Adenophostin A and analogues modified at the adenine moiety: Synthesis, conformational analysis and biological activity

Article abstract of DOI:10.1039/b415229h

The synthesis of adenophostin A (2) and two analogues [etheno adenophostin (4) and 8-bromo adenophostin (5)] modified at the adenine moiety, is reported. A combination of NMR analysis and molecular modelling was used to compare their structures in solutio

Full text of DOI:10.1039/b415229h

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A R T I C L E
Adenophostin A and analogues modified at the adenine moiety:
synthesis, conformational analysis and biological activity
Charles N. Borissow,a Steven J. Black,a Michael Paul,a Stephen C. Tovey,b
Skarlatos G. Dedos,b Colin W. Taylorb and Barry V. L. Potter*a
a Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology,
University of Bath, Claverton Down, Bath, UK BA2 7AY. E-mail: b.v.l.potter@bath.ac.uk;
Fax: +44 1225 386114; Tel: +44 1225 386639
b Department of Pharmacology, Tennis Court Road, University of Cambridge, Cambridge,
UK CB2 1PD
Received 1st October 2004, Accepted 3rd November 2004
First published as an Advance Article on the web 13th December 2004
The synthesis of adenophostin A (2) and two analogues [etheno adenophostin (4) and 8-bromo adenophostin
(5)] modified at the adenine moiety, is reported. A combination of NMR analysis and molecular modelling was used
to compare their structures in solution and determined that they all adopt very similar conformations. The analogues
were tested for their ability to mobilise Ca2+ from DT40 cells expressing recombinant Type 1 rat Ins(1,4,5)P3R which
reveals etheno adenophostin as a high affinity fluorescent probe of the Ins(1,4,5)P3R. 8-Bromo adenophostin was only
slightly less potent. The biological results support our current hypothesis regarding the binding mode of adenophostin
A at the Ins(1,4,5)P3R, i. e. that a cation–p interaction between the base moiety and Arg 504 of the receptor
in combination with H-bonding may be responsible for the high potency of adenophostin A relative to Ins(1,4,5)P3.
Since their discovery5 and structural elucidation6,7 in 1993,
adenophostin A 2 and B 3 have been the focus of much interest.
Introduction
D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] (1) (Fig. 1)
Both molecules have been shown to exhibit a 10-fold greater
is a second messenger involved in the release of Ca2+ from
affinity for the Ins(1,4,5)P3R8–13 than Ins(1,4,5)P3 itself and are
intracellular stores upon interaction with its specific receptor
currently the most potent agonists of Ins(1,4,5)P3R.7 These
[Ins(1,4,5)P3R].1
interesting properties have led to the widespread use of adeno-
phostin A as a biological tool in Ca2+ signalling studies and
prompted many groups, including our own, to develop synthetic
routes to 2 and its analogues.9,10,14–32 None of these synthetic
analogues has exceeded the potency of adenophostin A, but
a great deal of information has been learned about adeno-
phostin’s structure–activity relationship at the Ins(1,4,5)P3R
(see Fig. 2).
It has been demonstrated that the adenine base (or equivalent)
of adenophostin A is an important structural feature necessary
for high potency, although the exact reason for this has still to
be determined.8,15,20,33,34 Our current explanation for this arose
Fig. 1 Structures of D-myo-inositol 1,4,5-trisphosphate (1), adeno-
phostin A (2) and adenophostin B (3).
from modelling studies20 based on the crystal structure of the
binding domain of type 1 Ins(1,4,5)P3R,35 showing a possible
Due to the biological importance of Ca2+ release2 a number of
analogues of Ins(1,4,5)P3 have been synthesised and evaluated to
determine the structural features required for interaction at its
receptor.3 However, to date, none of these synthetic analogues
has improved upon Ins(1,4,5)P3 in terms of receptor binding
affinity or Ca2+ release,3 with the exception of the dimeric species
synthesised in our laboratories.4
cation–p interaction between the adenine ring and Arg504 when
adenophostin A binds in the anti-conformation. This interaction
may enhance binding affinity and improve the orientation of the
2ꢀ-phosphate resulting in an increase in potency. In an attempt
to further elucidate these binding interactions, we report here
the synthesis and conformational analysis of two analogues 4
and 5 (Fig. 3) modified at the adenine moiety.
Fig. 2 Summary of the structure–activity relationships of adenophostin A at the Ins(1,4,5)P3 receptor.
T h i s j o u r n a l i s T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5 O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 5 – 2 5 2
2 4 5
©

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has been reacted with a range of nucleophiles to produce a
variety of N-6 base substituted adenophostin analogues20 and is
a convenient precursor for an improved route to adenophostin
A. Treatment of 6 with ethanolic ammonia38 gave the required
6-amino functionality with concomitant removal of the acetate
protecting groups to afford 7 in 92% yield.
The introduction of the protected phosphates was achieved
using freshly prepared imidazolium triflate and bis(benzyloxy)
(diisopropylamino)phosphine.16,39 Oxidation of the trisphos-
phite with mCPBA furnished, after purification, the fully pro-
tected adenophostin intermediate 8 in 53% yield. Deprotection
was achieved using catalytic transfer hydrogenation16,40 with the
product being purified by ion exchange chromatography to give 2
in 75% yield. 1H, 13C, 31P NMR and FAB mass spectral analysis
were concurrent with those previously reported.16,32,41 Having
synthesised adenophostin A, our attention now turned to its
modification to both the etheno 4 and 8-bromo 5 analogues
(Scheme 2).
Fig. 3 Structures of etheno adenophostin (4) and 8-bromo adeno-
phostin (5).
The etheno analogue 4 should provide an easily accessible
fluorescent probe, which could be used for investigation of
Ins(1,4,5)P3Rs. A study by Rosenberg et al.20 demonstrated
that the Ins(1,4,5)P3R is tolerant to the introduction of large
hydrophobic groups at N-6 of adenophostin. This, coupled
with work by Leonard and co-workers36,37 showing that etheno
derivatives bind like endogenous substrates to several pro-
teins, indicated that 4 should be a good replacement for
adenophostin A, hopefully retaining its high affinity binding
to the Ins(1,4,5)P3R. Earlier modelling studies by Hotoda
et al.33 suggest that adenophostin binds to the Ins(1,4,5)P3R
in the syn-conformation as opposed to anti as devised from our
docking experiments.20 The 8-brominated analogue 5 is sterically
constrained in the syn-conformation and might therefore exhibit
a similar potency to adenophostin, or even higher, if the syn-
conformation is maintained upon binding to the Ins(1,4,5)P3R.
The 8-bromo analogue also provides the versatility to produce
potentially useful photoaffinity labels by either introducing an
azido functionality directly at the 8-position or via an alkyl
spacer. These compounds could be used for further investigation
of Ins(1,4,5)P3Rs.
Results and discussion
Synthesis
The synthesis of adenophostin A 2 and analogues 4 and 5
employs the 6-chloropurine building block 6 (Scheme 1), which
we have previously shown to be a versatile intermediate. This
Scheme 2 Reagents and conditions: (a) chloroacetaldehyde (50% aq.),
NaOAc, 2 days; (b) 0.5 M NaOAc, Br2, 60 h.
The synthesis of etheno nucleotides and nucleosides has been
accomplished numerous times using slight modifications of a
general procedure.36,37,42–45 Thus, adenophostin A was dissolved
in a 50% aqueous solution of chloroacetaldehyde (pH 4.0–4.5)
and was stirred for 48 h at room temperature. Purification using
an MP1 AG ion exchange column gave the fluorescent etheno
adenophostin analogue 4 showing characteristic doublets for
H-10 and H-11 in the 1H NMR spectrum at 8.22 and 8.87 ppm.
Like the etheno derivatives, a number of groups have
previously synthesised 8-bromo analogues of nucleotides and
nucleosides46–50 with the critical point being the need for a pH
of between 4.0–4.5 for the reaction to proceed efficiently. This
can be achieved using a sodium acetate buffer. In our case, an
optimum concentration of 0.5 M sodium acetate was found to
be sufficient, with the pH changing by only ca. 0.2 units over the
course of the reaction. Adenophostin A was dissolved in 0.5 M
sodium acetate buffer and a solution of bromine in buffer was
slowly added. After stirring for 60 h, purification of the reaction
mixture was achieved using an MP1 AG ion exchange column
eluting with 150 mM TFA to give the desired 8-bromo analogue
showing only one singlet in the 1H NMR spectrum at 8.01 ppm
representing H-2. The purity of analogues 4 and 5 was deter-
mined by HPLC showing >96% and >99% purity, respectively.
Conformational analysis in solution
Following the procedure of Hotoda et al.,33 the 1H and NOESY
spectra of compounds 2, 4 and 5 were recorded in 50 mM
phosphate buffer (p2H 6.8) at 25 ◦C in an attempt to explore their
conformation in solution. The results obtained for adenophostin
Scheme 1 Reagents and conditions: (a) EtOH, NH3, 60 ◦C, 4 days; (b)
i) (BnO)2PNiPr2, imidazolium triflate, DCM, RT, 1.5 h; ii) mCPBA,
−78 ◦C, 10 min; (c) moist 20 % Pd(OH)2/C, cyclohexene–MeOH–H2O,
80 ◦C, 3 h.
2 4 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 5 – 2 5 2

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Fig. 4 NOESY spectra of (a) synthetic adenophostin Aa 2; (b) etheno adenophostin 4b; (c) 8-bromo adenophostin 5b. The structures of each
compound are shown on the bottom right with the important NOE correlations highlighted. a Spectrum recorded in 50 mM phosphate buffer in D2O
(p2H 6.8) at 600 MHz with a mixing time of 800 ms at 298 K. b Spectra recorded in DMSO-d6 at 400 MHz with a mixing time of 800 ms at 298 K.
vacuo study which found the C-3ꢀ endo conformation in their
Table 1 Selected coupling constants of adenophostin A 2, and ana-
logues 4 and 5 and their calculated C-2ꢀ endo percentagea.
lowest energy structure (although they state that the C-2ꢀ endo
conformation is more likely). The NOESY experimental data is
,2ꢀꢀ
,3ꢀꢀ
,4ꢀꢀ
,5ꢀꢀ
J1ꢀ,2ꢀ
J3ꢀ,4ꢀ
[C-2ꢀ endo] %
1ꢀ,2ꢀ /(J1ꢀ,2ꢀ +J3ꢀ,4ꢀ
J1ꢀꢀ
J2ꢀꢀ
J3ꢀꢀ
J4ꢀꢀ
ꢀꢀ
ꢀ
˚
also supported, with the H-1 to H-3 distance being 2.37 A,
suitably close for a strong NOE correlation. The torsional
angle about the N-glycosidic bond (v†) is 6.4◦, showing a syn-
conformation of the adenine moiety which is required for the
NOE correlations between H-8 and H-1ꢀ and H-2ꢀ. Although
there is a difference in v between our model and Hotoda’s (56◦),
both show the expected syn-conformation of the adenine moiety.
This is observed because the energy barriers preventing rotation
around the N-glycosidic bond are not high when compounds
are in the C-2ꢀ endo conformation.51
Etheno adenophostin 4 gave markedly similar results to
adenophostin A (Table 1), with the ribose moiety showing a
preference for the C-2ꢀ endo conformer as determined by the
Davies and Danyluk equation.52 The NOESY spectrum (see
Fig. 4b) shows correlations between H-8 of the base with H-1ꢀ
and H-2ꢀ of the ribose moiety suggesting a syn conformation
in solution, similar to that of adenophostin A. Further NOE’s
show the expected interactions of H-10 with both H-11 and H-2
(Hz)
(Hz)
J
)
(Hz)
(Hz)
(Hz)
(Hz)
2
4
5
6.2
5.7
7.4
3.3
3.5
5.3
65%
62%
58%
3.7
3.2
3.8
9.5
9.1
9.4
9.5
9.1
9.4
9.5
9.1
9.4
a All values were recorded in 50 mM phosphate buffer in D2O (p2H 6.8)
at 400 MHz.
A (Table 1) paralleled those of Hotoda,33 confirming: a chair
conformation of the glucose ring with 2 equatorial phosphates;
the close proximity of H-1ꢀꢀ to H-3ꢀ as determined by NOE
correlation; a preference for the C-2ꢀ-endo conformation of
the ribose ring and a syn-orientation of the adenine moiety as
determined from NOE correlations between H-8 and both H-1ꢀ
and H-2ꢀ (Fig. 4a).
In order to model adenophostin A in solution, its structure
(based on H and NOESY NMR data) was surrounded by a
1
“layer” of water molecules and energy minimisation calculations
were performed to determine the lowest energy conformation.
The model obtained (Fig. 5a) supports the data from the H
NMR experiments, showing a chair conformation of the glucose
ring with 2 equatorial phosphates linked to a C-2ꢀ endo puckered
ribose moiety. This is the major difference to the Hotoda in
† The torsional angle about the N-glycosidic bond (C1ꢀ–N) is the angl◦e
between the C1ꢀ–O bond and the N9–C4 bond. Therefore, when v = 0
the O–C1ꢀ bond is eclipsed by the N9–C4 bond. When v = 0 90◦ the
base is said to be in the syn conformation with the anti conformation
being v = 180 90◦.
1
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 5 – 2 5 2
2 4 7

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Fig. 5 Minimum energy solution phase conformations of (a) adenophostin A 2; (b) etheno adenophostin 4; (c) 8-bromo adenophostin 5. For ease
of viewing the water molecules have been removed.
and also the correlation between H-3ꢀ and H-1ꢀꢀ. The proton–
proton coupling constants of the glucose ring confirm the
presence of a chair conformation with two equatorial phosphate
groups. Modelling studies were performed in the same manner
as described previously for adenophostin A, resulting in the low
energy structure seen in Fig. 5b. The carbohydrate backbone of
the molecule remains essentially unchanged; however, there is a
substantial change in v from 6.4 to −76.0◦ probably resulting
from the increase in electron density of the aromatic system being
repelled by the C-2ꢀ phosphate. Despite this large change, the
adenine moiety remains in the syn-conformation which would
allow the observed NOE correlations of H-8 with H-1ꢀ and H-2ꢀ.
8-Bromo adenophostin (5), not unexpectedly, follows the
same pattern as adenophostin A (2) and etheno adenophostin (4)
(Table 1), assuming the same conformation in terms of glucose
and ribose moieties. The NOESY spectrum (Fig. 4c) shows no
visible correlations between H-2 and any other protons suggest-
ing the same syn-conformation of the base moiety. Modelling
of the 8-bromo analogue (Fig. 5c) confirms the retention of
conformation within the glucose and ribose moieties, as expected
from NMR data, but again a change in v is observed from
6.4◦ in 2 to 63.2◦ in 5. This rotation about the N-glycosidic
linkage is caused by steric clashes between the bulky bromine
atom and the ribose moiety making the syn-conformation more
energetically favourable. Similar observations were made in the
crystal structure of 8-bromo adenosine.53
Despite the syn-conformation of adenophostin A in solution,
our proposed model for its binding to the Ins(1,4,5)P3R20 places
the adenine moiety of adenophostin in the anti-conformation
allowing the possibility of a cation–p interaction between the
base and the guanidinium sidechain of Arg 504 (Fig. 6a).
Cation–p interactions have been observed numerous
times,54–56 with calculations predicting that the interaction may
Biological evaluation
Compounds 4 and 5 were evaluated in DT40 cells, expressing
recombinant Type 1 rat Ins(1,4,5)P3R, for their ability to
release Ca2+ from intracellular stores (Table 2). The results for
Ins(1,4,5)P3 (1) and adenophostin A (2), synthesised by this
present route, are also shown and all values are shown relative
to adenophostin A measured in parallel experiments for a more
accurate comparison.
The EC50 value of adenophostin A was ca. 10 times lower than
that of Ins(1,4,5)P3 (EC50 24.8 and 2.1 nM, respectively), consis-
tent with previous results and confirming its higher potency. The
drop in potency of the etheno analogue 4 was modest, its EC50
being 65% that of adenophostin A. Interestingly, the 8-bromo
compound 5 was still half as potent as adenophostin A (2), with
a 4 fold increase in potency over Ins(1,4,5)P3 (1).
Fig. 6 Predicted binding modes of (a) adenophostin A and (b)
etheno adenophostin with the Ins(1,4,5)P3R20. The adenine moiety (or
equivalent) is in the anti-conformation showing a possible cation–p
interaction with Arg 504. Adapted from ref. 20
Table 2 45Ca2+ Release data for 1, 2 4 and 5 from DT40 cellsa
EC50/nM
max. Response/%
h
EC50 with respect to 2b
n
1
2
4
5
24.8 2.1
2.1 0.6
4.0 0.6
5.4 0.6
78
76
80
79
2
1
1
3
1.21 0.06
1.54 0.13
1.69 0.16
1.93 0.11
0.09 0.01
1.0
0.65 0.05
0.49 0.05
11
12
3
4
a Results (means SEM of n independent experiments) show the concentration of each compound causing half the maximal response (EC50), the
Hill coefficient (h) and the maximal response (% of intracellular Ca2+ stores released) for each of the four compounds. b Results are expressed relative
to the effect of 2 in parallel, with values lower than 1 denoting compounds of lower potency than 2.
2 4 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 5 – 2 5 2

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be stronger than a typical salt bridge in an exposed aqueous
environment57 such as we see in the Ins(1,4,5)P3R. The crystal
structures of the NADP+-dependent aldehyde dehydrogenase,58
tropinone reductase I59 and 6-phosphogluconate dehydroge-
nase60 are of particular interest due to the structural similarities
of parts of the cofactor for these enzymes and adenophostin
A. All three examples show interactions of an arginine residue
with both the adenine moiety (cation–p) and the 2ꢀ-phosphate
group (from NADP+ for the first two and 2ꢀ-AMP for the
last), corresponding well with our current model. Earlier work
on ribophostin,61 an adenophostin analogue without the base,
had shown that the adenine moiety (or equivalent) is essential
for high potency with the ribophostin EC50 for Ca2+ release
increasing compared to that of Ins(1,4,5)P3. This suggests that
a synergistic effect of the 2ꢀ-phosphate group and the adenine
moiety interacting with Arg 504, combined with H-bonding
at N-3 from Arg 269, may be the reason for the 10-fold
increase in potency of adenophostin over Ins(1,4,5)P3. The
energy gain from these strong interactions should be more than
sufficient to overcome the weak energy barriers imposed upon
interconversion from a syn- to anti-conformation.51
The slight decrease in potency of etheno adenophostin 4
relative to 2 may be caused by the increase in electron density
of the base repelling the C-2ꢀ phosphate therefore disrupting its
optimal orientation in the receptor. A more likely explanation
is that the additional ring of the etheno analogue disrupts
the electrostatic potential of the base moiety which, in turn,
lowers the energy of the cation–p interaction. Studies concerning
these interactions have shown that aromatics with areas of high
electrostatic potential at their centre form the strongest cation–p
interactions and that electron rich heterocycles are quite poor
cation–p binders.62,63 However, docking experiments have shown
a virtually identical orientation of the etheno analogue (Fig. 6b)
when compared to adenophostin A which would allow cation–p
interactions with Arg 504. Fig. 6b illustrates the space available
around the proposed adenine binding pocket for occupation by
modified bases, such as etheno adenine.
The lower activity of 8-bromo adenophostin 5 relative to 2 was
not expected based on Hotoda’s study33 but corresponds well
with our model. However, an EC50 value higher than Ins(1,4,5)P3
(1) was surprising due to the requirement of an anti-conforma-
tion for high affinity binding and the steric restraints imposed to
prevent this. This suggests that the ligand–receptor complex
could be of sufficiently low energy to overcome the steric repul-
sion in the transition between the syn- and anti-conformations.
The energy required to overcome this transitional barrier
leads to a loss in potency, with 5 being approximately half
as active as adenophostin A. Similar findings have been
observed for ligand–receptor complexes of 8-BrADP64,65 and
8-BrNAD+,65 both ligands binding as the anti-conformer de-
spite being in the syn-conformation when unbound. Interest-
ingly, protein bound adenosine derivatives binding in the syn-
conformation have been found, however, there are only a few
reported cases.66–68
Fig. 7 Variation in fluorescence emission intensity of etheno adeno-
phostin 4 with pH in 0.1 M Hepes buffer. Excitation at 296 nm.
further applications in the synthesis of potential photoaffinity
analogues and other 8-modified adenophostin analogues. We
propose that two conformations of adenophostin A exist
dependent on whether in solution or bound to the Ins(1,4,5)P3R
with NMR and modelling data supporting this. We suspect that
the proposed cation–p interaction between the base moiety of
adenophostin and its analogues with Arg 504 is at least partly
responsible for their increased potency relative to Ins(1,4,5)P3
and this model warrants further investigation.
Experimental
General
Chemicals were purchased from Acros, Aldrich, Sigma and
Fluka. All anhydrous solvents were purchased from either
Aldrich or Fluka. RPMI 1640 medium, L-glutamine, 2-
mercaptoethanol and G-418 were from Invitrogen, sera were
from Sigma and Mag-fluo-4AM was from Molecular Probes.
TLC was performed on precoated plates (Merck aluminum
sheets silica 60 F254, Art. No. 5554). Products were visualised
under UV light and by dipping into phosphomolybdic acid in
MeOH followed by heating or by dipping into anisaldehyde in
EtOH followed by heating. Radial-band chromatography (RBC)
was performed using a Chromatotron (7924T, TC Research,
UK) with Adsorbosil Plus-P (6–15 lm) (Alltech) as the adsor-
bent. Ion exchange chromatography was performed on an LKB-
Pharmacia medium pressure ion-exchange chromatograph using
MP1 AG ion-exchange resin and a gradient of 0–100% 150 mM
TFA as eluent. 1H NMR, NOESY and 13C spectra were
recorded on JEOL JMN GX-270, EX-400 or Varian Inova-
600 NMR spectrometers. 31P NMR spectra were recorded
on JEOL JMN GX-270 or EX-400 NMR spectrometers and
chemical shifts were measured in ppm and denoted positive
downfield from external 85% H3PO4. All coupling constants
are quoted in Hz. Melting points were determined using a
Reichert-Jung Therm Galen Kofler block and are uncorrected.
Low resolution mass spectra were recorded at the University of
Bath Mass Spectrometry Service using +ve and −ve fast atom
bombardment (FAB) with m-nitrobenzyl alcohol as the matrix.
High resolution accurate mass spectra were recorded at the
University of Bath Mass Spectrometry Service. Optical rotations
were measured at ambient temperature using an Optical Activity
Ltd AA-10 polarimeter in a cell volume of 1 cm3 or 5 cm3 and
specific rotation are given in 10−1 deg cm3 g−1. Fluorescence
measurements were made on a Perkin Elmer LS 50B fluorimeter.
UV spectra were recorded using a Perkin Elmer Lambda EZ201
spectrometer. HPLC analysis was carried out on a Hewlett-
Packard series chromatograph with a strong anion-exchange
resin (MP1 AG, column size 3 × 150 mm). A linear gradient
of 0–50% 150 mM TFA was used as eluent at 1 cm3 min−1
Fluorescence properties
The fluorescence emission of etheno adenophostin in 0.1M
HEPES buffer at varing pH is shown in Fig. 7. The fluorescence
emission is greatest at pH 7.0, forming a single unresolved band
with a maximum at 412 nm, indicating that etheno adenophostin
could be a valuable tool for the investigation of Ins(1,4,5)P3Rs
at physiological pH.
Conclusions
We have synthesised two adenophostin analogues 4 and 5, modi-
fied at the adenine moiety. Etheno adenophostin 4 is a high affin-
ity fluorescent probe which could be used at physiological pH for
the investigation of Ins(1,4,5)P3Rs. 8-Bromo adenophostin 5 has
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 5 – 2 5 2
2 4 9

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over 60 min, with the UV detector set at 254 nm. Synthetic
phosphates were assayed using an adaptation of the Briggs
phosphate test.69
intracellular stores of permeabilized cells. DT40/Ins(1,4,5)P3R1
cells were harvested by centrifugation (650 × g; 2 min) and re-
suspended (2–3 × 107 cells ml−1) in Hepes-buffered saline (HBS:
135 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.5 mM CaCl2,
11.6 mM Hepes, 11.5 mM D-glucose, pH 7.3) supplemented with
Mag-fluo-4AM (20 lM), Pluronic F-127 (0.05%) and bovine
serum albumin (1 mg ml−1). After 1 h at 20 ◦C in the dark, the
Mag-fluo-4-loaded cells were harvested (650 × g; 2 min) and
re-suspended (ca. 2 × 106 cells ml−1) in Ca2+-free cytosolic-like
medium (CLM:140mM KCl, 20 mM NaCl, 2mM MgCl2, 1 mM
EGTA, 20 mM Pipes, pH 7.0). The cells were permeabilized by
incubation with saponin (10 lg ml−1, 4 min at 37 ◦C), harvested
(650 × g; 2 min) and resuspended in Mg2+-free CLM (140 mM
KCl, 20 mM NaCl, 1 mM EGTA, 375 lM CaCl2 (ca. 200 nM
free [Ca2+]), 20 mM Pipes, pH 7.0). The permeabilized cells
(with Mag-fluo-4 trapped within the lumen of the ER) were
then attached to 96-well plates (ca. 8 × 105 cells per well)
coated with poly-L-lysine (0.01%) and centrifuged onto the
plate (300 × g; 2 min). Immediately before an experiment, the
cells were washed twice in Mg2+-free CLM to remove cytosolic
Mag-fluo-4 and the plates were then mounted in a Flexstation
fluorescence plate reader (Molecular Devices, Sunnyvale, CA),
which allows automated additions to the sample wells while
recording fluorescence. Mag-fluo-4 fluorescence was monitored
by excitation at 485 nm with emission detected at 520 nm. Active
Ca2+ uptake into the ER was initiated by addition of Mg2+-ATP
(1.5 mM) and after 150 s, when the stores had loaded to a
steady-state Ca2+ content, Ins(1,4,5)P3 was added. The amount
of Ca2+ released by Ins(1,4,5)P3 was expressed as a fraction of
the total Ca2+ content of the ER as assessed by addition of 1 lM
ionomycin. Data are presented as means s.e. means from n
independent experiments, each performed in triplicate.
Molecular modelling experiments
Initially, force field modelling and stochastic conformational
searching with MOE (2003.2) did not find global minimum
structures that could easily be reconciled with the NOE and
1H NMR data. Ab initio modelling with Gaussian 98-RevA.6,
running on a SGI Octane2, IRIX 6.54 was attempted with
HF/3–21G*, B3LYP/3–21G* and CPCM/B3LYP/6–31+G(d)
but failed to find satisfactory minima structures with proton
steric clashes in accordance with the NOE data. Modelling the
phosphate anion moieties with SCRF continuum models may
result in an energetic conflict between electrostatic repulsions
between phosphate anion and the tendency of SCRF envelopes
to minimise the volume of solvent cavities.
Favourable results were obtained when explicit water
molecules were included. Using MOE, Linux RedHat10, the
structures were drawn and dihedral angles adjusted to reflect
1
˚
the NOE and H NMR data. A 6 A spherical water envelope
was then placed around the solutes with the water soak facility in
MOE. The solutes were fixed and the water molecules minimised
˚
with MMFF94 with non bonding cut-off adjusted to 12 A. Once
complete, the structures were minimised using Mopac2002,
implemented in Cache-Pro6.11 running windows98rev2, on a
Desktop (Dell) PC without constraints. The semi-empirical
method PM5 was used as implemented in Mopac2002. Extra
keywords were used in Mopac2002 to enable reasonable con-
vergence, PRECISE, LET DDMIN = 0.00 GEO-OK, GRAD,
GNORM = 0.1. Cosmo solvation was not used and the clusters
were simply treated as gas phase entities. Minimised solute
structures are shown with explicit solvent molecules hidden.
Concentration–effect relationships were fitted to four-
parameter logistic equations using non-linear curve-fitting pro-
cedures (GraphPad Prism, San Diego, CA).
Stable transfection of DT40 cells with rat Ins(1,4,5)P3R1
2ꢀꢀ,5ꢀ,6ꢀꢀ-Tri-O-benzyl-3ꢀ-O-a-D-glucopyranosyl adenosine (7).
A mixture of 2ꢀ,3ꢀꢀ,4ꢀꢀ-tri-O-acetyl-2ꢀꢀ,5ꢀ,6ꢀꢀ-tri-O-benzyl-3ꢀ-O-a-D-
glucopyranosyl-6-chloro-9-b-D-ribofuranosidopurine 6 (44 mg,
52.1 lmol) in ethanolic ammonia (3 cm3) was heated at 60 ◦C in
a pressure tube for 72 h. The mixture was concentrated under re-
duced pressure and was purified by radial band chromatography
(RBC) (ethyl acetate → ethyl acetate–methanol 14 : 1) to give
the title compound 7 (33 mg, 92%). mp 93–97 ◦C (from ethanol)
(lit.16 mp 95–115 ◦C); [a]D22 5.7 (c = 1.06, CHCl3) (lit.16 [a]2D5 −2.3
The open reading frame of rat Ins(1,4,5)P3R1 was amplified
by PCR from the expression vector pCMVI-9-Ins(1,4,5)P3R170
using the following primers: 5ꢀ–AGGAATTCG-CCACCATG-
TCTGACAAAATG-3ꢀ and 5ꢀ–CCGGTACCG-AATTCTTA-
GGCTGGCTGCTGT-3ꢀ and cloned as an EcoRI fragment into
pcDNA3 (Invitrogen). The chicken b-actin hybrid promoter71
was excised from the vector pAneo72 and cloned in place of the
CMV promoter upstream of the Ins(1,4,5)P3R1 open reading
frame to create the construct pcDNA3-Ins(1,4,5)P3R1. DT40
cells in which the genes for all three endogenous Ins(1,4,5)P3R
subtypes have been deleted (DT40/InsP3R–KO)73 were sta-
bly transfected by electroporation with linearized pcDNA3-
Ins(1,4,5)P3R1 using a Gene Pulser apparatus (Bio-Rad
Laboratories) at 330 V, 500 lF with 5 lg DNA/106 cells. Clonal
isolation was carried out in the presence of 2 mg ml−1 G-418
and positive clones were amplified and screened for the presence
of rat Ins(1,4,5)P3R1 by western blotting using an anti-peptide
antiserum74 corresponding to the C-terminal 15-residues of rat
Ins(1,4,5)P3R1.
(c 2.6, CHCl3)); dH(270 MHz; CDCl3; Me4Si) 3.43 (1 H, dd, J1ꢀꢀ ꢀꢀ
,2
3.5, J2ꢀꢀ ꢀꢀ 9.9, H-2ꢀꢀ), 3.52–3.78 (5 H, m, H-5ꢀA, H-5ꢀB, H-4ꢀꢀ, H-
,3
6ꢀꢀA, H-6ꢀꢀ ), 3.86–3.96 (1 H, m, H-5ꢀꢀ), 4.09 (1 H, t, J2ꢀꢀ ꢀꢀ = J3ꢀꢀ ꢀꢀ
B
,3
,4
9.9, H-3ꢀꢀ), 4.24–4.30 (1 H, m, H-3ꢀ), 4.35–4.40 (1 H, m, H-4ꢀ),
4.45–4.56 (4 H, m, 2 × OCH2Ar), 4.59–4.83 (3 H, m, OCH2Ar,
H-2ꢀ), 4.90 (1H, d, J1ꢀꢀ ꢀꢀ 3.5, H-1ꢀꢀ), 6.10 (1 H, d, J1 ,2 6.7, H-1ꢀ),
ꢀ
ꢀ
,2
6.34 (2 H, s, NH2), 7.21–7.39 (15 H, m, ArH), 8.05, 8.26 (2 H,
2 s, H-2, H-8); dC(100.5 MHz; CDCl3; Me4Si) 69.85 (C-6ꢀꢀ), 70.19
(C-5ꢀ and C-4ꢀꢀ), 72.58 (C-5ꢀꢀ), 72.98 (C-3ꢀꢀ), 73.90, 73.93, 74.30
(3 × OCH2Ar), 75.93 (C-2ꢀ), 79.64 (C-2ꢀꢀ), 80.28 (C-3ꢀ), 83.21
(C-4ꢀ), 87.97 (C-1ꢀ), 99.77 (C-1ꢀꢀ), 119.47 (C-5), 127.84, 127.97,
128.17, 128.54, 128.61, 128.78, 128.83, 128.96 (ArCH), 137.04,
137.65, 138.17 (3 × ipso-C of Bn ring), 139.04 (C-8), 149.62
(C-4), 152.90 (C-2), 155.68 (C-6); m/z (FAB) 700.1 [(M + H)+,
70%]; (mass calcd for C37H42N5O9 (M + H)+, 700.29826; found
700.29749.
2ꢀꢀ,5ꢀ,6ꢀꢀ-Tri-O-benzyl-2ꢀ,3ꢀꢀ,4ꢀꢀ-tris(dibenzyloxyphosphoryl)-3ꢀ-
O-a-D-glucopyranosyl adenosine (8). A mixture of the triol 7
(382 mg, 0.55 mmol), bis(benzyloxy) (diisopropylamino)-
phosphine (631 mg, 1.83 mmol) and imidazolium triflate39
(393 mg, 1.82 mmol) in anhydrous DCM (4 cm3) was stirred
at room temperature under N2 for 90 min. TLC (ethyl
acetate–hexane, 7 : 3) indicated the complete conversion to the
trisphosphite so water (2 drops) was added and the reaction
was cooled to −78 ◦C. mCPBA (331 mg, 1.93 mmol) was
Cell culture
DT40/Ins(1,4,5)P3R–KO cells stably expressing recombinant
rat InsP3R1 (DT40/Ins(1,4,5)P3R1 cells) were cultured in sus-
pension in RPMI 1640 medium supplemented with foetal bovine
serum (10%), L-glutamine (2 mM), 2-mercaptoethanol (50 lM)
and heat-inactivated chicken serum (1%). Cells were incubated
in a humidified atmosphere (95% O2; 5% CO2 at 37 ◦C) and
passaged every 2–3 days when they had reached a density of ca.
2 × 106 cells ml−1.
Measurement of Ca2+ release from permeabilized cells
The effects of Ins(1,4,5)P3 on intracellular Ca2+ stores were
measured using a low-affinity Ca2+-indicator trapped within the
2 5 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 5 – 2 5 2

View Article Online
added, followed after 10 min by 10% aq. Na2SO3 (10 cm3) and
ethyl acetate (20 cm3). The reaction was allowed to attain room
temperature with the organic phase being washed consecutively
with sat. NaHCO3 (20 cm3) and brine (20 cm3), dried (MgSO4)
and concentrated under reduced pressure. Purification by RBC
(CHCl3-acetone 4 : 1 → 6:4) furnished the fully protected
trisphosphate 8 (431 mg, 53%). dH(270 MHz; CDCl3; Me4Si)
121.88 (C-11), 137.76 (C-8, C-2), 143.05 (C-4), 144.59 (C-6);
dP(109.2 MHz; D2O; 1H decoupled) −0.17, 0.39, 0.77 (3 s); m/z
(FAB) 694.0 [(M + H)+, 75%]; mass calcd for C18H27N5O18P3
(M + H)+, 694.05640; found 694.05368.
8-Bromo-3ꢀ-O-a-D-glucopyranosyl adenosine 2ꢀ, 3ꢀꢀ,4ꢀꢀ-trisphos-
phate (5). To a stirred solution of adenophostin A 2 (22 mg,
32.9 lmol) in 0.5 M sodium acetate buffer (1 cm3, pH 4.3) was
added a solution of Br2 (23 mg, 0.14 mmol) in 0.5 M sodium
acetate buffer (2 cm3, pH 4.3) dropwise. The reaction mixture
was stirred in the dark at room temperature for 60 h monitoring
the pH occasionally and adding additional buffer if necessary.
The solution was partitioned between CHCl3 (5 cm3) and water
with the aqueous layer being treated with solid NaHSO3 to
remove excess Br2. The aqueous layer was further extracted with
CHCl3 (3 × 5 cm3) and concentrated under reduced pressure.
Purification using an MP1 AG ion-exchange column eluting
with 0–100% 150 mM TFA (elution between 40–70%) gave the
desired compound 5 (9.6 mg, 41%). dH(400 MHz; D2O) 3.58–
3.50–3.70 (5 H, m, H-5ꢀA, H-5ꢀB, H-2ꢀꢀ, H-6ꢀꢀA, H-6ꢀꢀ ), 3.79–3.88
B
(1 H, m, H-5ꢀꢀ), 4.12 (1 H, AB, JAB 7.3, 0.5 × OCH2Ar)
4.26–4.86 (14 H, m, 5.5 × OCH2Ar, H-3ꢀ, H-4ꢀ, H-4ꢀꢀ), 4.87–5.06
(8 H, m, 3.5 ×; OCH2Ar, H-3ꢀꢀ), 5.32 (1H, d, J1ꢀꢀ ꢀꢀ 3.7, H-1ꢀꢀ),
,2
5.56–5.66 (1 H, m, H-2ꢀ), 5.70 (2 H, br s, NH2), 6.34 (1 H, d, J1 ,2
ꢀ
ꢀ
6.6, H-1ꢀ), 6.94–7.41 (45 H, m, ArH), 7.90, 8.25 (2 H, 2 s, H-8,
H-2); dC(100.5 MHz; CDCl3; Me4Si) 68.64 (C-6ꢀꢀ), 69.44–70.41
(C-5ꢀ, 6 × POCH2Ar with C–P coupling), 70.16 (C-5ꢀꢀ), 71.91,
73.66, 73.90 (3 × OCH2Ar), 74.10 (C-4ꢀꢀ), 74.68 (C-3ꢀ), 77.04
(C-2ꢀꢀ), 77.55 (C-2ꢀ with C–P coupling), 78.39 (C-3ꢀꢀ with C–P
coupling), 82.72 (C-4ꢀ), 85.89 (C-1ꢀ), 95.67 (C-1ꢀꢀ), 119.98 (C-5),
127.15, 127.81, 127.89, 127.93, 127.96, 128.04, 128.16, 128.56,
128.49, 128.51, 128.62, 128.65, 128.69, 128.74, 128.80 (ArCH),
135.23–136.37 (6 × ipso-C of benzylphospho ring with C–P
coupling) 137.52, 137.77, 138.18 (3 × ipso-C of Bn ring), 139.64
(C-8), 150.25 (C-4), 153.11 (C-2), 155.33 (C-6); dP(161.8 MHz;
3.78 (6 H, m, H-2ꢀꢀ, H-5ꢀꢀ, H-5ꢀA, H-5ꢀB, H-6ꢀꢀA, H-6ꢀꢀ ), 3.83 (1 H,
B
q, J3ꢀꢀ ꢀꢀ = J4ꢀꢀ ꢀꢀ 9.4, H-4ꢀꢀ), 4.32 (2 H, m, H-4ꢀ, H-3ꢀꢀ), 4.53 (1 H, m,
,4
,5
J3 ,4
ꢀ
ꢀ
5.3, H-3ꢀ), 5.26 (1 H, d, J1ꢀꢀ ꢀꢀ 3.5, H-1ꢀꢀ), 5.30 (1 H, m, H-2ꢀ),
,2
6.16 (1 H, d, J1 ,2
7.4, H-1ꢀ), 8.01 (1 H, s, H-2); dC(150.8 MHz;
ꢀ
ꢀ
D2O) 60.41 (C-6ꢀꢀ), 62.00 (C-5ꢀ), 71.32 (C-2ꢀꢀ), 72.16 (C-4ꢀꢀ) 72.30
(C-5ꢀꢀ), 73.67 (C-3ꢀ), 74.37 (C-2ꢀ with C–P coupling), 77.187 (C-
3ꢀꢀ with C–P coupling), 85.56 (C-4ꢀ), 89.46 (C-1ꢀ), 98.12 (C-1ꢀꢀ),
119.89 (C-5), 128.86 (C-8), 149.75 (C-4), 152.39 (C-2), 154.68
1
CDCl3; H decoupled) −0.13, −0.86, −0.01 (3 s); m/z (FAB)
1480.4 [(M + H)+, 90%]; mass calcd for C79H81N5O18P3 (M +
H)+, 1480.47895; found 1480.47537.
3ꢀ-O-a-D-Glucopyranosyl adenosine 2ꢀ,3ꢀꢀ,4ꢀꢀ-trisphosphate
(adenophostin A) (2). A mixture of 8 (256 mg, 0.17 mmol)
and moist 20% Pd(OH)2 on carbon (ca. 300 mg) in methanol
(10.5 cm3), cyclohexene (5.5 cm3) and milliQ water (0.75 cm3)
1
(C-6); dP(161.8 MHz; D2O; H decoupled) 3.67 and 2.54 (2 ×
brs integrating as 1 : 2 respectively); m/z (FAB) 746.0, 748.0
[(M–H)−, 45%]; mass calcd for C16H2479BrN5O18P3 (M–H)−,
745.95126 and C16H2481BrN5O18P3 (M–H)−, 747.94922; found
745.94998 and 747.94975.
◦
was heated at 80 C for 4 h. The mixture was passed through
a membrane filter to remove the catalyst washing well with
MeOH and water. The filtrate was concentrated under reduced
pressure with the resulting residue being purified on an MP1 AG
ion-exchange column eluting with 0–100% 150 mM TFA. The
relevant fractions (elution between 55–85%) were concentrated
to give adenophostin A 2 (105 mg, 90%) as the free acid.
dH(270 MHz; D2O) 3.59–3.77 (6 H, m, H-2ꢀꢀ, H-5ꢀꢀ, H-5ꢀA,
H-5ꢀB, H-6ꢀꢀA, H-6ꢀꢀ ), 3.99 (1 H, q, J3ꢀꢀ ꢀꢀ = J4ꢀꢀ ꢀꢀ 9.5, H-4ꢀꢀ), 4.28
Acknowledgements
We thank the Wellcome Trust for Programme Grant Support
(060554 to B.V.L.P. and 039662 to C.W.T.). We also thank Mr
James J. Robinson for useful discussions on molecular modelling
and Dr Andrew M. Riley, University of Bath, UK, for his
invaluable advice on all aspects of this work.
B
,4
,5
(1 H, m, J3 ,4
ꢀ
ꢀ
3.3, H-4ꢀ), 4.39 (1 H, q, J2ꢀꢀ ꢀꢀ = J3ꢀꢀ ꢀꢀ 9.5, H-3ꢀꢀ) 4.50
,3
,4
(1 H, m, H-3ꢀ), 5.13 (1 H, d, J = 3.7 Hz, H-1ꢀꢀ), 5.16 (1 H, m,
References
H-2ꢀ), 6.20 (1 H, d, J1 ,2 6.2, H-1ꢀ), 8.25, 8.35 (2 H, 2 s, H-8,
ꢀ
ꢀ
H-2); dC(150.8 MHz; D2O) 60.25 (C-6ꢀꢀ), 61.10 (C-5ꢀ), 70.50
(C-2ꢀꢀ), 71.61 (C-5ꢀꢀ), 73.13 (C-4ꢀꢀ), 73.99 (C-3ꢀ), 75.94 (C-2ꢀ with
C–P coupling), 78.14 (C-3ꢀꢀ with C–P coupling), 84.56 (C-4ꢀ),
87.24 (C-1ꢀ), 98.30 (C-1ꢀꢀ), 119.08 (C-5), 143.50 (C-8), 144.62
(C-2), 148.39 (C-4), 150.03 (C-6); dP(161.8 MHz; D2O; 1H
decoupled) 0.25, 0.81, 1.17 (3 s); m/z (FAB) 668.1 [(M–H)−,
100%]; mass calcd for C16H27N5O18P3 (M + H)+, 670.05585;
found 670.05635.
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(C-4ꢀ), 87.67 (C-1ꢀ), 98.15 (C-1ꢀꢀ), 114.38 (C-10). 119.08 (C-5),
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 5 – 2 5 2
2 5 1

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