9
(1 H, ddd, J ) 12.09, 12.09, 12.09 Hz, H-2 (ax)), 2.24 (1 H,
ddd, J ) 12.09, 4.31, 4.31 Hz, H-2 (eq)), 4.79 (1 H, ddd, J )
12.09, 8.77, 4.31 Hz, H-1), 5.14 (1 H, ddd, J ) 9.25, 9.25, 8.83
2
3
1
1
m, 8 × CH
2 3
Ph). P NMR (CDCl , H decoupled, 145.8 MHz):

3
642 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Rudolf et al.
ion FAB) 595 [(M + H) , 1], 91 [Bn , 100]. MS: m/z 235.120
Hz, H-5), 5.18-5.73 (3 H, m, H-3, H-4, H-6). 31P NMR (D
O,
+
+
2
1
+
H decoupled, 145.8 MHz): -0.08 (1 P, s), 0.28 (1 P, s), 0.34 (1
19 6
(M + H) (calcd for C10H O , 235.118).
+
P, s), 0.90 (1 P, s). MS: m/z (+ve ion FAB) 485 [(M + H) ,
r a c-2-O-Bu tyr yl-1-O-d eoxy-m yo-in ositol (r a c-19). Com-
pound rac-18 (75 mg, 126 mol) was hydrogenated with pal-
ladium (10%) on charcoal under hydrogen as described for
+
-
1
4
00]. MS: m/z (-ve ion FAB) 483 [(M - H ) , 100]. MS: m/z
+
6 15 17 4
82.928(M - H) (calcd for C H O P , 482.926).
D-2-Deoxy-m yo-in ositol 1,4,5,6-Tetr a k isp h osp h a te (en t-
). The free acid ent-14 (19 mg, 34 µmol) was saponified as
compound rac-12 to give tetrol rac-19 (28 mg, 99%) as a solid
1
2
after freeze-drying. Mp: 138.7-139.5 °C (from ethanol).
NMR (DMSO-d
1.41 (1 H, ddd, J ) 14.14, 11.90, 2.34 Hz, H-1 (ax)), 1.50-1.59
H
described for compound rac-2 to give the title compound ent-2
(
6
3
, 360 MHz): 0.89 (3 H, t, J ) 7.53 Hz, CH ),
2
4
15 mg, 91%) as a solid. [R]
D
+1.7 (c ) 0.58 in H
2
O). MS:
+
, 482.926). 1H and
(2 H, m, -CH ), 1.89 (1 H, ddd, J ) 14.14, 4.27, 4.27 Hz, H-1
m/z 482.927 (M - H) (calcd for C
P NMR data were in accordance with those obtained for rac-
6
H
15
O
17
P
4
2
3
1
(eq)), 2.26 (2 H, m, -CH ), 2.96 (1 H, dd, J ) 8.75, 5.90 Hz,
2
H-3), 3.25-3.32 (2 H, m, H-4, H-5), 3.39 (1 H, m, H-6), 4.65 (1
H, d, J ) 4.48 Hz, OH), 4.73 (1 H, s(br), OH), 4.77 (1 H, s(br),
OH), 5.00 (1 H, ddd, J ) 4.27, 2.34, 2.34 Hz, H-2). MS: m/z
2
.
r a c-3,4,5,6-Tetr a -O-ben zyl-1-O-p h en oxy(th ioca r bon yl)-
m yo-in ositol (r a c-16). A solution of rac-15 (1 g, 2 mmol),
O-phenyl chlorothionoformate (270 µL, 2 mmol), and DMAP
122 mg, 1 mmol) in dry pyridine (6 mL) was stirred at room
temperature for 20 min. The solvents were evaporated under
high vacuum to give an oil. Residual pyridine was removed
by evaporating three times with octane. The residue was
dissolved in tert-butyl methyl ether (50 mL) and was washed
twice with phosphate buffer (40 mL) and then with brine (40
+
-
+ -
(
(
(
+ve ion FAB) 235 [(M + H ) , 18], 71 [(Bt ) , 100]. MS: m/z
+
-
-
-ve ion FAB) 233 [(M - H ) , 18], 87 [BtO , 100]. Anal.
) C: calcd, 51.27; found, 51.09. H: calcd, 7.75; found,
(
10 18 6
C H O
7
.57.
r a c-2-O-Bu tyr yl-1-d eoxy-m yo-in ositol 3,4,5,6-Tetr a k is-
(d iben zyl)p h osp h a te (r a c-20). A solution of tetrol rac-19
(20 mg, 85 mol) and tetrazole (74 mg, 1.02 mmol) in acetonitrile
(2 mL) was treated with dibenzyl N,N-diisopropylphosphora-
mL). The organic layer was dried over Na
Purification by preparative HPLC (91% MeOH; 37.5 mL/min;
) 40.00 min) yielded compound rac-16 (843 mg, 63%). Mp:
2 4
SO and filtered.
midite (345 µL, 1.02 mmol) for 18 h, oxidized with peracetic
acid at -40 °C, and worked up as described for compound rac-
13. Purification by preparative HPLC (93% MeOH; 40 mL/
t
1
R
1
22.7-123.1 °C (from methanol). H NMR (CDCl
MHz): 3.59 (1 H, dd, J ) 9.58, 2.45 Hz, H-3), 3.60 (1 H, dd, J
9.36, 9.36 Hz, H-5), 4.01 (1 H, dd, J ) 9.58, 9.36 Hz, H-4),
.28 (1 H, dd, J ) 10.25, 9.36 Hz, H-6), 4.59 (1 H, dd, J )
.45, 2.45 Hz, H-2), 4.72-4.91 (8 H, m, 4 × CH Ph), 5.35 (1
Ph
3
, 360
min; t ) 15.50 min) gave compound rac-20 (80 mg, 73%) as
R
1
an oil. H NMR (CDCl
3
, 360 MHz): 0.90 (3 H, t, J ) 7.52 Hz,
), 1.51-1.63 (2 H, m, -CH ), 1.70 (1 H, ddd, J ) 12.98,
0.48, 2.73 Hz, H-1 (ax)), 2.22 (2 H, t, J ) 7.52 Hz, -CH ),
.50 (1 H, ddd, J ) 12.98, 4.90, 4.90 Hz, H-1 (eq)), 4.47 (1 H,
)
CH
3
2
4
2
1
2
2
2
H, dd, J ) 10.25, 2.45 Hz, H-1), 7.03-7.44 (25 H, m, 4 × CH
2
ddd, J ) 9.87, 9.81, 2.73 Hz, H-3), 4.59-4.71 (2 H, m, H-4,
H-5), 4.91-5.09 (17 H, m, 8 × CH Ph, H-6), 5.55 (1 H, ddd, J
4.90, 2.73, 2.73 Hz, H-2), 7.15-7.30 (40 H, m, 8 × CH Ph).
, H decoupled, 145.8 MHz): -1.58 (1 P, s),
1.12 (1 P, s), -0.92 (2 P, s). MS: m/z (+ve ion FAB) 1275
+
and C(S)OPh ArH). MS: m/z (+ve ion FAB) 677 [(M + H) ,
+
2
1
7
], 91 [Bn , 100]. Anal. (C41
2.43. H: calcd, 5.96; found, 5.93.
r a c-3,4,5,6-Tet r a -O-b en zyl-2-O-b u t yr yl-1-O-p h en oxy-
40 7
H O S) C: calcd, 72.76; found,
)
2
3
1
1
P NMR (CDCl
3
-
(
(
th ioca r bon yl)-m yo-in ositol (r a c-17). A solution of rac-16
532 mg, 787 mol), butyric anhydride (140 µL, 850 mol), and
+
+
[
[
(
(M + H) , 2], 91 [Bn , 100]. MS: m/z (-ve ion FAB) 1183
+
-
-
(M - Bn ) , 14], 277 [OPO(OBn) , 100]. MS: m/z 1275.358
M + H) (calcd for C66
DMAP (19 mg, 15 mol) in dry pyridine (5 mL) was stirred at
room temperature for 1 h. The solvents were evaporated
under high vacuum to give an oil. Residual pyridine was
removed by evaporating three times with octane. The residue
was dissolved in tert-butyl methyl ether (30 mL) and was
washed with phosphate buffer (20 mL), with sodium hydrogen
carbonate (20 mL), with sodium hydrogen sulfate (20 mL),
again with phosphate buffer (20 mL), and finally with brine
+
71 18 4
H O P , 1275.359).
r a c-2-O-Bu t yr yl-1-d eoxy-m yo-in osit ol 3,4,5,6-Tet r a k -
isp h osp h a te (r a c-21). Compound rac-20 (165 mg, 130 mol)
was hydrogenated with palladium (10%) on charcoal as
described for compound rac-12 to give title compound rac-21
1
(
65 mg, 90%) as a solid after freeze-drying. H NMR (D
2
O,
3
60 MHz): 0.85 (3 H, t, J ) 7.39 Hz, CH
CH ), 1.82 (1 H, ddd, J ) 13.52, 2.58, 2.21 Hz, H-1 (ax)),
.31-2.37 (3 H, m, H-1(eq), -CH ), 4.26 (1 H, ddd, J ) 9.29,
.18, 9.18 Hz, H-5), 4.27-4.37 (2 H, m, H-3, H-4), 4.48 (1 H,
3
), 1.51-1.61 (2 H, m,
-
2
9
2
(
20 mL). The organic layer was dried over Na
2 4
SO and filtered.
2
Evaporation of the solvent gave pure rac-17 (476 mg, 82%) as
1
an oil. H NMR (CDCl
CH ), 1.71 (2 H, m, -CH
J ) 9.45, 9.45 Hz, H-5), 3.63 (1 H, dd, J ) 9.67, 2.58 Hz, H-3),
3
, 360 MHz): 0.99 (3 H, t, J ) 7.31 Hz,
ddd, J ) 9.36, 9.36, 9.12 Hz, H-6), 5.38 (1 H, ddd, J ) 4.76,
2
), 2.41 (2 H, m, -CH ), 3.62 (1 H, dd,
2
31
1
3
2
.21, 2.21 Hz, H-2). P NMR (D
2
O, H decoupled, 145.8 MHz):
-
0.21 (1 P, s), -0.01 (1 P, s), 0.49 (1 P, s), 0.53 (1 P, s). MS:
3
9
.92 (1 H, dd, J ) 9.67, 9.45 Hz, H-4), 4.11 (1 H, dd, J ) 10.32,
.45 Hz, H-6), 4.71-4.94 (8 H, m, 4 × CH Ph), 5.38 (1 H, dd,
+
+
m/z (+ve ion FAB) 555 [(M + H) , 100], 485 [(M - Bt + 2
+
+
+
-
2
H
)
, 30]. MS: m/z (-ve ion FAB) 553 [(M - H
)
, 100].
J ) 10.32, 2.58 Hz, H-1), 6.08 (1 H, dd, J ) 2.58, 2.58 Hz,
MS: m/z 552.960 (M - H (calcd for C H O P , 552.967).
+ -
)
1
0
21 18
4
H-2), 7.04-7.43 (25 H, m, 4 × CH
2
Ph and C(S)OPh). MS: m/z
+ve ion FAB) 747 [(M + H) , 1], 91 [Bn , 100]. MS: m/z
r a c-2-O-Bu t yr yl-1-d eoxy-m yo-in osit ol 3,4,5,6-Tet r a k -
isph osph ate Octakis(acetoxym eth yl) Ester (r a c-6). DIEA
+
+
(
7
+
31.310 (M + H) (calcd for C45
47 7
H O S, 731.304).
(139 µL, 816 mol) and acetoxymethyl bromide (82 µL, 820 mol)
r a c-3,4,5,6-Tetr a -O-ben zyl-2-O-bu tyr yl-1-O-d eoxy-m yo-
in ositol (r a c-18). Compound rac-17 (476 mg, 638 mol) was
dissolved in dry toluene (100 mL), and AIBN (30 mg, 150 mol)
were added to a suspension of compound rac-21 (18 mg, 32
mol) in acetonitrile (1 mL) as described for compound rac-2.
Purification by preparative HPLC (69% MeOH; 40.00 mL/min;
and n-Bu
3
SnH (305 µL, 1.15 mmol) were added. The solution
t ) 11.50) gave compound rac-6 (21 mg, 55%) as a clear syrup.
R
1
was heated to reflux under an argon atmosphere for 2 h. The
reaction mixture was cooled and then washed with phosphate
buffer (30 mL) and brine (30 mL). The organic layer was dried
H NMR (toluene-d
CH ), 1.51-1.68 (3 H, m, H-1 (ax), -CH
), 2.57 (1 H, ddd, J )
8
, 360 MHz): 0.85 (3 H, t, J ) 7.48 Hz,
3
2
), 1.78-1.86 (24 H, 8
s, 8 × OAc), 2.08-2.17 (2 H, m, -CH
2
over Na
2
SO
4
and filtered. Evaporation of the solvent and
14.37, 4.53, 4.53 Hz, H-1 (eq)), 4.70 (1 H, ddd, J ) 9.06, 9.06,
2.34 Hz, H-3), 4.73-4.84 (2 H, m, H-4, H-5), 5.02 (1 H, ddd, J
purification by preparative HPLC (89% MeOH; 37.5 mL/min;
1
t
(
R
) 50.30 min) gave rac-18 (134 mg, 35%) as an oil. H NMR
CDCl , 360 MHz): 0.96 (3 H, t, J ) 7.45 Hz, CH ), 1.50 (1 H,
) 9.06, 9.06, 9.06 Hz, H-6), 5.59-5.92 (17 H, m, 8 × CH
2
OAc,
3
1
1
H-2). P NMR (toluene-d , H decoupled, 145.8 MHz): -4.75
8
3
3
ddd, J ) 14.21, 11.89, 2.33 Hz, H-1 (ax)), 1.61-1.71 (2 H, m,
CH ), 2.25 (1 H, ddd, J ) 14.21, 4.31, 4.31 Hz, H-1 (eq)), 2.33
2 H, m, -CH ), 3.54 (1 H, dd, J ) 10.01, 2.49 Hz, H-3), 3.56
1 H, dd, J ) 9.32, 9.32 Hz, H-5), 3.82 (1 H, ddd, J ) 11.89,
(1 P, s), -4.56 (1 P, s), -3.72 (1 P, s), -3.69 (1 P, s). MS: m/z
+
+
-
2
(+ve ion FAB) 1131 [(M + H) , 2], 987 [(M - 2 CH
2
+
OAc + 3
+
-
(
(
2
H) , 100], MS: m/z (-ve ion FAB) 1059 [(M - Bt ) , 6], 241
-
+
[OP(OCH
2 H) (calcd for C31H O P
2
OAc)
2
, 100]. MS: m/z 1059.133 (M - CH
, 1059.131).
2
OAc +
+
9
4
2
.32, 4.31 Hz, H-6), 3.84 (1 H, dd, J ) 9.32, 9.32 Hz, H-4),
.55-4.95 (8 H, m, 4 × CH Ph), 5.55 (1 H, ddd, J ) 4.31, 2.49,
.33 Hz, H-2), 7.26-7.39 (20 H, m, 4 × CH Ph). MS: m/z (+ve
51 32 4
2
r a c-1-Deoxy-m yo-in osit ol 3,4,5,6-Tet r a k isp h osp h a t e
(r a c-3). Compound rac-21 (20 mg, 35 µmol) was treated with
2

2
-Deoxy Derivative as Partial Agonist of Ins(3,4,5,6)P
4
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3643
1
M KOH (285 µL) to adjust the pH value to 12.8. The solution
(20) Barrett, K. E. Positive and negative regulation of chloride
secretion in T84 cells. Am. J . Physiol. 1993, 265, C859-C868.
was stirred at room temperature for 2 days. The reaction
mixture was directly poured onto an ion-exchange column
(
21) Barrett, K. E. Integrated regulation of intestinal epithelial
transport: intercellular and intracellular pathways. Am. J .
Physiol. 1997, 272, C1069-C1076.
+
(
Dowex 50 WX 8, H ) for purification. Lyophilization gave
1
compound rac-3 (18 mg, 94%). H NMR (D
2
O, 360 MHz): 1.63
(
22) Montrose, M. H.; Keely, S. J .; Barrett, K. E. Electrolyte secretion
and absorption: small intestine and colon. In Textbook of
Gastroenterology, 3th ed.; Yamada, T., Alpers, D., Laine, L.,
Powell, D., Owyang, C., Eds.; Lippincott, Inc.: Philadelphia, in
press.
(
1 H, ddd, J ) 13.72, 10.50, 2.31 Hz, H-1 (ax)), 2.27 (1 H, ddd,
J ) 13.72, 2.45, 2.31 Hz, H-1 (eq)), 4.62 (1 H, ddd, J ) 9.45,
9
.45, 9.45 Hz, H-5), 4.68 (1 H, ddd, J ) 10.50, 2.31, 2.31 Hz,
31
H-2), 4.95-5.05 (2 H, m, H-3, H-4), 5.18 (1 H, m, H-6).
NMR (D
P
(
23) Clarke, L. L.; Boucher, R. C. Ion and water transport across
airway epithelia. In Pharmacology of the Respiratory Tract;
Chung, K. F., Barnes, P. J ., Eds.; Marcel Dekker: New York,
1993; pp 505-550.
1
2
O, H decoupled, 145.8 MHz): -0.28 (1 P, s), -0.06
(1 P, s), 0.14 (1 P, s), 0.27 (1 P, s). MS: m/z (+ve ion FAB)
+
+ -
4
1
4
85 [(M + H) , 100]. MS: m/z (-ve ion FAB) 483 [(M - H ) ,
+
00]. MS: m/z 482.924 (M - H) (calcd for C
6
H
15
O
17
P
4
,
(24) Tan, Z.; Bruzik, K. S.; Shears, S. B. Properties of the inositol
3,4,5,6-tetrakisphosphate 1-kinase purified from rat liver. J .
Biol. Chem. 1997, 272, 2285-2290.
82.926).
(
25) Xie, W.; Kaetzel, M. A.; Bruzik, K. S.; Dedman, J . R.; Shears, S.
B.; Nelson, D. J . Inositol 3,4,5,6-tetrakisphosphate inhibits the
calmodulin-dependent protein kinase II-activated chloride con-
ductance in T84 colonic epithelial cells. J . Biol. Chem. 1996, 271,
Ack n ow led gm en t. We thank J . Stelten for record-
ing the NMR spectra, Dr. P. Schulze for providing FAB-
MS data, Dr. Roger Y. Tsien for giving us access to his
Ca -imaging facilities, and Dr. Stephen B. Shears for
reading the manuscript. We are indebted to the Deut-
sche Forschungsgemeinschaft (Schu 943/1-5 u. 1-6) and
NIH RO1 DK 47240 (to A.T.K.) for financial support.
1
4092-14097.
2
+
(
(
26) Ho, M. W. Y.; Shears, S. B.; Bruzik, K. S.; Duszyk, M.; French,
2
+
A. S. Ins(3,4,5,6)P
dependent Cl- current in CFPAC-1 cells. Am. J . Physiol. 1997,
72, C1160-C1168.
4
specifically inhibits a receptor-mediated Ca -
2
27) Ismailov, I. I.; Fuller, C. M.; Berdiev, B. K.; Shlyonsky, V. G.;
Benos, D. J .; Barrett, K. E. A biologic function for an “orphan”
messenger: D-myo-inositol 3,4,5,6-tetrakisphosphate selectively
blocks epithelial calcium-activated chloride channels. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 10505-10509.
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(
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(
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