A synthetic cyclitol-nucleoside conjugate polyphosphate is a highly potent second messenger mimic

Article abstract of DOI:10.1039/c9sc00445a

Reactions that form sec-sec ethers are well known, but few lead to compounds with dense functionality around the O-linkage. Replacement of the α-glucopyranosyl unit of adenophostin A, a potent d-myo-inositol 1,4,5-trisphosphate (IP₃R) agonist, with a d-chiro-inositol surrogate acting substantially as a pseudosugar, leads to "d-chiro-inositol adenophostin". At its core, this cyclitol-nucleoside trisphosphate comprises an ether linkage between the axial 1-hydroxyl position of d-chiro-inositol and the 3′-hydroxyl group of an adenosine ribose sugar. A divergent synthesis of d-chiro-inositol adenophostin has been achieved. Key features of the synthetic strategy to produce a triol for phosphorylation include a new selective mono-tosylation of racemic 1,2:4,5-di-O-isopropylidene-myo-inositol using tosyl imidazole; subsequent conversion of the product into separable camphanate ester derivatives, one leading to a chiral myo-inositol triflate used as a synthetic building block and the other to l-1-O-methyl-myo-inositol [l-(+)-bornesitol] to assign the absolute configuration; the nucleophilic coupling of an alkoxide of a ribose pent-4-ene orthoester unit with a structurally rigid chiral myo-inositol triflate derivative, representing the first sec-sec ether formation between a cyclitol and ribose. Reaction of the coupled product with a silylated nucleobase completes the assembly of the core structure. Further protecting group manipulation, mixed O- and N-phosphorylation, and subsequent removal of all protecting groups in a single step achieves the final product, avoiding a separate N6 protection/deprotection strategy. d-chiro-Inositol adenophostin evoked Ca²⁺ release through IP₃Rs at lower concentrations than adenophostin A, hitherto the most potent known agonist of IP₃Rs.

Full text of DOI:10.1039/c9sc00445a

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Chemical Science
ARTICLE
A synthetic cyclitol-nucleoside conjugate polyphosphate is a
highly potent second messenger mimic†
Wolfgang Dohle,a¶ Xiangdong Su,a¶ Stephen J. Mills,a¶ Ana M. Rossi,b Colin W. Taylor,b
Received 00th January 20xx,
Accepted 00th January 20xx
and Barry V. L. Potter *a
DOI: 10.1039/x0xx00000x
Reactions that form sec-sec ethers are well known, but few lead to compounds with dense functionality around the O-
linkage. Replacement of the α-glucopyranosyl unit of adenophostin A, a potent D-myo-inositol 1,4,5-trisphosphate (IP3R)
agonist, with a D-chiro-inositol surrogate acting substantially as a pseudosugar, leads to “D-chiro-inositol adenophostin”. At
its core, this cyclitol-nucleoside trisphosphate comprises a nucleoside sugar linked via an axial D-chiro-inositol 1-hydroxyl-
adenosine 3'-ribose ether linkage. A divergent synthesis of D-chiro-inositol adenophostin has been achieved. Key features of
the synthetic strategy to produce a triol for phosphorylation include a new selective mono-tosylation of racemic 1,2:4,5-di-
O-isopropylidene-myo-inositol using tosyl imidazole; subsequent conversion of the product into separable camphanate
ester derivatives, one leading to a chiral myo-inositol triflate used as a synthetic building block and the other to L-5-O-methyl-
myo-inositol [L-(+)-bornesitol] to assign the absolute configuration; the nucleophilic coupling of an alkoxide of a ribose pent-
4-ene orthoester unit with a structurally rigid chiral myo-inositol triflate derivative, representing the first sec-sec ether
formation between a cyclitol and ribose. Reaction of the coupled product with a silylated nucleobase completes the
assembly of the core structure. Further protecting group manipulation, mixed O- and N-phosphorylation, and subsequent
removal of all protecting groups in a single step achieves the final product, avoiding a separate N6 protection/deprotection
strategy. D-chiro-Inositol adenophostin evoked Ca2+ release through IP3Rs at lower concentrations than adenophostin A,
hitherto the most potent known agonist of IP3Rs.
www.rsc.org/
not been widely investigated.14 Beyond GPIs, derivatives of myo-
inositol can make simple conjugated derivatives with sugars; these
are much less common and only a few natural products are known.
Introduction
Inositols are present in cells from every domain of life (archaea,
bacteria and eukaryotes), with myo-inositol the most abundant of
the nine inositol stereoisomers.1 Polyphosphorylated derivatives of
myo-inositol are common in biology and have many functions2,
including roles in regulating ion-channels,3 phosphate levels,4
metabolic flux,5 transcription, mRNA export and translation,6 insulin
signalling, embryonic development7 and stress responses.8 D-myo-
Inositol 1,4,5-trisphosphate (IP3, 1, Figure 1), which is an intracellular
messenger that evokes Ca2+ signals after binding to its own receptor
(IP3R)1a,9 and higher myo-inositol polyphosphates are subjects of
intense interest.1a,10 Such compounds are implicated in diverse areas
of biology and disease.11 myo-Inositol is also a component of
glycosylphosphatidylinositols (GPIs, see compound 2, Figure 1),12
which anchor proteins to the cell surface and are present in all
eukaryotic cells and some bacteria.13 Other stereoisomers of inositol
also have biological activities,1a but these are less common or have
In general, an equatorial hydroxyl group of an inositol is linked to the
glycosidic C1 of a sugar. Examples of the rarer inositol derivatives are
fagopyritol (3 & 4) and pinitol derivatives (5 & 6) which contain a D-
chiro-inositol-galactose conjugate, and are isolated from various
plant sources (Figure 1).15 Most of the conjugates have a galactose
unit connected via its glycosidic C1 to an equatorially configured
oxygen at either C3 (3), C2 (4 & 5) or C5 (not shown) of the D-chiro-
inositol unit, with further conjugates sometimes added at the
primary C6 hydroxyl group of galactose (see compounds 3 & 4, Figure
1). Only one example was found where the axial configured oxygen
at C1 of D-chiro-inositol forms an axial/axial configured glycosidic
bond (see compound 6, Figure 1).
Complex, but feasible, synthetic routes to such compounds and GPIs
require conjugation at the sugar C1 glycosidic centre. GPIs are sugar
conjugates, where a D-myo-inositol derivative is usually oxygen-
linked from its C6 positon to a D-glucosamine unit at its glycosidic C1
position. A phospholipid moiety at C1 of the D-myo-inositol unit
enables GPIs to insert into the plasma membrane. The difficulty of
isolating and purifying GPIs from biological sources lead to the
development of methodologies for the synthesis of GPIs, GPI-
anchored proteins and glycoproteins.16-19 To our knowledge, such
sec-sec inositol-hexose sugar conjugates are rare, apart from those
at the glycosidic centre. Only one example of a pseudo-disaccharide
a.Medicinal Chemistry & Drug Discovery, Department of Pharmacology, University of
Oxford, Mansfield Road, Oxford, OX1 3QT, UK.E-mail: barry.potter@pharm.ox.ac.uk;
Tel: +44-1865-271945
b.Department of Pharmacology, University of Cambridge, Tennis Court Road,
Cambridge, CB2 1PD, UK.
†Electronic Supplementary Information (ESI) available: Experimental protocols and
NMR spectra. See DOI: 10.1039/x0xx00000x
¶Equal contribution
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could be found20 and there were no examples of inositol-pentose pharmacophore and which are more potent than IP3 1 in evoking Ca2+
sugar conjugates. However, one example of a prim-sec ether linkage release through IP3 receptors (IP3Rs).
is known, for the antibiotic adenomycin 7 (Figure 2).21 This
IP3Rs are intracellular Ca2+ channels that allow IP3, produced when
compound contains one adenosine unit linked at its primary C5 to C3
extracellular stimuli promote hydrolysis of phosphatidylinositol 4,5-
of an L-chiro-inositol derivative to form a prim-sec ether bond.
bisphosphate, to release Ca2+ from intracellular stores.23 The
Additionally, a hemi-acetal is located at the glycosidic C1 of an L-
resulting Ca2+ signals regulate diverse cellular activities.24 Opening of
gulosamine linked to the L-chiro-inositol C1. Interestingly, contrary to
the Ca2+-permeable channel of the IP3R is initiated when IP3 binds to
glycosidic connections in GPIs and in 3-6, the latter linkage in 7 is
reported to be β-glycosidic.21 Given the extensive biology of myo-
inositol sugar conjugates and growing interest in the other eight
inositol isomers1a, there is a need to generate analogues to construct
the IP3-binding core (IBC, residues 224-604) of each of the four
subunits of the tetrameric IP3R (Figure 3A and B).25,26 Structure-
activity analyses,27 structures of N-terminal fragments of IP3R25,28,29
and of complete IP3R,9b alongside mutagenesis studies have
prim-sec or more particularly sec-sec ether linkages. In the latter
established that IP3 initiates IP3R activation by causing partial closure
case, an example of particular interest can be drawn from the Ca2+-
of the clam-like IBC. The 1- and 5-phosphates of IP3 interact primarily
signalling field in the form of the adenophostins 8 & 9 (Figure 2).22
with residues in the α-domain of the IBC, while the 4-phosphate
These are naturally occurring fungal metabolites with
a
phosphorylated glucose component that resembles part of the IP3
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interacts with IBC- (Figure 3A). Adenophostin
ARTICLE
(8) and and the ribose sugar. Linkages are usually made betwVeieewnAartirceleaOcntilivnee
A
adenophostin B (9) bind to IP3R with greater affinity than IP3 and they sugar glycosyl acceptor at the 1-positionDOanI:d10.i1n0o3s9i/tCo9l,SCb0o0t4h45iAn
are 6-10-fold more potent than IP3 in evoking Ca2+ release.30,31 protected form, to give inositol-sugar conjugates after deprotection
Hitherto, adenophostin A is the most potent known agonist of IP3R.31 (Figure 1). Non-glycosidic conjugates of this type are quite rare.
Our model for the interaction of adenophostin A with the IBC,32 However, some years ago in the synthesis of a pseudo-disaccharide
which is supported by mutagenesis33 and SAR analyses,34,35 suggests a cyclitol derivative was used as the nucleophile and a triflate on a
that the glucose 3'',4''-bisphosphate motif of adenophostin A mimics rigid sugar unit as the electrophile.20 This strategy would not easily
the 4,5-bisphosphate motif of IP3, while the 2''-hydroxyl and 2'- work in our case because a D-chiro-inositol derivative would need to
phosphate of adenophostin A mimic the non-essential 6-hydroxyl react as a nucleophile ideally with a triflate at the 3-position of a
and 1-phosphate groups of IP3.25,36 A recently published cryo-EM xylose derivative. SN2 Reactions using 3-trifluoromethanesulfonyl-
structure of IP3R1 with and without adenophostin A bound37 hints at and 3-tosyl-xylose derivatives are described, but coupled products
substantially different binding modes for adenophostin A. However, are only achieved when using non-alkaline nucleophiles like azide or
the resolution of ligands in these structures is far from optimal and thiophenolate.38,39 With the hydrogen at C4 in xylose placed almost
the ligand structure itself is chemically incorrect.
anti-periplanar to the leaving group, the less reactive 3-tosyl
derivative undergoes mainly elimination to form an olefin when
reacted with alkoxides, but cleavage of the tosyl group is also
observed.39
We focused on developing a synthetic route to “D-chiro-inositol
adenophostin” 10. The aim was to replace the α-glucopyranosyl unit
of 8 with the most similarly-configured inositol unit (D-chiro-inositol,
acting substantially as a pseudo-sugar). The resulting ligand would We report here a divergent strategy for the synthesis of 10 that
allow us to study any potentially novel effects of this modification on includes the first sec-sec ether formation between a cyclitol and a
Ca2+ release. Since the phosphorylated inositol-nucleoside hybrid ribose sugar as one key feature. Others are a selective mono-
closely resembles adenophostin A, it would establish whether nature tosylation of a racemic inositol diol derivative and subsequent
evolved a phosphorylated glucose moiety as the best mimic of the conversion of the product into diastereoisomeric camphanate esters
inositol bisphosphate motif or whether the activity of adenophostin separable by column chromatography. One diastereoisomer
A can be still further improved by replacing glucose with a motif more provides the chiral inositol triflate to be used as a building block, and
similar to the cyclitol in IP3. Finally, and importantly, if high the other provides a route to L-(+)-bornesitol in order to confirm
adenophostin A-like potency were achieved in 10, the hybrid would absolute configurations. Coupling of the inositol triflate with the
offer an axial hydroxyl group at the D-chiro-inositol C6 position (C2 ribose alkoxide of
a pent-4-ene orthoester derivative and
position in myo-inositol), not present in adenophostin A. This subsequent reaction with a silylated nucleobase assembles the core
position could be modified (eg via extended substituents) to target structure for phosphorylation. Pan-phosphorylation of all hydroxyl
the cleft between the IBC domains (Figure 3B) and might facilitate and amino groups and removal of all protecting groups inventively in
the design of high-affinity ligands as potential antagonists by virtue a single step affords the final product 10 without recourse to an
of their ability to prevent closure of the IBC clam. Such a strategy adenine N6-protection strategy.
cannot be envisaged for adenophostin A.
Examining all the bonds that need to be formed to assemble 10, it
became clear that its total synthesis would be challenging, especially
the formation of the sec-sec ether bond between the inositol ring
Results and Discussion
Chemistry
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DOI: 10.1039/C9SC00445A
The retrosynthetic analysis of D-chiro-inositol adenophostin 10
(Scheme 1), leads to 14 as the first compound containing all three
components, D-chiro-inositol, ribose and a purine base as derivatives.
An initial analysis for the formation of the sec-sec ether suggested
that myo-inositol triflates may be used. Three such potential triflates
were synthesised in turn, with increasing rigidity of the inositol ring
(18-20, Figure 4).
Initially, although triflate 18 in the chair conformation did not have
an anti-periplanar H-Ins-OTf motif, the triflate eliminated to form a
double bond in the presence of an alkoxide, demonstrating that the
inositol framework needed to be more rigid. 13C NMR assignment
indicated a quaternary carbon derived from proton abstraction
located at δc = 154.1 and an olefinic C-H at δc = 97.0. The rigidity of
the inositol ring was further increased to give derivative 19.
However, elimination in the presence of base was still observed and
similar olefinic fingerprint signals in the 13C NMR were seen as above.
A still more constrained structure was an obvious further choice, so
no elimination should occur. A third compound 20, provided a more
rigid inositol triflate and the crude product from alkoxide treatment
was purified by column chromatography. However, elimination still
occurred and two products were observed in the 1H NMR spectrum.
After a number of inositol triflates eliminated to form cyclic vinyl
ethers, triflate 16 (and the most difficult to manipulate to the final
product) was prepared (Scheme 5) and successfully used as a
functionalised chiral myo-inositol intermediate for the preparation
of the D-chiro-inositol derivative 14. An inositol triflate with a similar
protecting group pattern has been previously reported in SN2
reactions. However, only non-basic nucleophiles such as fluoride,
azide and thioacetate were used, but no alkoxides.40
problem in the required SN2 type reaction is the use of highly
substituted, sterically hindered starting materials. A limited number
of electrophiles, such as a triflate within a rigid organic framework
have been used successfully.20,41 Compound 14 could be assembled
in two steps from the ribose orthoester derivative 15. Note that
although 15 is depicted as one isomer at the orthoester moiety,
which is indeed the case, in fact the exact stereochemistry at the
quaternary carbon has not been proven. Diverse attempts to
derivatise 15 and 23 to give a crystalline product for X-ray studies
were made, but all of these failed. Compound 15 was coupled to the
L-myo-inositol triflate derivative 16. Subsequent anomeric coupling
of the orthoester group with silylated 6-chloropurine 17 gave
compound 14.
The ribose fragment of 10 was synthesised in four steps from D-(-)-
ribose 21 to give the pent-4-ene orthoester 22.43 Reaction of 22 with
sodium methoxide in methanol furnished diol 23 in high yield.43
Selective benzylation of the primary hydroxyl group using silver
carbonate and benzyl bromide in toluene gave compound 15 in good
yield (Scheme 2).44
Synthesis of the chiral L-myo-inositol triflate derivative 16 started
from racemic 1,2:4,5-di-O-isopropylidene-myo-inositol 2445
(Schemes 3 & 4). In order to convert an L-myo-inositol derivative into
the D-chiro-inositol derivative via an SN2 reaction a triflate was
Although the formation of sec-sec ethers is well known, only a few
examples lead to densely functionalised compounds.20,41,42 The main
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required as a leaving group at C3. The hydroxyl group at C6 was NMR and optical rotation data.50 The chiral myo-inositol triflate 16
protected as p-methoxybenzyl (PMB) ether, but selective was then synthesised in good overall yield from 29 by ester hydrolysis
a
protection at the more reactive 3-hydroxyl group was required. and reaction of chiral alcohol 34 with triflic anhydride and pyridine in
Selective mono-tosylation of the hydroxyl group at C3 of 31 using dichloromethane (Scheme 4).
tosyl chloride in pyridine lead to low yields of compound 25.46
However, a new method was devised for this conversion using tosyl
imidazole47 in the presence of cesium fluoride in DMF at room
temperature. Compound 25 was obtained in good yield along with a
minor amount of the bis-tosylated product 26. Reaction of 25 with
sodium hydride and p-methoxybenzyl chloride in DMF gave
compound 27 in high yield. Subsequent removal of the tosyl group
Compound 15 was first dissolved in anhydrous THF/HMPA before
adding sodium hydride to generate the alkoxide. The chiral triflate 16
was then added to the alkoxide to give 35 in 86% yield, transforming
the L-myo-inositol unit into the desired D-chiro-inositol derivative by
inversion of configuration at C3. DMPU could also be used to replace
HMPA as a co-solvent, but gave overall slightly lower yields (about
10% less). However, no reaction was observed when DMF alone was
used as a solvent. Subsequent coupling of 35 via its pent-4-ene
orthoester group with silylated 6-chloropurine51 in the presence of
an activator under anhydrous conditions gave compound 14 in good
was achieved by dissolving 27 in dichloromethane followed by the
addition of methanol and magnesium turnings.48 Compound 28 was
treated with (1S)-(-)-camphanic chloride49 to afford diastereoisomers
29 and 30, which were separated by column chromatography and
yield.43b The trans-isopropylidene group was then selectively
both were isolated in good yield (Scheme 3).
removed by treatment with ethylene glycol and p-toluenesulfonic
The absolute configuration was determined using diastereoisomer acid in dichloromethane for no longer than 20 minutes to give
30, which was converted in several steps via ester hydrolysis, compound 13 in almost quantitative yield. After various attempts,
methylation and protecting group removal via 31 and 32 into the we found that a one-pot reaction procedure strategy was unsuitable
known natural product L-(+)-bornesitol 33; the configuration of this to mono-benzylate compound 13, since the harsh reaction
product was confirmed by comparison to previously reported 1H conditions resulted in complex mixtures and the desired compound
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36 was not observed.52 Selective mono-benzylation of 13 was best have been reported to be labile under acidic and/or hydrogenation
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achieved in a two-stage procedure.53 First, 13 was reacted with n- reaction conditions55 such a contaminant should therefore convert
DOI: 10.1039/C9SC00445A
Bu2SnO in refluxing acetonitrile using a Soxhlet condenser containing into the free amino group during the final step of our synthesis. This
activated 3Å MS. The resulting tin acetal was isolated and directly idea thus argued for a pan-phosphitylation approach, whereby 11
converted into the desired mono-benzylated compound 36 in 26% was first phosphitylated with an excess of reagent that easily leads
yield using benzyl bromide, cesium fluoride and tetra-n- to the tris-O-phosphitylated mono-N-phosphitylated product that
butylammonium iodide in anhydrous DMF. However, no could be converted to the mixed tris-phosphate/mono-
regioselectivity was observed and the other mono-benzylated phosphoramidate 38 upon oxidation. To illustrate this further, the
compound 37 was isolated in a similar yield together with conversion of 11 to the fully deblocked 10 was carried out on a <10
unconverted starting material 13 (Scheme 5).
mg scale. Compound 11 was treated with 5-phenyl-1H-tetrazole and
dibenzyl diisopropylphosphoramidite56 in dichloromethane followed
by oxidation of the product with t-butyl hydroperoxide to achieve the
fully protected compound 38 in good overall yield (Scheme 6). The
N-phosphate group was readily distinguished from the O-phosphate
by its broad 31P NMR resonance peak at δ -0.9 in comparison to sharp
peaks at δ -1.2 and -1.5 (two overlapping peaks) respectively.
Subsequent treatment of 38 with hydrogen generated from
cyclohexene in the presence of palladium hydroxide on carbon in
methanol and water at 70 °C overnight led to an acidic reaction
mixture (pH 3), as all O- and N-benzylated phosphate groups were
converted to their free acids. Subsequently, this also lead to removal
of the cis-isopropylidene group and degradation of the in situ
generated acid-labile free N-phosphate by nucleophilic cleavage of
its P-N bond.55 As a pleasing result, therefore, all different types of
protecting group were removed in a single step to give D-chiro-
inositol adenophostin 10 (Scheme 6). Yields of 10 were routinely
quite variable and possibly complicated by retention of compound
on the hydrogenation catalyst. The best yield achieved so far is 37%.
We imagine that this mixed O- and N-phosphitylation methodology,
avoiding a sometimes cumbersome extra N-protection strategy, will
find application in many nucleotide synthesis situations where only
small amounts of compound are available. For biological evaluation,
10 was purified by semi-prep HPLC (purity >99%) and quantified by
UV spectroscopy. Final structural characterisation was additionally
confirmed by 1H-31P NMR correlation (see SI).
Small samples of both chromatographically separable regioisomers
36 and 37 were treated with TFA/DCM to remove the p-
methoxybenzyl group and the products then analysed by H NMR
1
and COSY without and with the addition of D2O. All hydroxyl protons
and the corresponding adjacent cyclitol ring protons were identified.
The 1,2-diol relationship was indicated by a strong coupling between
these two adjacent ring protons. For the 1,3-diol relationship that
coupling was not found. Compound 36 led to a 1,2-diol, whereas 37
led to a 1,3-diol. Therefore, 36 thus was identified as the required 2-
O-benzylated derivative that was then treated with ammonia in
ethanol at 70 °C to give 12 in 69% yield with concomitant removal of
the benzoyl ester (Scheme 6). After trying various methods we found
that removal of the PMB protecting group was best achieved using
TFA in anhydrous dichloromethane for no longer than 5 minutes.54
Longer reaction times and/or traces of moisture usually lead to
increasing amounts of more polar by-products where the cis-
isopropylidene protecting group had also been removed. For the
final phosphorylation of triol 11 we had originally envisaged use of
imidazolium triflate33,35d as a means of achieving O-selective
phosphitylation over N-phosphitylation. While this approach has
been used well in the past, we found it to be very cumbersome for
the phosphorylation of low milligram quantities of triol. It was not
possible to achieve complete selective phosphitylation and to
completely exclude water, then titrate in the required amount of
phosphitylating reagent without achieving substantial amounts of
concomitant N-phosphitylation, although products from this could
be easily separated. We therefore reasoned that since N-phosphates
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Biology
-chiro-Inositol adenophostin 10 was investigated as an agonist
at the IP3R using an intracellular Ca2+ mobilisation assay in
comparison to adenophostin A and IP3 . We used a low-
affinity Ca2+ indicator within the intracellular stores of
permeabilized HEK cells expressing IP3R1 to record Ca2+ release
evoked by synthetic ligands. Maximally effective concentrations
myo-inositol diol derivative and subsequent elaboration of the
product into separable camphanate derivatives of a fully
protected intermediate. One such diastereoisomer is converted
to L-(+)-bornesitol to confirm the absolute configuration and the
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DOI: 10.1039/C9SC00445A
D
8
1
other leads to the required chiral myo-inositol triflate that is
used as a synthetic building block. Critical formation of a sec-sec
ether used a rigidly structured chiral inositol triflate with an
alkoxide of a suitable protected ribose derivative; subsequent
reaction of the fully protected coupled product with a silylated
nucleobase assembled the core structure and base amination
and protecting group manipulations finally afforded a triol for
phosphorylation. After phosphitylation of both the adenine
amino group and inositol hydroxyl groups, removal of all
protecting groups was accomplished in a single step to afford
the final product. Thus, replacement of the α-glucopyranosyl
of IP3, adenophostin A
caused release of a similar fraction (~70%) of the Ca2+
sequestered by intracellular stores. However, adenophostin A
and 10 were 5.3 and 8.9-fold more potent than IP3, respectively.
Moreover, -chiro-inositol adenophostin 10 was 1.7-fold more
potent than AdA (Figure 5 and Table 1).
8 or D-chiro-inositol adenophostin 10
8
D
8
unit in adenophostin A with the equivalent
motif leads to the trisphosphate -chiro-Inositol adenophostin
10
10 Was evaluated as an agonist for intracellular Ca2+ release
through the IP3R and its EC50 of 21 nM exceeded that of (35
D-chiro-inositol
D
.
8
nM). It is the most potent agonist of IP3Rs so far identified and
may offer new opportunities to develop high-affinity
antagonists of IP3R.
Conflicts of interest
There are no conflicts to declare.
Figure 5
adenophostin A
means ± SEM, n = 6) show the Ca2+ release evoked by the indicated
concentrations of IP3 adenophostin or -chiro-inositol
adenophostin 10 from the intracellular stores of permeabilized HEK cells
expressing IP3R1.
D
-chiro-Inositol adenophostin 10 is more potent than
Acknowledgements
8
in evoking Ca2+ release through IP3R. Results (%,
This research was supported by the Wellcome Trust. B.V.L.P.
and C.W.T. are Wellcome Trust Senior Investigators (grants no.
101010 and 101844). A.M.R. is a Fellow at Queens’ College,
Cambridge. We thank Dr J.M. Watt for HPLC support and Dr
1
,
A
8
D
Table
1
Effects of IP3, adenophostin A 8 and D-chiro-inositol A.M. Riley for discussions and help with graphics.
adenophostin 10 on Ca2+ release from the intracellular stores of
permeabilized HEK-IP3R1 cells.
Ca2+ release
Notes and references
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Graphical abstract
A densely functionalised phosphorylated chiro-inositol-nucleoside ether conjugate
constructed from cyclic fragments is the most potent IP3 receptor ligand discovered.