Synthesis and characterization of non-hydrolysable diphosphoinositol polyphosphate messengers

Article abstract of DOI:10.1039/c2sc21553e

The diphosphoinositol polyphosphates (PP-IPs) are a central group of eukaryotic messengers. They regulate numerous processes, including cellular energy homeostasis and adaptation to environmental stresses. To date, most of the molecular details in PP-IP signalling have remained elusive, due to a lack of appropriate methods and reagents. Here we describe the expedient synthesis of methylene-bisphosphonate PP-IP analogues. Their characterization revealed that the analogues exhibit significant stability and mimic their natural counterparts very well. This was further confirmed in two independent biochemical assays, in which our analogues potently inhibited phosphorylation of the protein kinase Akt and hydrolytic activity of the Ddp1 phosphohydrolase. The non-hydrolysable PP-IPs thus emerge as important tools and hold great promise for a variety of applications.

Full text of DOI:10.1039/c2sc21553e

Chemical Science
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Synthesis and characterization of non-hydrolysable
diphosphoinositol polyphosphate messengers†
Cite this: Chem. Sci., 2013, 4, 405
*
Mingxuan Wu, Barbara E. Dul,‡ Alexandra J. Trevisan‡ and Dorothea Fiedler
The diphosphoinositol polyphosphates (PP-IPs) are a central group of eukaryotic messengers. They regulate
numerous processes, including cellular energy homeostasis and adaptation to environmental stresses. To
date, most of the molecular details in PP-IP signalling have remained elusive, due to a lack of
appropriate methods and reagents. Here we describe the expedient synthesis of methylene-
bisphosphonate PP-IP analogues. Their characterization revealed that the analogues exhibit significant
stability and mimic their natural counterparts very well. This was further confirmed in two independent
biochemical assays, in which our analogues potently inhibited phosphorylation of the protein kinase Akt
and hydrolytic activity of the Ddp1 phosphohydrolase. The non-hydrolysable PP-IPs thus emerge as
important tools and hold great promise for a variety of applications.
Received 19th September 2012
Accepted 4th October 2012
DOI: 10.1039/c2sc21553e
www.rsc.org/chemicalscience
Traditionally, small diffusible messengers bind to particular
target proteins to control their activity or localization. It is
Introduction
Cellular decision-making is governed by the concerted actions
of signalling proteins and small molecule second messengers.
Among the eukaryotic second messengers, the diphosphoino-
sitol polyphosphates (PP-IPs) constitute an important class.
These molecules have captured the attention of both chemists
and biologists, because of their unique chemical structure and
their ability to mediate a plethora of interesting phenotypes.1
The PP-IPs are a group of highly phosphorylated signalling
molecules, based on the myo-inositol scaffold, that contain one
or two high-energy pyrophosphate groups. These phosphate
groups are installed in a biosynthetic pathway that is well
conserved from yeast to mammals (a simplied pathway
diagram is shown in Fig. 1).1
therefore commonly assumed that PP-IPs utilize an allosteric
mechanism to regulate protein function. However, only a
handful of targets have been identied to date, and in some
cases the biological relevance is not clear.4,5b,7 An alternative
signalling mechanism for the PP-IPs involves the transfer of the
high-energy b-phosphate group onto a phospho-serine residue,
yielding a pyrophosphorylated protein.8 But due to technical
challenges, it has only been possible to identify pyrophos-
phorylated proteins in biochemical assays and not from
complex cell lysates.9 Overall, progress in decoding PP-IP
The successive phosphorylation reactions are carried out by a
set of dedicated small molecule kinases. Genetic perturbation of
these kinases in yeast revealed important functions for PP-IPs in
many cellular processes, including telomere maintenance,2
response to oxidative stress,3 and nutrient sensing.4 Mice that
lack the kinase IP6K1 (inositol hexakisphosphate kinase 1) are
not able to produce sufficient amounts of 5PP-IP5 – the best
characterized PP-IP to date – in various tissues (Fig. 1).5 These
mice exhibit severe defects in insulin secretion, increased
peripheral insulin sensitivity, and resistance to age and diet-
induced obesity.5,6 While these phenotypes are truly remarkable,
the underlying molecularmechanisms have remainedenigmatic.
Fig. 1 The diphosphoinositol polyphosphate biosynthetic pathway, in abbrevi-
ated form. Phosphorylation of inositol 1,4,5-triphosphate (IP3) by inositol multi-
kinase (IPMK) and inositol pentakisphosphate 2-kinase (IPPK) results in formation
of inositol pentakisphosphate (IP5) and inositol hexakisphosphate (IP6). Both IP5
and IP6 are substrates for IP6K (inositol hexakisphosphate kinase) to yield the
diphosphoinositol polyphosphates 5PP-IP4 and 5PP-IP5, respectively. The
numbering of the ring positions is indicated in the IP3 structure.
Department of Chemistry, Princeton University, Washington Rd, Princeton, NJ, 08544,
USA. E-mail: dedler@princeton.edu; Tel: +1 609 258 1025
† Electronic supplementary information (ESI) available: Experimental procedures,
spectroscopic data, and supporting gures. See DOI: 10.1039/c2sc21553e
‡ These authors contributed equally to this work.
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Fig. 2 Comparison of the naturally occurring signalling molecule 5PP-IP5 (1)
with the analogues described in this study.
signalling has been hindered by a lack of suitable methods and
reagents. As current approaches rely on standard biochemical
and genetic techniques, there is a pressing need for chemical
tools that can help to decipher the discrete PP-IP signalling
functions.
Here we report the synthesis and characterization of a
number of non-hydrolysable methylene-bisphosphonate PP-IP
analogues (Fig. 2). We demonstrate that the analogues are good
mimics of the natural counterpart with regard to their confor-
mation in solution and their protein binding properties. Our
analogues thus represent an important set of mechanistic
probes that will be of great use for the inositol signalling
community, and we highlight
applications.
a
number of possible
Scheme
1
Synthesis of non-hydrolysable PP-IP analogues. Reagents and
conditions: (i) Benzyl((bis(benzyloxy)phosphoryl)methyl)phosphonochloridate,
KHMDS, THF, ꢀ78 ꢁC to rt, overnight; (ii) NaOMe, MeOH, rt, overnight; (iii) H2O, p-
TsOH, acetone, overnight; (iv) N,N-diethyl-1,5-dihydro-2,4,3-benzodiox-
aphosphepin-3-amine, 1H-tetrazole, CH3CN, 0 ꢁC to rt, 36 h; (v) mCPBA, CH3CN,
Results and discussion
Expedient synthesis of PP-IP analogues
0
ꢁC to rt, 3 h; (vi) H2, Pd black, NaHCO3, t-BuOH/H2O, rt, overnight; (vii) conc. aq.
NH3, rt, 4 days; (viii) Dowex-H+.
The methylene-bisphosphonate moiety has been used widely as
a stable substitute for diphosphate groups; an important
example is AMPPCP (b,g-methyleneadenosine 50-triphosphate),
a non-hydrolysable analogue of ATP.10 Among the PP-IP family
members, the 5PP-IP5 messenger is the most prominent one
and we therefore decided to focus on the synthesis of the non-
hydrolysable methylene-bisphosphonate 5PCP-IP5 (2, Fig. 2)
using the synthetic route outlined in Scheme 1.11 The protected
myo-inositol 5 can be obtained from myo-inositol in three
steps.12 The bisphosphonate group was then installed in the 5-
aqueous ammonia and yielded the non-hydrolysable analogue
5PCP-IP4 (3). Both 5PCP-IP4 (3) and 5PCP-IP5 (2) showed no
signs of decomposition in aqueous solution at neutral pH, aer
40 days at room temperature.
5PP-IP5 and 5PCP-IP5 exhibit a similar pH dependent
conformational change
position, to provide intermediate 6a in good yield. Removal of The pKa’s of a pyrophosphate group differ slightly from those
the benzoyl- and acetonide protecting groups furnished the of a bisphosphonate group,14 and it was important to deter-
pentaol 7b, which was subsequently phosphitylated and mine that 5PP-IP5 (1), and the non-hydrolysable analogue
oxidized (8b). The phosphate groups were unveiled as the free 5PCP-IP5 (2), exhibit the same properties in solution. Since the
phosphates via hydrogenation in the presence of NaHCO3, and conformation of the inositol ring depends on the ionization
5PCP-IP5 (2) was isolated as the sodium salt. Compared to the state of the individual phosphate groups, 1H and 31P NMR
synthesis of the natural molecule 5PP-IP5,13 the non-hydro- titration curves can be used to compare the solution properties
lysable analogue is accessible in good quantity and high purity, of 1 and 2.15
while requiring fewer synthetic steps.
To do so, 5PP-IP5 (1) was synthesized according to a modied
Another PP-IP family member is 5PP-IP4 (Fig. 1), a molecule literature procedure (Scheme S1†),13,16 and both 5PP-IP5 and
that has been linked to telomere maintenance and DNA damage 5PCP-IP5 were titrated in D2O (containing 140 mM K+, 10 mM
repair.2 The corresponding bisphosphonate analogue 5PCP-IP4 Na+, and 1 mM Mg2+ to mimic cellular metal cation content) at
(3) was synthesized using a slightly modied route: Removal of room temperature. Fig. 3 depicts a subset of the 1H NMR
the acetonide groups from 6a was followed by phosphitylation, titration data of the two molecules (for all data see Fig. S1 and
and oxidation (8a). Subsequent hydrogenation afforded a S2†). The ring inversion, which converts 5 equatorial/1 axial
diphosphoinositol phosphate analogue that is benzoyl-pro- substituents to 1 equatorial/5 axial substituents, occurred
tected in the 2-position (4). The benzoyl group was cleaved using around pD 8.8 for both molecules.17 While these data do not
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Recently, it was reported that 5PP-IP5 binds to the pleckstrin
homology (PH) domain of the protein kinase Akt (also known as
protein kinase B).5b This binding event was proposed to stabilize
Akt in an inactive conformation, which precludes Akt from
becoming phosphorylated on threonine 308 by the upstream
kinase PDK1 (3-phosphoinositide-dependent protein kinase,
Fig. 4a). We tested the inhibitory activity of both 5PP-IP5 and
5PCP-IP5 in a biochemical assay, using puried inactive Akt and
activated PDK1. The two kinases were incubated in the presence
of varying concentrations of 5PP-IP5 or 5PCP-IP5, and inhibition
of Akt phosphorylation at threonine 308 was monitored using a
phosphospecic antibody (Fig. 4b). As previously reported, 5PP-
IP5 potently inhibited Akt phosphorylation; under our assay
conditions, 5PP-IP5 displayed an IC50 of 217 nM.20 5PCP-IP5
closely resembled its natural counterpart with an IC50 of 129
nM. These data illustrate that changing the pyrophosphate
moiety to a methylene-bisphosphonate group did not signi-
cantly alter the affinity of the small molecule for a known
protein binding partner. Consequently, it should be feasible to
utilize 5PCP-IP5 as an affinity reagent to identify PP-IP protein
binding partners.
From a synthetic perspective, the 2-position of 5PCP-IP5
could most easily be derivatized for attachment to a solid
support. To determine how important the phosphate group in
the 2-position is for the binding interaction with Akt, we eval-
uated the analogues 5PCP-IP4 (3) and 2Bz-5PCP-IP4 (4), which
contain a hydroxyl group or a benzoyl group in the 2-position,
respectively. Remarkably, those structural changes did not
diminish their affinity for Akt (Fig. 4b).21 Encouraged by these
results, we are currently exploring a number of attachment
strategies for these molecules.
Fig. 3 (a) 5PP-IP5 and 5PCP-IP5 undergo a conformational change around pD
8.8. (b) 1H NMR titration curves for 5PP-IP5 (hollow circles for H4/6 and hollow
diamonds for H1/3) and 5PCP-IP5 (filled circles for H4/6 and filled diamonds for
H
1/3). Between pD 8.4 and pD 9.2 peaks could not be assigned due to severe
broadening of the resonances (see Fig. S1†). (c) 31P NMR titration curves for 5PP-
IP5 (hollow triangles for P2) and 5PCP-IP5 (filled triangles for P2).
provide a direct measurement of the pKa values for 1 and 2, the
titration curves highlight the similarity of the overall ionization
state of the two molecules.18
5PCP-IP5 exhibited much higher stability compared to 5PP-
IP5. Even at a low or high pD (2.0 or 13.0), no detectable
decomposition occurred aer 40 days at room temperature. The
non-hydrolysable PP-IP analogues will therefore be of great use
to characterize the physicochemical properties of the PP-IP
messengers. Furthermore, our lab is interested in the rich
metal-coordination chemistry of these molecules.19 The pres-
ence of highly Lewis acidic metal cations, such as Mg2+ and
Fe3+, promotes hydrolysis of the PP-IPs, but our bisphosphonate
analogues circumvent this problem and can be used as surro-
gates to delineate the metal-binding properties of PP-IP
molecules.
PP-IP analogues potently inhibit Akt phosphorylation in vitro
Fig. 4 (a) Binding of 5PP-IP5 is proposed to stabilize Akt in an inactive confor-
mation that cannot become phosphorylated by PDK1. (b) Western blots for Akt
inhibition experiments. Akt phosphorylation at threonine 308 (p-Akt) was
measured using a phosphospecific antibody. A Western blot for total Akt was
used as a loading control. IC50 values were determined in three independent
experiments, and the errors are indicated.
It is thought that PP-IPs can control protein activity or locali-
zation via allosteric regulation.4,5b,7 If 5PCP-IP5 indeed closely
mimics 5PP-IP5, it should exert the same allosteric control over
a given protein binding partner.
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submicromolar range (0.16 mM). As was also observed for Akt,
the benzoyl substituent in the 2-position does not lower the
binding affinity; in fact, it made the molecule signicantly more
potent. Possible explanations for the increased potency include
a favourable interaction of the benzoyl group with the protein,
and/or changes in solvation of the unbound 2Bz-5PCP-IP4,
which may decrease its ground-state stabilization compared to
the other analogues.
Based on its high potency, 2Bz-5PCP-IP4 may become useful
to inhibit Ddp1 activity in cell lysates to prevent hydrolysis of
Ddp1 substrates, thereby facilitating their identication and
analysis. Overall, the Ddp1 inhibition studies paralleled our
observations from the Akt assay, further validating that the
bisphosphonate analogues are adequate surrogates for the
natural molecules.
PP-IP analogues inhibit the Ddp1 phosphohydrolase in vitro
We next sought to validate the non-hydrolysable analogues in
a different biochemical assay, but examples of PP-IP binding
proteins in the literature are scarce.4,5b,7 Since the enzymes
involved in PP-IP metabolism must bind to their substrates,
we chose to investigate the binding of our analogues to the
yeast polyphosphate phosphatase Ddp1. Ddp1 hydrolyses
diadenosine polyphosphates, inorganic polyphosphates, and a
subset of PP-IPs.22 A recent report from Saiardi and co-workers
demonstrated that Ddp1 exhibited highly varied activity
towards different PP-IP isomers: while 1PP-IP5 – a PP-IP family
member with the pyrophosphate group in the 1-position – was
completely hydrolysed to IP6 aer 30 minutes at 37 ꢁC, 5PP-IP5
showed no signs of hydrolysis under the same conditions.22c
We therefore reasoned that 5PP-IP5 could be a competitive
inhibitor for Ddp1 when used in combination with other
Ddp1 substrates, such as diadenosine pentakisphosphate
(Ap5A).
Conclusions
Ddp1 was expressed and puried using an N-terminal poly-
histidine tag (His). When His–Ddp1 was incubated with Ap5A, it
displayed robust phosphohydrolase activity, as determined by
HPLC analysis of the reaction mixture (Fig. 5a).23 Next, varying
concentrations of 5PP-IP5 were added to the Ap5A hydrolysis
reaction. As expected, 5PP-IP5 acted as a competitive inhibitor
with an IC50 of 2.5 mM (Fig. 5b).24 Testing of 5PCP-IP5 and 5PCP-
IP4 revealed very similar inhibition proles (Fig. 5b and S4†),
which corroborates that the bisphosphonate moiety is a suit-
able mimic for the pyrophosphate group, as it is bound by the
protein with similar affinity. Interestingly, 2Bz-5PCP-IP4 dis-
played the highest potency with an IC50 value in the
Even though the PP-IPs were discovered almost 20 years ago, we
are just beginning to appreciate their complex functions as
central regulators of cell homeostasis. This gap in our under-
standing is due to the dearth of reagents available to study PP-IP
function in vivo and in vitro. The PP-IP analogues described in
this paper hold great promise for lling several of these gaps.
We have shown that the bisphosphonate group adequately
mimics the pyrophosphate group in PP-IPs, while providing
easier synthetic accessibility and increased stability. The non-
hydrolysable analogues can therefore be developed into affinity
reagents for the systematic identication of PP-IP interacting
partners. Subsequently, the increased stability of the analogues
will be advantageous for co-crystallizations of PP-IPs with their
binding partners.
The bisphosphonate analogues will also be highly informa-
tive with respect to the other PP-IP signalling mechanism,
protein pyrophosphorylation. Our compounds are not able to
transfer their b-phosphoryl group onto protein substrates,
which allow us to distinguish the two signalling mechanisms in
biochemical assays. Progress on using the analogues as mech-
anistic probes will be described in due course.
Until now there has been a reliance on genetic and molecular
biology techniques to study PP-IP function. A major focus,
however, should be on the molecules themselves. We believe
that the non-hydrolysable PP-IPs describe in this paper will help
to shi this imbalance, and will provide valuable insight into
the biological functions of PP-IP molecules.
Acknowledgements
We would like to thank Dr Adolfo Saiardi for providing the
constructs for Ddp1 expression, and the Muir, Doyle, MacMil-
lan, and Sorensen groups for use of their chemicals and
instruments. We would also like to thank Dr. Adam Resnick for
helpful advice, and Dr. Istvan Pelczer and Dr. Simona Ghizdavu
Pellascio for assistance with the NMR titration experiments.
Financial support was provided by Princeton University, and the
NIH (R00 GM087306).
Fig. 5 (a) Ddp1 hydrolyzes diadenosine pentakisphosphate (Ap5A) to adenosine
monophosphate (AMP) and adenosine tetrakisphosphate (p4A), which is subse-
quently further degraded. (b) Ddp1 mediated Ap5A hydrolysis is inhibited by 5PP-
IP5 and the analogues. IC50 values for 5PP-IP5, 5PCP-IP5, 5PCP-IP4 and 2Bz-5PCP-
IP4 were determined in three independent experiments and the inhibition curves
are shown (see also Fig. S4†).
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11 We have also considered the incorporation of
a
diuoromethylene-bisphosphonate (PCF2P) group, because
the PCF2P group more closely resembles the pKa values of
the pyrophosphate group.10a However, the PCF2P analogues
posed a signicant synthetic challenge, and we ultimately
focused our efforts on the methylene-bisphosphonates. We
acknowledge that the differences in the pKa’s of 5PP-IP5
versus 5PCP-IP5 could be an issue, but, as our subsequent
biochemical studies show, these differences in pKa do not
appear to be a signicant limitation in the two examples
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16 An HPLC purication step before the nal deprotection
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17 The assignments of conformations was based on previous
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1H–31P HSQC (see ESI†).
18 Interestingly, there is a noticeable difference in the chemical
shis of H4/6 for 1 versus 2 at basic pH. We believe that subtle
changes in Mg2+ coordination may be responsible for this, as
the difference in chemical shi is less pronounced when the
NMR spectra were recorded in the absence of Mg2+ (see
ESI†).
19 Nothing is known about the coordination chemistry of PP-
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20 The published IC50 value is 20 nM. Presumably differences
in buffer composition and/or incubation times are
responsible for this discrepancy.
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21 2Bz-5PCP-IP4 appeared to be the most potent. This trend
was paralleled in subsequent experiments using the yeast
D. Bosch, O. Loss, C. Azevedo and A. Saiardi, J. Biol. Chem.,
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Ddp1 phosphohydrolase and is discussed in the following 23 We ruled out the possibility that the reaction product IP6 may
section.
be responsible for this inhibition, as the IC50 value for IP6 in
this reaction is more than ten times higher (data not shown).
22 (a) J. L. Cartwright and A. G. McLennan, J. Biol. Chem., 1999,
274, 8604; (b) S. T. Safrany, S. W. Ingram, J. L. Cartwright, 24 As a control, we conrmed that Ap5A showed no signs of
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hydrolysis when incubated with His–Ddp1 EE/AQ,
catalytically inactive form of Ddp1 (Fig. S3†).
a
410 | Chem. Sci., 2013, 4, 405–410
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