Contribution of phosphates and adenine to the potency of adenophostins at the IP3 receptor: Synthesis of all possible bisphosphates of adenophostin A

Article abstract of DOI:10.1021/jm201571p

Although adenophostin A (AdA), the most potent agonist of d-myo-inositol 1,4,5-trisphosphate receptors (IP₃R), is thought to mimic IP ₃, the relative roles of the different phosphate groups and the adenosine motif have not been established. We synthesized all three possible bisphosphate analogues of AdA and glucose 3,4-bisphosphate (7, AdA lacking the 2′-AMP). 2′-Dephospho-AdA (6) was prepared via a novel regioselective dephosphorylation strategy. Assessment of the abilities of these bisphosphates to stimulate intracellular Ca²⁺ release using recombinant rat type 1 IP₃R (IP₃R1) revealed that 6, a mimic of Ins(4,5)P₂, is only 4-fold less potent than IP₃, while 7 is some 400-fold weaker and even 3′3-dephospho-AdA (5) is measurably active, despite missing one of the vicinal bisphosphate groups normally thought to be crucial for IP₃-like activity. Compound 6 is the most potent bisphosphate yet discovered with activity at IP₃R. Thus, adenosine has a direct role independent of the 2′-phosphate group in contributing toward the potency of adenophostins, the vicinal bisphosphate motif is not essential for activity at the IP₃R, as always thought, and it is possible to design potent agonists with just two of the three phosphates. A model with a possible adenine-R504 interaction supports the activity of 5 and 6 and also allows a reappraisal of the unexpected activity previously reported for the AdA regioisomer 2′3-phospho-3′3- dephospho-AdA 40.

Full text of DOI:10.1021/jm201571p

Article
pubs.acs.org/jmc
Contribution of Phosphates and Adenine to the Potency of
Adenophostins at the IP3 Receptor: Synthesis of All Possible
Bisphosphates of Adenophostin A
Kana M. Sureshan,†,§ Andrew M. Riley,† Mark P. Thomas,† Stephen C. Tovey,‡ Colin W. Taylor,‡
and Barry V. L. Potter*,†
†Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down,
Bath BA2 7AY, United Kingdom
‡Department of Pharmacology, Tennis Court Road, University of Cambridge, CB2 1PD, United Kingdom
S
* Supporting Information
ABSTRACT: Although adenophostin A (AdA), the most potent agonist of D-myo-
inositol 1,4,5-trisphosphate receptors (IP3R), is thought to mimic IP3, the relative
roles of the different phosphate groups and the adenosine motif have not been
established. We synthesized all three possible bisphosphate analogues of AdA and
glucose 3,4-bisphosphate (7, AdA lacking the 2′-AMP). 2′-Dephospho-AdA (6) was
prepared via a novel regioselective dephosphorylation strategy. Assessment of the
abilities of these bisphosphates to stimulate intracellular Ca2+ release using
recombinant rat type 1 IP3R (IP3R1) revealed that 6, a mimic of Ins(4,5)P2, is only
4-fold less potent than IP3, while 7 is some 400-fold weaker and even 3″-
dephospho-AdA (5) is measurably active, despite missing one of the vicinal
bisphosphate groups normally thought to be crucial for IP3-like activity. Compound
6 is the most potent bisphosphate yet discovered with activity at IP3R. Thus,
adenosine has a direct role independent of the 2′-phosphate group in contributing
toward the potency of adenophostins, the vicinal bisphosphate motif is not essential
for activity at the IP3R, as always thought, and it is possible to design potent agonists with just two of the three phosphates. A
model with a possible adenine−R504 interaction supports the activity of 5 and 6 and also allows a reappraisal of the unexpected
activity previously reported for the AdA regioisomer 2″-phospho-3″-dephospho-AdA 40.
release.6 This finding has stimulated many syntheses of various
INTRODUCTION
■
analogues of the adenophostins and studies of their SARs.7−10
SAR studies with synthetic analogues of adenophostin A
(AdA) with and without a purine ring established that the
presence of the adenine ring (or an aromatic surrogate) is
crucial for enhanced affinity.10 This suggests that the adenosine
moiety either disposes the 2′-phosphate in a special spatial
arrangement to strengthen its interactions with the receptor
(indirect role)11 or that the adenine moiety itself is involved in
supplementary binding interactions with a nearby region of the
binding site (direct role).12 It has been shown that the glucose
2″-OH group (analogous to 6-OH in IP3) is important, while
the glucose 5″-CH2OH (analogous to the 3-OH in IP3) and
ribose 4′-CH2OH are less important for AdA activity.13,14 The
roles of the phosphate groups of IP3 seem to be established,4,14
but their relative importance in the adenophostins has not been
examined systematically using synthetic chemistry. We have
proposed a model for AdA binding to the IP3-binding core
(IBC) of the receptor in which the glucose 2″-OH and 3″,4″-
bisphosphate triad mimic the 6-OH and 4,5-bisphosphate triad
D-myo-Inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+
channels located on the endoplasmic reticulum.1 IP3 [Ins-
(1,4,5)P3, 1] is a second messenger produced by the action of
phospholipase C on phosphatidylinositol 4,5-bisphosphate in
response to various extracellular signals. IP3 binds to its
receptor and opens its intrinsic Ca2+ channel, allowing Ca2+ to
leak into the cytosol and so cause the increase in cytosolic
[Ca2+] that regulates many cellular events.2 To study the
interaction of 1 with its receptor and to understand structure−
activity relationships (SARs), many synthetic analogues of IP3
have been synthesized.3 These studies revealed that the 4,5-
bisphosphate functionality and 6-OH group are apparently
crucial for activity, while the 1-phosphate has a supplementary
role, and the 2-OH and 3-OH groups are much less important.4
Although the relative importance of all of the hydroxyl and
phosphate groups has been established, none of the synthetic
analogues has proven to be more potent than the natural ligand.
In 1993, two potent agonists of IP3 receptors, adenophostin A
(3a, Figure 1) and adenophostin B (3b), were isolated from
culture broths of Penicillium brevicompactum.5 Both 3a and 3b
bind to IP3 receptors with much greater affinity than IP3 and
are 10−100 times more potent than IP3 in evoking Ca2+
Received: November 22, 2011
Published: January 16, 2012
© 2012 American Chemical Society
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Figure 1. Structures of IP3 (1), Ins(4,5)P2 (2), and adenophostins (3a and 3b).
Figure 2. Bisphosphate analogues of AdA.
a
Scheme 1. Synthesis of 4″-Dephospho-adenophostin A (4)
a
Reagents and conditions: (a) BnBr, NaH, DMF, 0 °C−room temperature, 2 h. (b) 80% aqueous HOAc, reflux, 1 h. (c) Bu2SnO, BnBr, Bu4NBr,
MeCN, 3 Å molecular sieves, reflux, 24 h. (d) Bu4NHSO4, BnBr, DCM: 5% aqueous NaOH (1:1), room temperature. (e) Ac2O, pyr, room
temperature, 12 h. (f) Compound 16, NIS, TMSOTf, dioxane:toluene (3:1 v/v), 3 Å molecular sieves, room temperature, 30 min. (g) 90% TFA,
room temperature, 15 min. (h) Ac2O, pyr, room temperature, 3 h. (i) 6-Chloropurine, BSA, TMSOTf, MeCN, 70 °C, overnight. (j) NH3, EtOH, 70
°C, 5 days. (k) (1) (BnO)2PN(iPr)2, ImOTf, DCM, room temperature, 30 min; (2) mCPBA, −78 °C−room temperature, 1 h. (l) Cyclohexene,
Pd(OH)2, MeOH, H2O, 80 °C, overnight.
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a
Scheme 2. Synthesis of 3″-Dephospho-adenophostin A (5)
a
Reagents and conditions: (a) MeOH, NaOMe, room temperature, 30 min. (b) Dowex-H+. (c) PhCH(OMe)2, CSA, DMF, 70 °C. (d) BnBr, NaH,
DMF, room temperature, 2 h. (e) TFA, Et3SiH, DCM, 0 °C. (f) Ac2O, pyr, room temperature, 3 h. (g) Compound 16, NIS, TMSOTf,
dioxane:toluene, 3 Å molecular sieves, room temperature, 30 min. (h) 90% TFA, room temperature, 10 min. (i) 6-Chloropurine, BSA, TMSOTf,
MeCN, 70 °C, overnight. (j) NH3, EtOH, 74 °C, 5 days. (k) (1) (BnO)2PN(iPr)2, ImOTf, DCM, room temperature, 30 min; (2) mCPBA, −78
°C−room temperature, 1 h. (l) Cyclohexene, Pd(OH)2, MeOH, H2O, 80 °C, overnight.
of IP3, while the adenine engages in a cation-π interaction with
Arg504 adjacent to the binding site. In this model, AdA takes
on a 2′-endo anti extended binding conformation.7 In addition,
the ribose 2′-phosphate may have a stabilizing helix−dipole
interaction with the N terminus of an α-helix in the IBC.15 We
recently reviewed aspects of the chemistry and biology of AdA
in its interaction with IP3R16 and examined the thermody-
namics of both IP3 and AdA binding to both the N terminus
and IBC of the IP3R.17 AdA binds to both proteins with
significantly higher affinity than does IP3.
Bisphosphate 6 (Figure 2) was previously made by enzymatic
hydrolysis of AdA using alkaline phosphatase and was reported
to be 1800 times weaker than AdA.18 This implied a critical role
for the 2′-phosphate group and led some workers to conclude
that the adenine had only an indirect role, by enhancing the
positioning of the 2′-phosphate group. However, this finding
seems incompatible with our suggestion based on detailed SAR
and molecular modeling7,19 that the adenine may form its own
interactions with the IP3R. Furthermore, inframolecular
protonation studies of AdA suggest that, at least in solution,
the 2′-P is close to the glucose ring and the three phosphate
groups behave similarly to those in IP3.20 Hence, we were
curious to re-examine the biological activity of 6 with a
chemically synthesized sample and to compare its activity with
a minimal motif of AdA, glucose 3,4-bisphosphate
[Gluc(3,4)P2, 7]. Quantitative insight into the contribution of
the adenosine moiety might be obtained by comparison of the
activities of 6 and 7, which differ only by the presence of the
adenosine moiety.
report the synthesis and biological evaluation of all three
possible bisphosphates of AdA. A preliminary communication
on this work has appeared,21 and we recently reported the
ability of 6 to evoke Ca2+ release via recombinant and mutant
IP3Rs.19
RESULTS AND DISCUSSION
■
For the synthesis of adenophostin analogues, an ideal and
economical strategy would be the Vorbruggen condensation of
a silylated purine with an appropriately protected disaccharide,
followed by deprotection of hydroxyl groups (to be later
phosphorylated), phosphorylation, and final deprotection.22,23
It is a prerequisite to have ester functionalities at 1-O- and 2-O-
̈
positions of the disaccharide for the Vorbruggen condensa-
̈
tion.24 Other hydroxyl groups on the disaccharide have to be
protected with orthogonal protecting groups, such that the
hydroxyls to be phosphorylated are protected with an easily
removable temporary protecting group and the other hydroxyls
are protected with a stable protecting group that can be cleaved
at the final step. Because the nucleoside after Vorbruggen
̈
condensation will have an ester protecting group at the 2′-O-
position (which has to be phosphorylated in 4 and 5) and the
fact that this ester can be removed under milder conditions, it is
ideal to protect the other hydroxyl to be phosphorylated as a
similar ester. Benzyl is the protecting group of choice for all
other hydroxyl groups as it is stable to various conditions and
also due to the convenience in its deprotection along with other
benzyl protecting groups on phosphate triesters in a single final
step.
Thus, to explore our model further and to investigate
whether the extra binding motif of AdA as compared to IP3
might compensate for removal of a phosphate interaction, we
The synthesis of 4 started from the cheaply available penta-
O-acetyl-β-D-glucose (8, Scheme 1). The glycosyl donor 15 was
prepared from 8 by a series of protecting group manipulations.
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Figure 3. Base-mediated dephosphorylation.
Figure 4. Various possible transesterification mechanisms for phosphate triesters. Only the first step is shown. The intermediates can undergo further
transesterification.
The butane 2,3-diacetal (BDA) derivative 9 was obtained from
8 as reported previously.25 Diol 9 on benzylation gave the
dibenzyl ether 10 in excellent yield. The acid-labile BDA
protecting group was removed by hydrolysis with 80% acetic
acid. Various conditions were tried to achieve selective
benzylation at the 2-OH in 11. Stannylene-mediated
benzylation was sluggish and gave a 1:1 mixture of isomeric
tribenzyl ethers 12 (27%) and 13 (23%) along with unreacted
11 (36%). Sodium hydride-mediated benzylation also gave a
1:1 mixture of tribenzyl ethers 12 (18%) and 13 (16%) in very
low yield along with fully benzylated 14 (32%) and starting
material 11 (24%). Fortunately, benzylation under biphasic
conditions with tetrabutylammonium hydrogen sulfate as phase
transfer catalyst gave predominantly the tribenzyl ether 12
(74%) along with minor amounts of 13 (8%) and 14 (10%).
Acetylation of 12 provided the acetate 15 in 95% yield. N-
Iodosuccinimide (NIS)-mediated glycosylation of 5-O-benzyl-
1,2-O-isopropylidene-α-D-ribofuranose (16), which was pre-
pared from 1,2-O-isopropylidene-α-D-xylofuranose by a known
procedure26 with glycosyl donor 15, gave the α-glycoside 17
selectively. No detectable amount of the β-isomer was obtained.
Such a high degree of selectivity has been observed before
when structurally similar compounds were used.25,27,28 To
then selectively phosphitylated with dibenzyl N,N-diisopropyl
phosphoramidite in the presence of imidazolium triflate as
catalyst,29 and the resulting bisphosphite was oxidized in situ to
bisphosphate 21. Finally, all of the benzyl protecting groups
(on both the phosphate and the sugar) were removed by
transfer hydrogenolysis30 to provide the bisphosphate 4, which
was purified by ion exchange chromatography.
Synthesis of bisphosphate 5 started with commercially
available tetra-acetate 22 (Scheme 2). The acetate protecting
groups on 22 were removed by methanolysis with a catalytic
amount of sodium methoxide in methanol, and the resulting
crude tetrol was converted into diol 23 by benzylidenation
using benzaldehyde dimethyl acetal and camphorsulphonic acid
(CSA). Crude 23 was then benzylated with an excess of benzyl
bromide and sodium hydride to afford the dibenzyl ether 24.
Reductive opening of the benzylidene acetal by following a
known procedure31 gave the tribenzyl ether 2532 selectively.
Glycosylation of ribose derivative 16 with the glycosyl donor
26, obtained by acetylation of the tribenzyl ether 25, gave α-
glycoside 27 selectively. As in the previous case, no detectable
amount of β-isomer was obtained. Disaccharide 27 was
converted into the triacetate 28 (α- and β-anomer) in a one
pot reaction of hydrolysis of acetonide with TFA followed by
make the disaccharide donor for Vorbruggen glycosylation, the
acetylation. Vorbruggen condensation with 6-chloropurine gave
̈
̈
isopropylidene group in 17 was hydrolyzed with 90%
trifluoroacetic acid (TFA), and the resulting mixture of diols
(anomeric mixture) was acetylated under standard conditions
to produce a chromatographically inseparable α/β mixture of
the nucleoside 29, which on ammonolysis provided the diol 30.
Chemoselective phosphitylation followed by in situ oxidation
afforded the fully protected bisphosphate 31. Transfer
hydrogenolysis removed all of the benzyl protecting groups
to provide the bisphosphate 5, which was purified by ion
exchange chromatography.
triacetates 18. Vorbruggen condensation of 18 with silylated 6-
̈
chloropurine gave the β-nucleoside 19 as the exclusive product.
The yield of the condensation depended on the nature of the
silylating agents used for the persilylation of purine. Among the
various silylating agents used, N,O-bis-trimethylsilyl-acetamide
(BSA) gave a very good yield for the subsequent same pot
glycosylation. Removal of the acetate protecting groups in
nucleoside 19 and substitution of chlorine with an amino group
were achieved concomitantly by ammonolysis. The diol 20 was
The requirement that the 1-O- and 2-O-positions of the
disaccharide should be protected as esters for Vorbruggen
̈
condensation leaves an ester group at the 2′-O-position of
nucleosides 19 and 29. With the next step being the
substitution of 6-Cl with NH2 and deprotection of the hydroxyl
groups to be phosphorylated, the ester functionality at 2′-O-
position is advantageous, as treatment with NH3 effects both of
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a
Scheme 3. Synthesis of 2′-Dephospho-adenophostin A (6)
a
Reagents and conditions: (a) BnOH, NaH, room temperature, 30 min. (b) BnOH, K2CO3, 70 °C, overnight. (c) Pd(OH)2, cyclohexene, MeOH,
H2O, 80 °C, overnight. (d) R−NH2 (R = H, Bu, Pr), EtOH, room temperature.
a
Scheme 4. Synthesis of Glucose 3,4-Bisphosphate (7)
a
Reagents and conditions: (a) (1) (BnO)2PN(iPr)2, 1H-tetrazole, DCM, room temperature, 1 h; (2) mCPBA, −78 °C, 10 min, 92%. (b) PdCl2,
MeOH, 81%. (c) H2, Pd(OH)2, MeOH, H2O, 85%.
the transformations. The other hydroxyl group to be
phosphorylated (3″ in 18 and 4″ in 28) was also protected as
its acetate for convenient one-step deprotection. This strategy
offered an economical synthesis of 4 and 5. However, it cannot
be applied to the synthesis of bisphosphate 6 as there is no
phosphate at the 2′-O-position. This prompted us to explore
other possibilities.
in situ-generated sodium benzoxide [benzyl alcohol (5 equiv)
and NaH (2 equiv)] in N,N-dimethylformamide (DMF) at
room temperature for 30 min gave a bisphosphate as major
product with minor amounts of other byproducts. Fortunately,
the use of the milder base potassium carbonate and BnOH as
solvent resulted in the exclusive formation of this bisphosphate
in excellent yield. A detailed spectral analysis revealed that the
bisphosphate is the expected 34. The fact that the 2′-phosphate
is comparatively less crowded and hence more prone to
nucleophilic attack than those in the 3″,4″ vicinal bisphosphate
functionality could be the reason for this high selectivity. This
assumption is supported by the fact that triacetate 35 on
aminolysis with various amines gives the 2′-O-deacetylated
derivative 36 exclusively. Finally, the benzyl protecting groups
on both sugar and phosphorus were removed by transfer
hydrogenolysis and purification of the product by ion exchange
column chromatography provided bisphosphate 6 quantita-
tively.
During our synthesis of the AdA analogue, guanophostin,15
several attempts to substitute the chlorine in the intermediate
32 with different oxygen nucleophiles under basic conditions
[4 M NaOH, dioxane; 3-hydroxypropiononitrile, 1,8-diaza-
bicycloundec-7-ene (DBU), dichloromethane (DCM);33 3-
hydroxypropiononitrile, NaH, tetrahydrofuran (THF); BnOH,
K2CO334] were marred by accompanied dephosphorylation.
For instance, when 3-hydroxypropiononitrile in the presence of
a base (DBU or NaH) was used, it was observed that at first all
of the benzyl groups were displaced by the 2-cyanoethoxy
group, which underwent β-elimination to give water-soluble
phosphate monoesters. Prolonged treatment with the oxygen
nucleophile resulted in P−O−sugar cleavage, also giving all of
the possible mono-, bis-, and tris-phosphates. Possible trans-
esterification pathways for a protected sugar phosphate are
shown in Figure 4. Because the P−O−sugar also underwent
cleavage, we were curious to investigate the possible selective
dephosphorylation at the 2′-position from a protected tri-
sphosphate such as 33 (Scheme 3) via transesterification with
an alcohol. We envisioned that if we used the incoming
nucleophile (RO−), the same as the protecting group on the
phosphorus (benzyl), the unwanted loss of protecting group on
phosphorus can be prevented. Thus, benzyl alcohol was our
agent of interest for transesterification.
For biological comparison with 6, the previously unknown
glucose 3,4-bisphosphate 7 was synthesized from allyl glycoside
37 (Scheme 4).36 Phosphorylation of 37 using standard
phosphoramidite methodology37gave the fully protected bis-
phosphate 38, and the allyl protecting group was removed
using PdCl2 in methanol38 to give bisphosphate 39 as a mixture
of α- and β-anomers. Hydrogenolysis of 39 then provided the
1
bisphosphate 7, and H NMR and 31P NMR spectroscopy
showed this to exist as a mixture of α- and β-anomers in
aqueous or methanolic solution. The relative proportions of the
two anomers were dependent on salt form and pH.
The ability of bisphosphates 4−7, IP3 (1), AdA (3a), and D-
myo-inositol 4,5-bisphosphate [Ins(4,5)P2 (2)] to stimulate the
IP3R of intracellular Ca2+ stores was measured using a low-
affinity Ca2+ indicator trapped within the intracellular stores of
Trisphosphate 33 was made in 12 steps from 8 by following
the reported procedure.35 Trisphosphate 33 on treatment with
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permeabilized DT40 cells expressing only recombinant rat
IP3R1, as previously reported.19 The amount of Ca2+ released
by each of these compounds was expressed as a fraction of the
total Ca2+ content of the endoplasmic reticulum (ER) as
assessed by the addition of ionomycin. Results are shown in
Table 1.
cells (Table 1), synthetic Ins(4,5)P2 from the present study is
ca. 375-fold less potent than IP3 but ca. 3-fold more potent than
7. The marginally weaker activity of 7 as compared to
Ins(4,5)P2 is not surprising, given the existence of 7 as a
mixture of anomers. The 7-β isomer mimics four of the IP3
carbon centers retaining the important triad binding motif (4,5-
bisphosphate and 6-OH); hence, 7-β might be more or less
active as IP2 (Figure 5). However, 7-α can orient in two
different ways (A and B; Figure 5), mimicking the orientation
of IP3. Although orientation A has the binding motif in full, it
mimics only three carbons of IP3, while orientation B mimics
four centers of IP3 but lacks the 6-OH mimic of the binding
motif. While it is reasonable to expect that the mode with
maximum resemblance to IP3 (B) would be the preferred fit,
the association constant could be less due to the lack of 6-OH
interaction with the receptor. Competition between these two
orientations (A and B) for receptor binding could reduce the
overall potency. The ratio of 7-α to 7-β at the receptor is
unknown.
AdA binds to a site that at least substantially overlaps the IP3-
binding site and in a manner that is thought to be broadly
similar to IP3.10 The crystal structure41 of IP3 bound to the IBC
(residues 224−604) of IP3R1 and our model7 for AdA binding
together allow a rationalization of the biological activities of the
bisphosphates of adenophostin A. The IBC comprises an α-
domain and β-domain, with IP3 sandwiched between the two
domains (Figure 6).
Table 1. Ca2+ Release by IP3R1 Mediated by Compounds 1−
a
7
ligand
EC50 (nM)
nHill
% release
IP3 (1)
23
4
1.00 0.12 74
0.87 0.09 73
1.79 0.41 67
2
2
3
AdA (3a)
2.8 0.4
107 15
2′-dephospho AdA
(6)
3″-dephospho AdA
(5)
53030 6249* 1.31 0.36 53 8 at 50 μM
4″-dephospho AdA
(4)
ND
ND
8% release at
50 μM
Gluc(3,4)P2 (7)
Ins(4,5)P2 (2)
25190 4782
8634 1005
1.04 0.17 72
4
0.86 0.07 73 10
a
Results show the half-maximal effective concentration of each ligand
(EC50), the Hill coefficient (nHill), and the percentage of the
intracellular stores released by a maximal concentration of each ligand.
Results are means SEMs from eight independent experiments. The
EC50 for 5, where even the maximal practicable concentration (50
μM) failed fully to release the IP3-sensitive Ca2+ stores, was calculated
by assuming that it was a full agonist capable of releasing the entire
IP3-sensitive Ca2+ store.
The 4,5-bisphosphate motif of IP3 makes more contacts with
the receptor than the more exposed 1-phosphate. The 1-
phosphate interacts only with R568 and the backbone NH of
K569 in the α-domain, while the 4-phosphate interacts mainly
with residues in the β-domain (R265, R269, and T267), and
the 5-phosphate interacts predominantly with amino acids in
the α-domain (R504, K508, R511, and Y567). We and others
have speculated1,42 that binding of IP3 pulls the domains
together and closes the “clam-like” binding core, in a manner
similar to glutamate binding to ionotropic glutamate
receptors.43 This is illustrated schematically in Figure 7 (top
left).
Recently published X-ray structures of the N-terminal ligand-
binding domain (LBD)44 and the N-terminal domain of rat
IP3R1 (residues 1−604) with and without IP3 bound support
the idea that IP3 causes domain closure within the IBC.45 As IP3
binds, side chains of nine residues within the α- and β-domains
of the IBC become organized around IP3, causing the clam-like
structure to partially close, reducing the angle between the two
domains by ∼8°. Unfortunately, the resolution of these
structures is not sufficient to provide further clues about how
AdA and its analogues might bind. The 4,5-bisphosphate of IP3
Among the bisphosphates, 6 is the most potent; it releases
the same fraction of the Ca2+ stores as other full agonists, and it
is only 40 times less potent than AdA and only four times less
potent than IP3. This contradicts an earlier report18 where 6
was found to be 1800-fold less potent than AdA, although this
is now thought to be in error (see ref 19 for discussion).
Bisphosphate 4 released only 8% of the Ca2+ stores at a
concentration of 50 μM. It was impracticable to assess the
activity at higher concentrations and so impossible to resolve
whether 4 is a full agonist with very low affinity or a partial
agonist. Bisphosphate 5 had some activity, but even at 50 μM, it
released only 53% of the intracellular stores. Again, it was
impracticable to increase the concentration of 5 sufficiently to
assess whether it is a full agonist, capable at a high enough
concentration, of mimicking the response to a maximal
concentration of AdA or IP3. Assuming 5 to be a full agonist,
then it is 2300 times less potent than IP3. Gluc(3,4)P2 (7) is
approximately 1000 times less potent than IP3. This is broadly
in agreement with an earlier report,39 where Ins(4,5)P2 was
460-fold less potent than IP3 in evoking Ca2+ release from rat
brain microsomes. When compared in permeabilized DT40
Figure 5. Structural comparison of Ins(4,5)P2 (2), 7, and IP3.
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Figure 6. Possible hydrogen bonds between IP3 and IP3R in the crystal structure (PDB: 1N4K) of the IBC of IP3R1. Waters not shown. Green
carbons, α-domain; purple carbons, β-domain.
Figure 7. Schematic representation of IP3R activation by inositol phosphates and adenophostin analogues. IP3 (1) and Ins(4,5)P2 (2) activate IP3R
by engaging residues in the α- (green) and β- (pink) domains of the IBC, stabilizing a closed conformation that favors opening of the C-terminal
Ca2+ channel.45 Phosphate groups may have strong (dark green) or weaker (light green) interactions with the α-domain. Molecular modeling
suggests that Ada (3a) has additional interactions (light green) with the α-domain of the IBC, accounting for the greater potency of AdA. 4″-
Dephospho-AdA (4) is essentially inactive because it cannot form effective β-domain interactions. However, 3″-dephospho-Ada (5) and 2′-
dephospho-AdA (6) retain activity because they can effectively engage both domains, even though 5 does not contain a vicinal bisphosphate pair.
The previously unexplained and relatively potent activity of AdA regiosomer 40 can now be explained by analogy with 5. P = phosphate group, and
Ad = adenine.
clearly plays a major role in cross-linking the two domains of
the IBC, and the 1-phosphate exerts its enhancing effect by
providing an additional, weaker interaction with the α-domain,
accounting for the greater potency of IP3 relative to Ins(4,5)P2
(Figure 7). The known inactivity of D-myo-inositol 1,4-
bisphosphate [Ins(1,4)P2] suggests that the 1-P alone cannot
interact strongly enough with the α-domain to pull the two
domains together. Our model for AdA binding7 shows how the
3″,4″-bisphosphate of AdA can mimic the 4,5-bisphosphate of
IP3, while both its 2′-phosphate group and adenine moiety have
additional interactions with the IBC (Figures 7 and 8A).
Specifically, we have proposed that while the 2′-phosphate of
AdA essentially mimics the 1-phosphate of IP3, the adenine can
engage in a cation-π interaction with the guanidinium side
chain of R504 in the α-domain.
The present study shows that bisphosphate analogues of AdA
lacking the 3″- or 4″-phosphate (4 and 5) are very weak
agonists, confirming the important role for the 3″,4″-vicinal
bisphosphate in AdA.40 However, our finding that 4 and 5 differ
in their potency suggests unequal contributions from the two
phosphates (4″-P and 3″-P). While bisphosphate 4 is almost
inactive, 5 is a weak agonist of IP3R, being some 2300-fold
weaker than IP3. This shows that between the vicinal
bisphosphates, the 3″-phosphate in AdA is less important
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than the 4″-phosphate. Loss of activity after deletion of the 4″-
phosphate from AdA is consistent with it providing the major
contact with the β-domain, which may be essential for “clam”
closure. Thus, 4 and 5 can be considered as analogues of D-myo-
inositol 1,5-bisphosphate [Ins(1,5)P2] and Ins(1,4)P2, respec-
tively, and the fact that 5 is a weak agonist while its inositol
equivalent Ins(1,4)P2 is inactive can be explained in terms of
our model by the enhancing role of the adenine in 5,
strengthening its α-domain interactions (Figures 7 and 8B).
2′-Dephospho-AdA (6) differs from Gluc(3,4)P2 (7) only in
having an adenosine moiety; yet, it is 200-fold more potent
than 7 (Table 1). This 200-fold enhancement of activity by the
adenosine moiety in the absence of a 2′-phosphate establishes
unequivocally that the adenosine moiety contributes directly to
enhanced activity independent of any effect on positioning of
that phosphate. In a recent report,19 we demonstrated, in a
study linking chemical modification with receptor mutagenesis,
that removal of the 2′-phosphate from AdA to give 6 has
significantly lesser effects on affinity for the IBC than did
removal of the 1-phosphate from IP3 to give Ins(4,5)P2. A cell
line was established that expresses only IP3R1, and mutation of
R504 more profoundly reduces the affinity of IP3R for AdA
(353-fold) than for IP3 (13-fold). The activities of other
adenophostin analogues and the corresponding IP3 analogues
were also comparable. Thus, when an amino acid thought to be
responsible for engaging the adenine unit (R504) is mutated,
the enhanced activity of adenophostin ligands disappears.
Bisphosphate 6 is the most potent known agonist of the IP3R
with only two phosphates, suggesting that it may be possible to
develop high-affinity ligands with fewer phosphates or even
nonphosphate moieties. The possibility to develop less charged
agonists or antagonists would be particularly attractive for
cellular or in vivo chemical biological studies. Even though 5 is
a weak agonist, its measurable activity suggests that a vicinal
bisphosphate is not absolutely required for a ligand to activate
IP3R. This now explains an apparent anomaly observed in
previous work. The regioisomeric AdA analogue 2″-phospho-3″-
dephospho-AdA 40 (Figure 9) with the 3″-phosphate group of
AdA transposed to the 2″-position was previously shown to
possess surprisingly potent activity that did not fit established
SAR considerations.
Compound 40, although lacking the vicinal 3″,4″-bisphos-
phate, is only 12−20 times less potent than IP3 in liver flux and
binding assays, respectively.46 Interestingly, D-myo-inositol
1,4,6-trisphosphate (41), which may be pictured as having a
similar arrangement of phosphate groups to 40 (Figure 9), is
also an agonist, only 2−3 times weaker than IP3.47 However, 41
may be able to present an IP3-like arrangement of phosphate
groups to the IBC simply by binding in an alternative
orientation (41b, Figure 9). Similar reasoning was used to
explain the unexpected activity of 40 in the original study,
where it was suggested that it too may be able to adopt an
inverted binding orientation.40 This may seem improbable
given the size and complexity of 40 as compared to 41, but the
alternative explanation, namely, that a vicinal bisphosphate is
not required, would have been totally unprecedented. Given
the new finding of activity for 5, which cannot present a vicinal
bisphosphate in any binding orientation, we can now suggest a
simpler explanation for the activity of both 5 and 40 that is also
consistent with more recent information on the structure of the
IBC and with the concept of a direct role for the adenine. We
propose that, contrary to previous assumptions, a vicinal
bisphosphate motif is not absolutely essential for activation of
Figure 8. Interactions of AdA (A), compound 5 (B), and compound 6
(C) with the IBC of IP3R as predicted by molecular docking
experiments. Waters not shown. Green carbons, α-domain; purple
carbons, β-domain. See the Experimental Section for details.
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Figure 9. Structural comparison of IP3R ligands lacking vicinal bisphosphates with IP3.
IP3R, given sufficiently strong compensating contributions from
other components of the ligand. Thus, we suggest that for
bisphosphate 5, interactions of the adenine with the α-domain
partially compensate the loss of one phosphate group in the
vicinal pair, while in 40, these interactions are supplemented by
additional contributions from the 2″-phosphate, presumably
also with the α-domain, strengthening binding still further
(Figure 7). Thus, it appears that two or more weak interactions
with the α-domain can, to some extent, compensate for the loss
of a strong interaction.
Glucose-3,4-bisphosphate essentially mimicks Ins(4,5)P2 in
activity as expected. A 200-fold enhancement in activity is
found in going from the simple glucose bisphosphate 7 to the
related 6 bearing an adenosyl moiety, establishing a direct role
for the adenosine of AdA in increasing affinity for the receptor.
Compound 6, the most potent among the bisphosphates
synthesized, approaches the potency of IP3. This is the first
report of a relatively potent agonist of IP3R devoid of one
phosphate group from the natural ligand and suggests the
possibility to develop other ligands having a fewer number of
phosphates. Such ligands, with less charge, could be useful for
pharmacological intervention in this cellular signaling system
and, at their simplest, might comprise two motifs that interact
with the IBC domains linked by a suitable spacer. The
reappraisal here of the relatively potent activity and surprising
activity of 40, published earlier, in the light of these new results
and particularly the activity of 5, begins to demonstrate the
potential of this approach, suggesting that it should be possible
in principle to develop IP3R ligands without a vicinal
bisphosphate moiety.
CONCLUSION
■
In summary, as a continuation of our efforts to understand
SARs of adenophostins at the IP3 receptor and to gain further
insight into the molecular mechanism of IP3-mediated signal
transduction pathways in general, we have synthesized all of the
bisphosphate analogues of AdA, excising one of its three
phosphates at a time. Bisphosphates 4 and 5 were prepared in a
series of reactions involving α-selective glycosylation of an
appropriately protected glucose (glycosyl donor) and protected
EXPERIMENTAL SECTION
ribose acceptor, Vorbruggen condensation of an appropriately
̈
■
protected disaccharide with 6-chloropurine, followed by further
manipulation and chemoselective phosphitylation. A novel
strategy for regioselective dephosphorylation by transesterifica-
tion has been introduced for the efficient synthesis of 6. The
glucose bisphosphate 7 was synthesized as a control. The ability
of these novel bisphosphates to stimulate IP3R-mediated Ca2+
release was measured using rat type I IP3R expressed in chicken
DT40 cells and compared to that of glucose 3,4-bisphosphate
(7) and Ins(4,5)P2. This study reveals that although the 3″,4″-
bisphosphate functionality is important for high affinity binding
and channel opening, it is still possible for molecules lacking
this feature to stimulate the IP3R, albeit to a lesser extent. Such
knowledge is important in designing novel ligands as tools for
pharmacological intervention. We conclude that a vicinal
bisphosphate moiety is not essential for IP3R activation. P-4
of IP3, which contacts the β-domain of the IBC, is required, but
P-5, which contacts the α-domain, can be replaced by a cation-π
interaction between the adenine of AdA and R504 in the α-
domain. The same interaction can substantially compensate the
loss of P-1 to provide the potent agonist 6 with only two
phosphate groups. Inositol polyphosphates often bind to sites
rich in Arg and Lys residues, and replacing such interactions
with a polar phosphate by a cation-π motif could have more
general applications in the chemical biology of inositol
phosphate signaling and probably also in other fields.
General. Chemicals were purchased from Aldrich, Sigma, and
Fluka. All anhydrous solvents were purchased from Aldrich or Fluka.
TLC was performed on precoated plates (Merck Aluminum sheets
silica 60 F254, art No. 5554). Chromatograms were visualized under
UV light and by dipping plates into either phosphomolybdic acid in
MeOH or anisaldehyde in ethanol, followed by heating. Ion exchange
chromatography was performed on an LKB-Pharmacia Gradifrac
medium pressure ion-exchange chromatograph using MP1 AG ion-
exchange resin and a gradient of 0−100% 150 mM TFA as eluent or Q
Sepharose Fast Flow resin and a gradient of 0−100% 1.0 M
1
triethylammonium bicarbonate (TEAB). H NMR, COSY, NOESY,
HMBC, and HMQC spectra were recorded on Varian EX-400 (400
MHz) and Bruker Avance III (400 and 500 MHz) spectrometers.
Proton chemical shifts are reported in ppm (δ) relative to internal
tetramethylsilane (TMS, δ 0.0 ppm) or with the solvent reference
relative to TMS employed as the internal standard (CDCl3, δ 7.26
ppm; D2O, δ 4.79 ppm). Data are reported as follows: chemical shift
(multiplicity [singlet (s), doublet (d), triplet (t), quartet (q), and
multiplet (m)], integration, coupling constants [Hz], annotation). 13C
and DEPT spectra were recorded on Varian EX-400 (100 MHz) and
Bruker Avance III (100 and 126 MHz) spectrometers with complete
proton decoupling. Carbon chemical shifts are reported in ppm (δ)
relative to TMS with the respective solvent resonance as the internal
standard (CDCl3, δ 77.0 ppm). 31P NMR spectra were recorded on
Varian EX-400 (162 MHz) or Bruker Avance III (162 MHz)
spectrometers with complete proton decoupling. Phosphorus chemical
shifts are reported in ppm (δ) relative to an 85% H3PO4 external
standard (H3PO4, δ 0.0 ppm). All NMR data were collected at 25 °C.
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Melting points were determined using a Reichert-Jung Thermo Galen
Kofler block and are uncorrected. Microanalysis was carried out at the
University of Bath microanalysis service. Mass spectra were recorded
at the SERC Mass Spectrometry Service Centre, Swansea, and at the
University of Bath on VG Autospec or MicroTOF instruments. The
method of ionization is given in parentheses. Optical rotations were
measured at ambient temperature using an Optical Activity Ltd. AA-10
polarimeter in a cell volume of 5 cm3, and specific rotation is given in
10−1 deg cm3 g−1. UV spectra were recorded using a Perkin-Elmer
Lambda EZ201 spectrometer. The purities of final compounds were
determined to be greater than 98% by HPLC analysis. HPLC analysis
was carried out on a Hewlett-Packard series chromatograph with a
strong anion-exchange resin (MP1 AG, column size 3 mm × 150
mm). A linear gradient of 0−50% 150 mM TFA was used as eluent at
1 cm3/min over 60 min, with the UV detector set at 254 nm. Synthetic
phosphates were assayed using an adaptation of the modified Brigg's
phosphate assay48 and/or Ames phosphate assay.49 Flash column
chromatography was performed using Silica Gel 60 A (32−63 μm). All
reactions were carried out under argon or nitrogen atmosphere
employing oven-dried glassware unless stated otherwise. Usual work
up refers to taking up the crude material in an organic solvent (ethyl
acetate, DCM, or CHCl3) followed by washing successively with water,
cold diluted HCl, saturated NaHCO3 solution, and brine and drying
over anhydrous MgSO4.
nucleoside 19 (103 mg, 77%) as a colorless gum. [α]D +47.5 (c 1.6,
CHCl3). 1H NMR (400 MHz, CDCl3): 1.84 (s, 3H, COCH3), 1.86 (s,
3H, COCH3), 3.41 (dd, 1H, 10.14 Hz, 3.48 Hz, H-2″), 3.44 (dd, 1H,
8.99 Hz, 1.74 Hz, HA-6″), 3.56 (dd, 1H, 10.72 Hz, 3.19 Hz, HB-6″),
3.56−3.61 (m, 1H, HA-5′), 3.60 (t, 1H, 9.27 Hz, H-4″), 3.70−3.80 (m,
2H, H-5″, HB-5′), 4.40 (AB q, 2H, 33.62 Hz, 11.01 Hz, PhCH2), 4.41
(AB q, 2H, 78.84 Hz, 11.88 Hz, PhCH2), 4.42−4.46 (m, 1H, H-4′),
4.47 (AB q, 2H, 14.49 Hz, 11.59 Hz, PhCH2), 4.49 (AB q, 2H, 64.92
Hz, 12.17 Hz, PhCH2), 4.68 (t, 4.93 Hz, H-3′), 4.95 (d, 1H, 3.48 Hz,
H-1″), 5.45 (dd, 1H, 10.14 Hz, 9.28 Hz, H-3″), 5.68 (dd, 1H, 5.22 Hz,
4.64 Hz, H-2′), 6.30 (d, 1H, 4.64 Hz, H-1′), 7.00−7.30 (m, 20H, 4 ×
Ph), 8.45 (s, 1H, H-8), 8.66 (s, 1H, H-2). m/z (ES+) = 915.44 [(M +
Na)+, 100%]. HRMS: mass calcd for C48H50ClN4O11 [M + H]+,
893.3159; found, 893.3160.
6-Amino-5′,2″,4″,6″-tetra-O-benzyl-3′-O-(α-D-glucopyrano-
syl)-9-β-D-ribofuranosylpurine (20). A solution of chloro-nucleo-
side 19 (100 mg, 0.112 mmol) in ethanol (10 mL) in a pressure tube
was saturated with NH3 (bubbled at 0 °C for 10−15 min). The tube
was then closed tightly and was heated at 74 °C for 5 days. Solvents
were evaporated off in vacuo, and the crude product was chromato-
graphed with 4% MeOH−CHCl3 to afford the diol 20 (86 mg, 97%)
1
as a colorless gum. H NMR (400 MHz, CDCl3): 3.51 (dd, 1H, 9.78
Hz, 3.52 Hz, H-2″), 3.56 (dd, 1H, 10.17 Hz, 9.39 Hz, H-4″), 3.55−3.61
(m, 2H, HA-5′ and HA-6″), 3.66 (dd, 2H, 10.57 Hz, 3.52 Hz, HB-5′ and
HB-6″), 3.82 (ddd, 1H, 10.17 Hz, 3.13 Hz, 1.96 Hz, H-5″), 4.04 (br,
1H, OH), 4.20 (dd, 1H, 9.39 Hz, 9.78 Hz, H-3″), 4.28 (dd, 1H, 6.26
Hz, 3.13 Hz, H-4′), 4.41 (dd, 1H, 5.87 Hz, 3.52 Hz, H-3′), 4.48−4.52
(br, 1H, OH), 4.48 (s, 2H, PhCH2), 4.51 (AB q, 2H, 43.82 Hz, 12.13
Hz, PhCH2), 4.67 (dd, 11.74 Hz, 5.87 Hz, H-2′), 4.69 (AB q, 2H,
116.21 Hz, 11.35 Hz, PhCH2), 4.80 (AB q, 2H, 17.61 Hz, 11.74 Hz,
PhCH2), 4.96 (d, 1H, 3.91 Hz, H-1″), 5.98 (d, 1H, 5.87 Hz, H-1′), 6.05
(br s, 2H, NH2), 7.15−7.35 (m, 20H, 4 × Ph), 7.98 (s, 1H, H-8), 8.39
(s, 1H, H-2). m/z (ES+) = 812.48 [(M + Na)+, 100%]. HRMS: mass
calcd for C44H48O9N5 [M + H]+, 790.3447; found, 790.3449.
6-Amino-5′,2″,4″,6″-tetra-O-benzyl-2′,3″-bis-O-(dibenzylox-
yphosphoryl)-3′-O-(α-D-glucopyranosyl)-9-β-D-ribofuranosyl-
purine (21). To a solution of diol 20 (123 mg, 0.156 mmol) and
dibenzyl-N,N-di-isopropyl phosphoramidite (113 mg, 0.327 mmol) in
DCM (5 mL) was added imidazolium triflate (75 mg, 0.343 mmol),
and the solution was stirred at room temperature for 30 min. When
TLC showed the disappearance of diol 20, the temperature was
reduced to −78 °C, and then, mCPBA (170 mg, 60−70%) was added,
and the solution was stirred for 1 h, gradually allowing the temperature
to attain room temperature. Workup was in ethyl acetate, and the
crude product was chromatographed with ethyl acetate:hexane:Et3N,
77:20:3 (v/v/v), to get pure bisphosphate 21 (165 mg, 81%) as a
colorless oil. [α]D +22.6 (c 1.15, CHCl3). 1H NMR (400 MHz,
CDCl3): 3.46 (dd, 1H, H-6A″), 3.56 (dd, 1H, H-6B″), 3.58−3.64 (m,
2H, H-2″, H-5A′), 3.67−3.76 (m, 3H, H-4″, H-5″, H-5B′), 4.35−4.54
(m, 7H, H-4′, 3 × CH2Ph), 4.62−4.77 (m, 5H, H-3′, 2 × CH2Ph),
4.79−4.98 (m, 7H, H-3″, 3 × CH2Ph), 5.40 (d, 1H, 3.52 Hz, H-1″),
5.66 (ddd, 1H, 8.22 Hz, 5.48 Hz, 4.90 Hz, H-2′), 5.70−6.00 (br, 2H,
NH2), 6.34 (d, 1H, 5.48 Hz, H-1′), 7.00−7.42 (m, 40H, 8 × Ph), 7.93
(s, 1H, H-8), 8.27 (s, 1H, H-2). 31P NMR (161.94 MHz, CDCl3):
−1.625, −1.101. Elemental analysis calcd for C72H73N5O15P2 C, 66.00;
H, 5.62; N, 5.34. Found: C, 65.8; H, 5.94; N, 5.21. m/z (ES+) =
1334.29 [(M + Na)+, 100%], 1311.38 [(M + 1)+, 100%]. HRMS
(FAB, CsI/glycerol): mass calcd for C72H73O15N5P2 [M]+, 1309.4573;
found, 1309.4585.
3′-O-Acetyl-5,2′,4′,6′-tetra-O-benzyl-3-O-(α-D-glucopyrano-
syl)-1,2-O-isopropylidene-α-D-ribo-furanoside (17). Both donor
15 (1.925 g, 3.587 mmol) and acceptor 16 (981 mg, 3.5 mmol) were
dissolved in toluene−dioxane (10 mL: 30 mL), and the mixture was
stirred with powdered 3 Å molecular sieves for 15 min. NIS (866 mg,
3.85 mmol) was then added, and when it had dissolved, TMSOTf
(100 μL, 0.55 mmol) was added dropwise, and the mixture was stirred
at room temperature. TLC after 30 min showed disappearance of the
starting material. NaHCO3 solution was added to quench the acidic
residue, and the mixture was diluted with ethyl acetate and washed
with saturated Na2S2O3 solution and then with brine. The solution was
dried over MgSO4 and concentrated under reduced pressure. The
residue thus obtained was chromatographed using 15% ethyl acetate−
hexane to afford the α-glycoside 17 (2 g, 76%) as a colorless crystalline
1
solid; mp 112 °C; [α]D +120.8 (c 1, CHCl3). H NMR (400 MHz,
CDCl3): 1.35 (s, 3H, CH3 isopropylidene), 1.50 (s, 3H, CH3
isopropylidene), 1.96 (s, 3H, COCH3), 3.46 (dd, 1H, 10.87 Hz,
1.00 Hz, H-6A′), 3.51 (dd, 1H, 9.88 Hz, 3.95 Hz, H-2′), 3.59 (dd, 1H,
10.87 Hz, 2.47 Hz, H-6B′), 3.67 (dd, 1H, 8.40 Hz, 2.97 Hz, H-5A),
3.67−3.74 (m, 2H, H-4′ and H-5′), 3.79 (dd, 1H, 11.61 Hz, 1.73 Hz,
H-5B), 4.15 (dd, 1H, 9.14 Hz, 3.95 Hz, H-3), 4.29 (ddd, 1H, 9.14 Hz,
3.21 Hz, 1.73 Hz, H-4), 4.44 (AB q, 2H, 25.69 Hz, 11.36 Hz, CH2Ph),
4.47 (AB q, 2H, 82.02 Hz, 11.86 Hz, CH2Ph), 4.53 (AB q, 2H, 46.44
Hz, 11.86 Hz, CH2Ph), 4.64 (AB q, 2H, 50.89 Hz, 12.35 Hz, CH2Ph),
4.71 (t, 1H, 3.95 Hz, H-2), 5.23 (d, 1H, 3.95 Hz, H-1′), 5.45 (t, 1H,
9.39 Hz, H-3′), 5.83 (d, 1H, 3.95 Hz, H-1), 7.11−7.35 (m, 20 H, 4 ×
C6H5). Elemental analysis calcd for C44H50O11: C, 70.01; H, 6.68.
Found: C, 69.8; H, 6.69. m/z (ES+) = 777.4 [(M + Na)+, 100%].
HRMS: mass calcd for C44H54O11N1 [M + NH4]+, 772.3691; found,
772.3691.
2′,3″-Di-O-acetyl-5′,2″,4″,6″-tetra-O-benzyl-6-chloro-3′-O-
(α-D-glucopyranosyl)-9-β-D-ribo-furanosylpurine (19). Disac-
charide 17 (128 mg, 0.17 mmol) was stirred with 90% TFA (0.5
mL) for 10−15 min at room temperature. The TFA was then
evaporated off, and the residue was coevaporated several times with
toluene to remove traces of water. The residue was then dissolved in
pyridine (2 mL), Ac2O (0.5 mL, 5.3 mmol) was then added, and the
mixture was stirred for 4 h at room temperature. Work up was as usual,
and the crude product was chromatographed to afford an inseparable
mixture of the α- and β-triacetates 18 (120.6 mg, 89%). A suspension
of triacetate 18 (120 mg, 0.15 mmol), 6-chloropurine (46 mg, 0.3
mmol), and BSA (182 μL, 0.74 mmol) in MeCN (5 mL) was refluxed
until the solution became clear. The mixture was cooled to room
temperature, and then, TMSOTf (60 μL, 0.33 mmol) was added, and
the solution was stirred at 70 °C overnight. Workup was in ethyl
acetate, and the crude product was chromatographed to afford the
3′-O-(α-D-Glucopyranosyl)-adenosine-2′,3″-bisphosphate
(4). A suspension of bisphosphate 21 (130 mg, 0.1 mmol) and 20%
Pd(OH)2 on carbon (300 mg) in a mixture of cyclohexene (4 mL),
MeOH (10.5 mL), and H2O (0.75 mL) was refluxed at 80 °C
overnight. After filtration through a membrane filter, the filtrate was
concentrated in vacuo. Purification by an AG column with 0−100%
gradient elution using 150 mM TFA as an eluent gave pure
1
bisphosphate 4 (53 mg, 91%). H NMR (400 MHz, D2O): 3.58 (t,
1H, 8.5 Hz, H-4″), 3.66−3.76 (m, 3H, H-2″, H-5″, H-6A″), 3.80−3.90
(m, 3H, H-5A′, H-5B′, H-6B″), 4.34 (ddd, 1H, 9.45 Hz, 8.50 Hz, 8.50
Hz, H-3″), 4.43 (dd, 1H, 7.09 Hz, 3.78 Hz, H-4′), 4.63 (dd, 1H, 5.20
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Hz, 3.31 Hz, H-3′), 5.24−5.29 (m, 1H, H-2′), 5.27 (d, 1H, 3.78 Hz, H-
1″), 6.35 (d, 1H, 5.61 Hz, H-1′), 8.40 (s, 1H, H-2), 8.50 (s, 1H, H-8).
31P NMR (161.94 MHz, D2O with excess of TEA): 4.25, 3.35. m/z
residue was chromatographed with 35% ethyl acetate−hexane to yield
the chloronucleoside 29 (241 mg, 92%) as a colorless gum. [α]D +14.1
(c 0.9, CHCl3). H NMR (400 MHz, CDCl3): 2.16 (s, 3H, COCH3),
1
(ES+) = 590.1 [(M + H)+, 90%]; 612.1 [(M + Na)+, 100%]. m/z
(ES−) 588.2 [(M − H)+, 100%]. HRMS: mass calcd for
C16H26O15N5P2 [M + H]+, 590.0895; found, 590.0895.
2.20 (s, 3H, COCH3), 3.67−3.75 (m, 2H, HA-6″ and HB-6″), 3.80−
3.92 (m, 3H, H-2″, HA-5′, HB-5′), 4.01 (m, 1H, H-5″), 4.27 (t, 1H, 9.50
Hz, H-3″), 4.72 (AB q, 2H, 29.72 Hz, 11.64 Hz, PhCH2), 4.80 (AB q,
2H, PhCH2), 4.91 (q, 1H, H-4′), 4.93−5.00 (m, 3H, H-3′, PhCH2),
5.10 (AB q, 2H, 87.33 Hz, 11.34 Hz, PhCH2), 5.23 (d, 1H, 3.68 Hz,
H-1″), 5.29 (dd, 1H, 10.11 Hz, 9.50 Hz, H-4″), 5.95 (dd, 1H, 6.44 Hz,
5.52 Hz, H-2′), 6.79 (d, 1H, 6.44 Hz, H-1′), 7.50−7.70 (m, 20H, 4 ×
Ph), 8.79 (s, 1H, H-8), 9.06 (s, 1H, H-2). m/z (ES+) = 915.4 [(M +
Na)+, 100%]. HRMS: mass calcd for C48H49O11N4Cl1Na1 [M + Na]+,
915.2979; found, 915.2984.
3-O-(4′-O-Acetyl-2′,3′,6′-tri-O-benzyl-α-D-glucopyranosyl)-
5-O-benzyl-1,2-O-isopropylidene-α-D-ribofuranoside (27). To a
solution of donor acetate 26 (prepared from the known dibenzyl ether
2450) (1.007 g, 1.876 mmol), acceptor 16 (527 mg, 1.88 mmol), and
N-iodosuccinimide (640 mg, 2.845 mmol) in a mixture of toluene (14
mL) and dioxane (19 mL) were added powdered 3 Å molecular sieves,
and the mixture was stirred for 15 min under argon atmosphere.
TMSOTf (100 μL, 0.55 mmol) was then added dropwise, and the
mixture was stirred at room temperature for 30 min. When TLC
showed disappearance of the starting material, the reaction mixture
was filtered through a small pad of Celite, and the filtrate was diluted
with ethyl acetate and washed successively with saturated Na2S2O3
solution, NaHCO3 solution, and brine. After it was dried over MgSO4,
chromatography using ethyl acetate−hexane gave pure α-disaccharide
27 (1 g, 71%) as a colorless gum. [α]D +82.6 (c 0.9, CHCl3). 1H NMR
(400 MHz, CDCl3): 1.38 (s, 3H, CH3 isopropylidene), 1.60 (s, 3H,
CH3 isopropylidene), 1.85 (s, 3H, COCH3), 3.33 (dd, 1H, 10.59 Hz,
4.33 Hz, H-6A′), 3.39 (dd, 1H, 10.59 Hz, 2.89 Hz, H-6B′), 3.65 (dd,
1H, 9.63 Hz, 3.85 Hz, H-2′), 3.69 (dd, 1H, 11.55 Hz, 3.85 Hz, H-5A),
3.77 (ddd, 1H, 10.11 Hz, 3.85 Hz, 3.85 Hz, H-5′), 3.80 (dd, 1H, 11.55
Hz, 1.93 Hz, H-5B), 3.88 (t, 1H, 9.63 Hz, H-3′), 4.15 (dd, 1H, 9.63 Hz,
4.33 Hz, H-3), 4.32−4.38 (m, 1H, H-4), 4.43 (AB q, 2H, 47.18 Hz,
12.04 Hz, CH2Ph), 4.57 (AB q, 2H, 47.66 Hz, 12.04 Hz, CH2Ph), 4.72
(AB q, 2H, 32.98 Hz, 11.80 Hz, CH2Ph), 4.73 (dd, 1H, 4.70 Hz, 1.17
Hz, H-2), 4.75 (AB q, 2H, 119.15 Hz, 11.31 Hz, CH2Ph), 5.09 (dd,
1H, 10.11 Hz, 9.63 Hz, H-4′), 5.21 (d, 1H, 3.37 Hz, H-1′), 5.83 (d, 1H,
3.85 Hz, H-1), 7.20−7.40 (m, 20 H, 4 × C6H5). Elemental analysis
calcd for C44H50O11: C, 70.00; H, 6.68. Found: C, 69.79; H, 6.69. m/z
(ES+) = 777.47 [(M + Na)+, 100%]. HRMS: mass calcd for
C44H54O11N [M + NH4]+, 772.3691; found, 772.3694.
6-Amino-5′,2″,3″,6″-tetra-O-benzyl-3′-O-(α-D-glucopyrano-
syl)-9-β-D-ribofuranosylpurine (30). A solution of chloronucleoside
29 (360 mg, 0.403 mmol) in ethanol was saturated with ammonia and
heated at 74 °C in a sealed pressure tube for 5 days. The solvents were
evaporated off, and the residue was dissolved in DCM and washed
with water and then with brine. The organic layer was dried over
MgSO4, and the solvents were evaporated under reduced pressure to
afford the pure diol 30 (318 mg, 100%) as a colorless gum. [α]D +6 (c
1, CHCl3). 1H NMR (400 MHz, CDCl3): 3.54 (dd, 1H, 9.39 Hz, 3.91
Hz, H-2″), 3.57−3.68 (m, 5H, H-4″, H-5A′, H-5B′, H-6A″ and H-6B″),
3.76−3.90 (m, 2H, H-3″, H-5″), 4.28−4.38 (m, 1H, H-4′), 4.38−4.52
(m, 5H, H-3′, 2 × CH2Ph), 4.64−4.72 (m, 1H, H-2′), 4.72 (ABq, 2H,
46.17 Hz, 11.74 Hz, CH2Ph), 4.86 (d, 3.52 Hz, H-1″), 4.89 (ABq, 2H,
66.52 Hz, 11.35 Hz, CH2Ph), 5.74−5.90 (br, 2H, NH2), 6.03 (d, 1H,
5.48 Hz, H-1′), 7.20−7.38 (m, 20 H, 4 × C6H5), 7.98 (s, 1H, H-8),
8.30 (s, 1H, H-2). m/z (ES+) = 812.70 [(M + Na)+, 100%]. HRMS:
mass calcd for C28H42O7NS [M + H]+, 790.3447; found, 790.3454.
6-Amino-5′,2″,3″,6″-tetra-O-benzyl-2′,4″-bis-O-(dibenzylox-
yphosphoryl)-3′-O-(α-D-glucopyranosyl)-9-β-D-ribofuranosyl-
purine (31). To a solution of 30 (165.5 mg, 0.21 mmol) and
dibenzyl-N,N-di-isopropyl phosphoramidite (152 mg, 0.44 mmol) in
DCM (5 mL) was added imidazolium triflate (102 mg, 0.468 mmol),
and the solution was stirred at room temperature for 30 min. When
TLC showed disappearance of the starting material, the temperature
was reduced to −78 °C, mCPBA (170 mg) was added, and the mixture
was stirred for 30 min, allowing the temperature to attain room
temperature. The mixture was taken up in ethyl acetate, and the
solution was washed successively with Na2SO3 solution, water, and
brine. The solution was dried over MgSO4, and the residue after
evaporation was chromatographed to yield bisphosphate 31 (233 mg,
85%) as a colorless oil.
3-O-(4′-O-Acetyl-2′,3′,6′-tri-O-benzyl-α-D-glucopyranosyl)-
5-O-benzyl-1,2-di-O-acetyl-α/β-D-ribofuranoside (28). Disac-
charide 27 (270 mg, 0.358 mmol) was treated with 90% TFA (2
mL) at room temperature for 15 min. TFA was evaporated off, and the
residue was coevaporated with toluene several times. The residue thus
obtained was dissolved in pyridine (5 mL), added Ac2O (2 mL, 21.2
mmol), and stirred at room temperature for 3 h. Workup was as usual,
and the product was crystallized from ethyl acetate−hexane to afford
pure crystals of α-triacetate 28-α (100 mg). The mother liquor was
concentrated and chromatographed using 25% ethyl acetate−hexane
to give an inseparable α/β mixture of the triacetate 28 (149 mg, total
yield = 249 mg, 87%). Data for 28-α: mp 127−128 °C; [α]D +46 (c 1,
3′-O-(α-D-Glucopyranosyl)-adenosine-2′,4″-bisphosphate
(5). A suspension of bisphosphate 31 (155 mg, 0.118 mmol) and 20%
Pd(OH)2 on carbon (400 mg) in a mixture of cyclohexene (5.5 mL),
MeOH (10.5 mL), and H2O (0.75 mL) was heated at 80 °C overnight.
After filtration through a membrane filter, the evaporated filtrate was
purified on an AG column using 0−100% gradient elution using 150
1
CHCl3). H NMR (400 MHz, CDCl3): 1.80 (s, 3H, CH3), 1.89 (s,
3H, CH3), 1.93 (s, 3H, CH3), 3.31 (dd, 1H, 10.77 Hz, 4.72 Hz, H-6A′),
3.38 (dd, 1H, 10.77 Hz, 2.94 Hz, H-6B′), 3.55 (dd, 1H, 9.78 Hz, 3.52
Hz, H-2′), 3.60 (dd, 1H, 11.16 Hz, 4.11 Hz, H-5A), 3.69 (dd, 1H, 11.35
Hz, 3.13 Hz, H-5B), 3.81 (ddd, 1H, 10.17 Hz, 4.70 Hz, 2.94 Hz, H-5′),
3.89 (t, 1H, 9.78 Hz, H-3′), 4.39 (AB q, 2H, 41.87 Hz, 11.74 Hz,
CH2Ph), 4.40 (t, 1H, 3.52 Hz, H-4), 4.50 (AB q, 2H, 14.87 Hz, 11.74
Hz, CH2Ph), 4.60 (t, 1H, 5.87 Hz, H-3), 4.65 (s, 2H, CH2Ph), 4.73
(AB q, 2H, 94.68 Hz, 11.74 Hz, CH2Ph), 4.95 (d, 1H, 3.52 Hz, H-1′),
5.02 (dd, 1H, 10.17 Hz, 9.40 Hz, H-4′), 5.31 (dd, 1H, 4.70 Hz, 1.17
Hz, H-2), 6.12 (d, 1H, 1.17 Hz, H-1), 7.22−7.33 (m, 20 H, 4 × C6H5).
Elemental analysis calcd for C45H50O13: C, 67.66; H, 6.31. Found: C,
67.5; H, 6.34. m/z (ES+) = 821.73 [(M + Na)+, 100%]. HRMS: mass
calcd for C45H54O13N [M + NH4]+, 816.3590; found, 816.3592.
2′,4″-Di-O-acetyl-5′,2″,3″,6″-tetra-O-benzyl-6-chloro-3′-O-
(α-D-glucopyranosyl)-9-β-D-ribofuranosylpurine (29). A suspen-
sion of triacetate 28 (234 mg, 0.293 mmol), 6-chloropurine (91 mg,
0.589 mmol), and BSA (356 μL, 1.46 mmol) in acetonitrile (9 mL)
was refluxed until the solution became clear. After it was cooled and
the addition of TMSOTf (115 μL, 0.635 mmol), the solution was
heated to 70 °C, stirred overnight, and quenched with a saturated
solution of NaHCO3, and the mixture worked up in ethyl acetate. The
1
mM TFA as eluent to give pure bisphosphate 5 (62 mg, 89%). H
NMR (400 MHz, D2O): 3.52 (dd, 1H, 9.78 Hz, 3.91 Hz, H-2″), 3.60−
3.79 (m, 5H, H-5″, H-5A′, H-5B′ H-6A″, H-6B″), 3.82 (dd, like a t, 9.29
Hz, 8.80 Hz, H-3″), 3.89 (ddd, like a q, 1H, 9.29 Hz, 8.80 Hz, 8.80 Hz,
H-4″), 4.31 (dd, 1H, 6.85 Hz, 3.42 Hz, H-4′), 4.52 (dd, 1H, 4.89 Hz,
3.42 Hz, H-3′), 5.11 (d, 1H, 3.91 Hz, H-1″), 5.18 (ddd, 1H, 9.78 Hz,
5.87 Hz, 5.38 Hz, H-2′), 6.23 (d, 1H, 6.36 Hz, H-1′), 8.28 (s, 1H, H-
2), 8.39 (s, 1H, H-8). 31P NMR (161.94 MHz, D2O with excess of
TEA): 4.22, 3.13. m/z (ES−) = 588.1 [(M − H), 50%]. HRMS
(ES−): mass calcd for C16H24O15N5P2 [M − H], 588.0750; found,
588.0751.
6-Amino-5′,2″,6″-tri-O-benzyl-3″,4″-bis-O-(dibenzyloxy-
phosphoryl)-3′-O-(α-D-glucopyranosyl)-9-β-D-ribofuranosyl-
purine (34). To a solution of trisphosphate 33 (60 mg, 0.0405 mmol)
in BnOH (2 mL) was added anhydrous K2CO3 (25 mg, 0.18 mmol),
and the mixture was stirred at 70 °C overnight. Chromatography with
30% ethyl acetate−hexane removed the benzyl alcohol, and then,
elution with 5% MeOH−CHCl3 gave the bisphosphate 34 (46 mg,
93%) as a colorless gum. 1H NMR (400 MHz, CDCl3): 3.46 (dd, 1H,
10.70 Hz, 3.30 Hz, H-5A′), 3.50 (dd, 1H, 9.78 Hz, 3.70 Hz, H-2″), 3.54
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(dd, 1H, 10.84 Hz, 2.38 Hz, H-5B′), 3.54−3.62 (m, 2H, H-6A″ and H-
6B″), 3.88 (ddd, 1H, 10.04 Hz, 3.83 Hz, 2.78 Hz, H-5″), 4.24−4.30 (m,
2H, H-3′ and H-4′), 4.27 (AB q, 2H, 47.56 Hz, 11.63 Hz, C−O−
CH2Ph), 4.35 (s, 2H, C−O−CH2Ph), 4.49 (ddd, like a q, 9.78 Hz,
9.51 Hz, 9.51 Hz, H-4″), 4.51 (AB q, 2H, 40.83 Hz, 12.03 Hz, C−O−
CH2Ph), 4.58−4.66 (m, 1H, H-2′), 4.72 (d, 1H, 3.44 Hz, H-1″), 4.82−
5.06 (m, 9H, 4 × P−O-CH2Ph, H-3″), 5.81 (br, 2H, NH2), 5.99 (d,
1H, 5.02 Hz, H-1′), 7.10−7.26 (m, 35 H, 7 × Ph), 7.91 (s, 1H, H-8),
8.17 (s, 1H, H-2). 31P NMR (161.94 MHz, CDCl3): −1.967, −1.223.
m/z (ES+) = 1243.16 [(M + Na)+, 100%], (ES−) 1218.64 [(M −
H)−, 100%]. HRMS (FAB, CsI/glycerol): mass calcd for
C65H67O15N5P2 [M]+, 1219.4103; found, 1219.4096.
(dd, 0.25H*, 8.9 Hz, 7.7 Hz, H-2 in β-anomer), 3.54, (dd, 0.75H, 9.5
Hz, 3.4 Hz, H-2 in α-anomer), 3.56 (m, 0.25H, buried, H-5 in β-
anomer), 3.64−3.79 (m, 2H, CH2-6 in α- and β-anomers), 4.09−4.14
(m, 1.5H, OH-1 and H-5 in α-anomer), 4.32−4.66 (m, 5.25H, H-4
and 2 × OCH2Ph in α- and β-anomers, H-3 in β-anomer), 4.69−4.76
(m, 0.75H, H-1 and POCH2Ph in β-anomer), 4.87−5.07 (m, 8.25H,
H-3 and 4 × POCH2Ph in α-anomer, 3 × POCH2Ph in β-anomer),
5.13 (dd, 0.75H, 3.2 Hz, 3.2 Hz, H-1 in α-anomer), 7.02−7.35 (m,
30H, Ph). 31P NMR (162 MHz, CDCl3): −2.27 (0.75P), −2.24
(0.25P), −1.99 (0.25P), −1.72 (0.75P). m/z (FAB+) 881 [(M + H)+,
+
90%], 91 [C7H7 , 100%]. HRMS: mass calcd for C48H50O12P2,
903.2670 [M + Na]+. Found, 903.2649. Elemental analysis calcd for
C48H50O12P2 (880.85): C, 65.45; H, 5.72. Found C, 65.4; H, 5.70.
*Approximately 3:1 mixture of α- and β-anomers; integrals are
therefore approximate.
3′-O-(α-D-Glucopyranosyl)-adenosine-3″,4″-bisphosphate
(6). A suspension of bisphosphate 34 (45 mg, 0.037 mmol) and 20%
Pd(OH)2 on carbon (120 mg) in a mixture of cyclohexene (1.6 mL),
MeOH (3 mL), and H2O (0.22 mL) was heated at 80 °C overnight.
After filtration through a membrane filter, the evaporated filtrate was
purified on an AG column using 0−100% gradient elution using 150
D-Glucopyranose 3,4-Bisphosphate (7). To a solution of 39
(225 mg, 0.255 mmol) in MeOH (20 mL) and water (5 mL) was
added Pd(OH)2−C (20%, 50% water, 600 mg). The mixture was
shaken in a Parr hydrogenator under H2 (50 psi) for 24 h. The catalyst
was removed by filtration through a PTFE syringe filter, and 1.0 M
TEAB (1 mL) was added. The solvents were removed by evaporation
under reduced pressure, and the residue was purified by ion-exchange
chromatography on Q-Sepharose Fast Flow resin eluting with a
gradient of triethylammonium bicarbonate (0−1 M). Fractions
containing the target compound were identified by a modification of
the Briggs phosphate test.48 The combined fractions were concen-
trated by evaporation in vacuo, and methanol was repeatedly added
and evaporated, eventually leaving the triethylammonium salt of 7 as a
colorless glass (0.216 mmol, 85%); [α]D +19 (c 1.5, MeOH). 1H
NMR (400 MHz, TEA+ salt, D2O, approximately 1:1 mixture of α- and
β-anomers): 3.41 (dd, 0.5H, 8.9 Hz, 8.5 Hz, H-2 in β-anomer), 3.55−
3.59 (m, 0.5H, H-5 in β-anomer), 3.70 (dd, 0.5H, 9.6 Hz, 3.8 Hz, H-2
in α-anomer), 3.77−3.88 (m, 2H, H-6A and H-6B in α- and β-
anomers), 3.92−3.96 (m, 0.5H, H-5 in α-anomer), 4.00−4.09 (m, 1H,
H-4 in α- and β-anomers), 4.23 (ddd, 0.5H, 9.0 Hz, 9.0 Hz, 9.0 Hz, H-
3 in β-anomer), 4.41 (ddd, 0.5 H, 9.0 Hz, 8.6 Hz, 8.6 Hz, H-3 in α-
anomer), 4.70 (d, 0.5H, 8.0 Hz, H-1 in β-anomer), 5.25 (d, 0.5H, 3.8
Hz, H-1 in α-anomer). 31P NMR (162 MHz, TEA+ salt, CD3OD, TEA
added, approximately 1:1 mixture of α- and β-anomers): 2.22 (0.5P),
2.63 (0.5P), 2.79 (0.5P), 3.04 (0.5P) m/z (FAB−) 338.9 [M−, 100%].
HRMS: mass calcd for C6H13O12P2, 338.9888 [M−]; found, 338.9894.
D-myo-Inositol 4,5-Bisphosphate (2). A sample of D-2,3,6-tri-O-
benzyl-myo-inositol 4,5-bis-O-(dibenzylphosphate)51 (94 mg, 0.10
mmol) was subjected to hydrogenolytic deprotection as described
for 39, above. Purification of the product by ion-exchange
chromatography on Q-Sepharose Fast Flow resin, as before, gave
the triethylammonium salt of 2 as a colorless glass (0.076 mmol, 76%);
[α]D −17 (c 1.0, MeOH), Lit.52 −15.4 (c 0.6, H2O, cyclo-
hexylammonium salt); Lit.39 −10 (c 1, H2O, tetrapotassium salt);
1
mM TFA as eluent to give pure bisphosphate 6 (20 mg, 92%). H
NMR (400 MHz, D2O): 3.74−3.90 (m, 6H, H-2″, H-5″, H-5A′, H-5B′
H-6A″, H-6B″), 4.10 (ddd, like a q, 1H, 9.66 Hz, 9.66 Hz, 9.66 Hz, H-
4″), 4.42 (dd, 1H, 7.24 Hz, 3.86 Hz, H-4′), 4.47−4.55 (m, 2H, H-3′, H-
3″), 4.87 (dd, like a t, 1H, 5.80 Hz, 5.31 Hz, H-2′), 5.19 (d, 1H, 3.38
Hz, H-1″), 6.19 (d, 1H, 5.80 Hz, H-1′), 8.40 (s, 1H, H-2), 8.50 (s, 1H,
H-8). 31P NMR (161.94 MHz, D2O with excess of TEA): 4.50, 3.62.
m/z (ES+) = 590.2 [(M + H)+, 100%]; 612.1 [(M + Na)+, 100%]; m/
z (ES−) 588.2 [(M − H)+, 100%]. HRMS: mass calcd for
C16H26O15N5P2 [M + H]+, 590.0895; found, 590.0895.
Allyl 2,6-Di-O-benzyl-3,4-bis-O-(dibenzyloxyphosphoryl)-α-
D-glucopyranoside (38). To a stirred solution of allyl 2,6-di-O-
benzyl-α-D-glucopyranoside (37) (400 mg, 1.00 mmol) and 1H-
tetrazole (280 mg, 4.00 mmol) in dry CH2Cl2 (5 mL) at room
temperature was added dibenzyl-N,N-diisopropylphosphoramidite (1.0
mL, 3.0 mmol). After 1 h, the mixture was cooled to −78 °C, and
mCPBA (1.0 g, 57%, 3.3 mmol) was added in portions over 1 min.
After a further 10 min at −78 °C, a solution of Na2SO3 (50 mL, 10%
W/V) was added, and the mixture was stirred vigorously for 2−3 min,
until the mixture began to freeze. The cooling bath was then removed,
and the mixture was allowed to reach room temperature and diluted
with CH2Cl2 (50 mL). The organic layer was separated, washed with a
saturated solution of NaHCO3 (50 mL), dried over MgSO4, and
concentrated. Purification of the residue by flash chromatography
(EtOAc/hexane 1:2 then 1:1) gave 38 as a colorless oil (846 mg, 0.919
mmol, 92%); [α]D +15.9 (c 2.3, CHCl3). 1H NMR (400 MHz,
CDCl3): 3.57 (dd, 1H, 9.7 Hz, 3.6 Hz, H-2), 3.72 (dd, 1H, 10.9 Hz,
2.1 Hz, H-6A), 3.76 (dd, 1H. 10.9 Hz, 4.4 Hz, H-6B), 3.84−3.90 (m,
2H, H-5 and OCH2CHCH2), 4.07−4.12 (m, 1H, one proton of
OCH2CHCH2), 4.38, 4.51 (ABq, 2H, JAB = 12.1 Hz, OCH2Ph),
4.47, 4.72 (ABq, 2H, JAB = 12.1 Hz, OCH2Ph), 4.61 (ddd, 1H, 9.8 Hz,
9.4 Hz, 9.4 Hz, H-4), 4.72 (d, 1H, 3.6 Hz, H-1), 4.90−5.08 (m, 9H, 4
× POCH2Ph and H-3), 5.16−5.21 (m, 1H, OCH2CHCHH cis),
5.26−5.32 (m, 1H, OCH2CHCHH trans), 5.82−5.93 (m, 1H,
OCH2CHCH2), 7.15−7.30 (m, 28H, Ph), 7.31−7.35 (m, 2H, Ph).
31P NMR (162 MHz, CDCl3): −2.28, −1.89. MS m/z (ES−); 919 [(M
Lit.53 −4.4 (c 0.3, H2O, pH 6); Lit.53 +7.9 (c 1.2, H2O, free acid). H
1
NMR (500 MHz, TEA+ salt, D2O): 3.60 (dd, 1H, 10.0 Hz, 2.8 Hz, H-
1), 3.70 (dd, 1H, 9.8 Hz, 2.8 Hz, H-3), 3.81 (dd, 1H, 9.7 Hz, 9.5 Hz,
H-6), 3.99 (ddd, 9.0 Hz, 9.0 Hz, 9.0 Hz, H-5), 4.07 (dd, 1H, 2.8 Hz,
2.8 Hz, H-2), 4.27 (dd, 1H, 9.3 Hz, 9.2 Hz, 9.2 Hz, H-4). 31P NMR
(162 MHz, D2O, TEA added): 4.50 (1P), 4.64 (1P). 31P NMR (162
MHz, TEA+ salt, D2O, TEA added): 4.50 (1P), 4.64 (1P). HRMS:
mass calcd for C6H13O12P2, 338.9888 [M−]; found, 338.9896.
− H)−, 45%], 829 [(M − C7H7)−, 100%]. HRMS: mass calcd for
C51H54O12P2, 943.2983 [M + Na]+; found, 943.2954. Elemental
analysis calcd for C51H54O12P2 (920.91): C, 66.51; H, 5.91. Found: C,
66.6; H, 5.97.
Measurement of Ca2+ Release from Permeabilized Cells. The
effects of AdA and its analogues on intracellular Ca2+ stores were
measured using a low-affinity Ca2+-indicator trapped within the
intracellular stores of permeabilized cells. DT40 cells stably expressing
only rat type 1 IP3R (DT40-IP3R1) were harvested by centrifugation
(650g; 2 min) and resuspended [(2−3) × 107 cells/mL] in hepes-
buffered saline (HBS: 135 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2,
1.5 mM CaCl2, 11.6 mM hepes, and 11.5 mM D-glucose, pH 7.3)
supplemented with Mag-fluo-4AM (20 μM), Pluronic F-127 (0.02%),
and bovine serum albumin (1 mg/mL). After 1 h at 20 °C in the dark,
the Mag-fluo-4-loaded cells were harvested (650g; 2 min) and
resuspended (∼2 × 106 cells/mL) in Ca2+-free cytosolic-like medium
(CLM: 140 mM KCl, 20 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and
2,6-Di-O-benzyl-3,4-bis-O-(dibenzyloxyphosphoryl)-D-glu-
copyranose (39). To a solution of 38 (400 mg, 0.434 mmol) in dry
methanol (5 mL) was added PdCl2 (20 mg, 0.11 mmol). The mixture
was stirred vigorously in a flask fitted with a drying tube (air is
required for the reaction) for 3 h, after which time TLC (ethyl acetate/
hexane 1:1) showed the reaction to be essentially complete, with
conversion of 38 (Rf 0.36) into two products (Rf 0.10 and 0.16). The
acidic solution was neutralized by stirring with excess NaHCO3 for 5
min, then filtered through Celite, and concentrated. Purification by
flash chromatography (EtOAc/hexane 2:3, then EtOAc) gave 39
(mixture of α and β anomers) as a colorless oil (311 mg, 0.353 mmol,
1
81%); [α]D +6.4 (c 1.1, CHCl3). H NMR (400 MHz, CDCl3): 3.46
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20 mM pipes, pH 7.0). The cells were permeabilized by incubation
with saponin (10 μg/mL, 4 min at 37 °C), harvested (650g; 2 min),
and resuspended in Mg2+-free CLM (140 mM KCl, 20 mM NaCl, 1
mM EGTA, 375 μM CaCl2 (∼200 nM free [Ca2+]), and 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 (∼8 × 105
cells/well) coated with poly-L-lysine (0.01%) and centrifuged onto the
plate (300g; 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
the addition of Mg-ATP (1.5 mM), and after 150 s, when the stores
had loaded to a steady-state Ca2+ content, AdA or its analogues were
added. The amount of Ca2+ released was expressed as a fraction of the
total Ca2+ content of the ER as assessed by addition of 1 μM
ionomycin. Data are presented as means SEMs from at least three
independent experiments, each performed in triplicate. Concentra-
tion−effect relationships were fitted to four-parameter logistic
equations using nonlinear curve-fitting procedures (GraphPad Prism,
San Diego, CA).
085295 to C.W.T.) and the EPSRC Mass Spectrometry Service
Centre, Swansea, for high-resolution accurate mass spectra.
ABBREVIATIONS USED
■
AdA, adenophostin A; IP3, D-myo-inositol 1,4,5-trisphosphate;
IP3R, D-myo-inositol 1,4,5-trisphosphate receptor; 2′-AMP, 2′-
adenosine monophosphate; SAR, structure−activity relation-
ship; IBC, IP3-binding core; BDA, butane 2,3-diacetal; NIS, N-
iodosuccinimide; BSA, N,O-bissilylacetamide; CSA, camphor-
sulphonic acid; TFA, trifluoroacetic acid; DBU, 1,8-diaza-
bicycloundec-7-ene; DCM, dichloromethane; THF, tetrahy-
drofuran; DMF, N,N-dimethylformamide; Ins(4,5)P2, D-myo-
inositol 4,5-bisphosphate; Ins(1,5)P2, D-myo-inositol 1,5-bi-
sphosphate; Ins(1,4)P2, D-myo-inositol 1,4-bisphosphate; ER,
endoplasmic reticulum
REFERENCES
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Maestro version 9.2.112 was used unless otherwise stated. Compounds
5, 6, and 40 were built and minimized. The 1N4K crystal structure and
the model of adenophostin A docked into IP3R were prepared using
the Protein Preparation Wizard, with exhaustive sampling, including
sampling the water orientations, for the hydrogen bond assignment,
and minimization of the resulting structures. GOLD version 5.1 was
used for docking experiments. In the structure with AdA, the
phosphates and the hexose were removed and replaced with hydroxyls.
The resulting adenine-ribose was used as a template when docking
compounds 5, 6, and 40. The compounds were docked 25 times, with
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orientation. The highest scoring solution had a sensible pose, and the
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five protein−ligand complexes were superimposed using the Protein
Structure Alignment tool. Ligand interaction diagrams were prepared
for all five ligands (IP3, adenophostins A, and compounds 5, 6, and 40)
with IP3R (see the Supporting Information). All other figures were
prepared using PyMOL (DeLano Scientific LLC).
ASSOCIATED CONTENT
■
S
* Supporting Information
Full synthetic details for compounds 10−12, 15, and 24−26;
13C NMR data for all compounds; overlays, molecular docking,
and ligand interaction diagrams for IP3, Ada, 5, 6, and 40 (SI-
1); and spectral charts of all compounds (SI-2). This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: ++44-1225-386639. Fax: ++44-1225-386114. E-mail:
B.V.L.Potter@bath.ac.uk.
Present Address
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§School of Chemistry, Indian Institute of Science Education
and Research, Thiruvananthapuram, Kerala-695016, India.
ACKNOWLEDGMENTS
■
We thank the Wellcome Trust for Programme Grant Support
(060544 to B.V.L.P., 082837 to B.V.L.P. and A.M.R., and
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