Synthesis of a Tethered myo -Inositol (1,3,4,5,6)Pentakisphosphate (IP5) Derivative as a Probe for Biological Studies

Article abstract of DOI:10.1055/s-0035-1560381

There is sufficient evidence to suggest that myo-inositol pentakisphosphate is a vital intermediate species in higher inositol phosphate metabolism, however, its biological roles and physiological function in cells remain uncertain. A tethered myo-inosito

Full text of DOI:10.1055/s-0035-1560381

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Synthesis of a Tethered myo-Inositol (1,3,4,5,6)Pentakisphos-
phate (IP5) Derivative as a Probe for Biological Studies
Mark Gregory*a
Bruno Catimelb
Meng-Xin Yina
Melanie Condronb
Antony W. Burgessb
Andrew B. Holmes*a
a School of Chemistry, Bio21 Institute, The University of
Melbourne, 30 Flemington Road, Parkville, Victoria 3052,
Australia
m.gregory4@pgrad.unimelb.edu.au
aholmes@unimelb.edu.au
b Walter and Eliza Hall Institute of Medical Research, 1G
Royal Parade, Parkville, Victoria 3052, Australia
This paper is dedicated to Prof. Steven Ley on the occasion
of his 70th birthday and in recognition of his pioneering
synthesis of the GPI anchor.
Received: 24.09.2015
Accepted after revision: 13.11.2015
Published online: 30.11.2015
part it is a stable cell constituent with turnover times of
many hours or even days in mammalian cells.6 However, it
can occasionally exhibit rapid changes in concentration in
response to stimuli.7 IP5 has been linked with many cellular
activities including chromatin remodelling,8 regulation of
Ca2+ channels,9 apoptosis10 and viral assembly.11 Interest-
ingly, there is growing evidence supporting the fact that IP5
acts as a regulator of important phosphoinositide interac-
tions.4 In addition, IP5 has been shown to mediate the
Wnt/β-catenin signalling pathway through canonical sig-
nalling.12 These combined findings place IP5 as a major reg-
ulator of disease related pathways in biological systems.
DOI: 10.1055/s-0035-1560381; Art ID: st-2015-d0764-l
Abstract There is sufficient evidence to suggest that myo-inositol pen-
takisphosphate is a vital intermediate species in higher inositol phos-
phate metabolism, however, its biological roles and physiological func-
tion in cells remain uncertain.
A
tethered myo-inositol
pentakisphosphate (IP5) derivative with a terminal amine group is syn-
thesised allowing facilitated immobilisation onto M-270 magnetic Dy-
nabeads for pull-down experiments and biosensor chip preparation for
surface plasmon resonance studies. The probes are validated by both
pull-down and surface plasmon resonance (SPR) studies of the known
binding protein GRP-1 (general receptor for phosphoinositides 1), and
furthermore by SPR studies of protein kinase B (PKB or AKT) binding.
Key words protecting groups, proteins, total synthesis, regioselectivi-
ty, carbohydrates, phosphates, bioorganic chemistry, hydrogenation
The family of inositol compounds has an extraordinary
range of biological functions and only slight changes in
their level of phosphorylation can have huge effects on
their biological roles.1,2 Since the initial discovery of myo-
inositol (1,4,5)triphosphate (IP3) and its role in the regula-
tion of Ca2+ signalling pathways,3 the function of both inosi-
tol phosphates (IPs) and their lipid counterparts (phospha-
tidylinositol phosphates, phosphoinositides or simply PIPs)
has become a widely studied topic in the scientific commu-
nity. Although a great deal is now known about the struc-
ture, binding interactions and disease association of phos-
phoinositides, gaps in the understanding of these incredible
molecules still remain.4
Figure 1 Structure of the most naturally abundant form of myo-inosi-
tol pentakisphosphate (IP5) with the carbons numbered 1–6 (in red)
and the structure of the target molecule 1 – a functionalised IP5 mole-
cule with a tether and terminal amine group for further manipulation
Synthetic chemistry has been a useful tool in assisting
biological researchers to further evaluate the properties of
these inositol-based molecules.4 One synthetic approach is
to modify the compound of interest with a tethered chain
allowing it to be covalently attached to reporter groups,2,13
other small molecules,14 or beads and surfaces.2 Previously,
we have synthesised all eight members of the PIP family as
simplified lipid analogues and also with terminal amino
groups.15 Conjugation through this amine onto affinity ma-
Of the six possible structural isomers of myo-inositol
pentakisphosphate, I(1,3,4,5,6)P5 (IP5, Figure 1) was the
first to be characterised from natural sources.5 For the most
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trices allowed us to identify binding proteins for PI(3,4,5)P3,
PI(4,5)P2, P(3,5)P2 and PI(3)P from the colorectal carcinoma
cell line LIM1215.16
was then reductively cleaved, selectively, with diisobutylal-
uminum hydride (DIBAL-H) to expose the C-5 positioned
hydroxy moiety, a method previously developed by our
group.15,21
The advantage when designing chemical probes to
mimic the PIPs is that coordination can take place on the
lipid chain away from the main head group. The inositol
sugar acts as the main anchoring point for most protein in-
teractions, although often the membrane environment
plays a significant role in the affinity of the head group for
the target protein. The only option when designing tethered
chemical probes of inositol phosphates is to connect the re-
porter group directly to the inositol ring.17 The syntheses of
tethered IP5 compounds have been previously explored.
Ozaki18 as well as Potter14 have synthesised substituted de-
rivatives from the C-2 oxygen. However, although the syn-
theses have been reported, only Potter proved that their
probe could be used for biological studies (although mainly
for studying IP3 rather than IP5).14 In addition, the impor-
tance of the axial hydroxy group at the C-2 position is not
fully understood. To simplify the synthetic effort, it is also
beneficial to attach the tether via the oxygens at C-2 or C-5
as this preserves a plane of symmetry through the com-
pound, allowing a meso structure to be maintained.
Here we report the synthesis of an amino-modified IP5
analogue, 1, with the tether linked through the phosphate
at the C-5 position, allowing immobilisation onto solid sup-
port/affinity matrices for biological investigation. The
probe was validated by both pull-down and surface plas-
mon resonance (SPR) studies of the known binding protein
GRP-1 (general receptor for phosphoinositides 1), and fur-
thermore by SPR studies of protein kinase B (PKB or AKT)
binding.
The free C-5 alcohol of the acetal 5 was subsequently
protected with an allyl group to give the fully protected
inositol 6, as confirmed by the distinctive signals of the allyl
1
group between δ 5–6 in the H NMR spectrum. The allyl
group was used as it can survive the strongly acidic condi-
tions required for the deprotection of the acetal and remov-
al of the p-methoxybenzyl (PMB) groups. This acid treat-
ment produced the tetraol 7, a vital intermediate, in quanti-
tative yield.
Phosphorylation of 7 using standard phosphoramidite
chemistry conditions followed by oxidation as previously
developed in the group15 proceeded as expected (Scheme
1), giving two singlets in the 31P NMR spectrum at δ 1.59
and δ –2.00, each representing two equivalent phosphorus
atoms. The next step required the removal of the allyl
group, which initially proved to be problematic, as regular
deprotection conditions, such as the use of Wilkinson’s cat-
alyst, were unsuccessful; the very slight acidic nature of
this method resulted in some dephosphorylation. However,
conditions using palladium(II) chloride as the catalyst in
methanol seemed to be an effective and simple, as well as a
very mild approach to this deprotection step giving the al-
cohol 9 in 79% yield.22 This transformation was monitored
by signals in the 31P NMR spectrum shifting to δ –0.43 and δ
–1.96.
There are various factors, such as length, polarity and
stability, that needed to be considered when selecting the
most appropriate linker for these experiments.23 A medium
length ethylene glycol derived structure, functionalised
with a terminal amine for later conjugation, was used due
to its capability of retaining rigidity. The replacement of
carbon atoms, in an alkyl chain, with oxygen atoms reduces
the lipophilicity and thus improves the water solubility of
the final compound. The linker was synthesised via the pro-
tection of the commercially available 2-aminoethoxyetha-
nol 10 with a benzyloxycarbonyl (Cbz) group followed by
coupling with a diphosphoramidite under inert conditions
to give the tethered phosphoramidite 12 (confirmed by the
presence of a single 31P NMR signal at δ 147.7) in 82% yield
(Scheme 2).
Selective protection of both axial hydroxy groups of or-
thoformate 219 gave the mono alcohol 3 in good yield
(Scheme 1).20 Benzylation at the C-2 alcohol afforded 4 in
good yield. This protection of the axial alcohol was carried
through to the end of the synthesis. The orthoformate 4
Scheme 2 Synthesis of phosphoramidite tether 12
Coupling of this freshly made phosphoramidite to the
now exposed C-5 alcohol of tetraphosphate 9 did not go ac-
cording to plan; no reaction was observed. It seems that the
limitations of this reaction were the result of the sheer
Scheme 1 Synthesis of intermediates en route to tethered IP5 deriva-
tive 1
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amount of steric hindrance owing to the surrounding ben-
zyl phosphate groups. Thus, an alternative phosphor-
amidite 13 was prepared.18,24
pound could also be functionalised with biotin for subse-
quent attachment onto chips for biosensor experiments. By
performing these biological evaluation techniques, poten-
tial new binding partners, their affinities and specificities,
and a further understanding of the role and function of IP5
in the cell might be discovered.
We previously used Affi-Gel beads, however, prelimi-
nary studies showed that coupling to the Affi-Gel beads
with the much more polar and charged amino-IP5 com-
pound 1 was not as efficient as with the more lipophilic
ω-amino PIP derivatives. After testing a number of alterna-
tives, magnetic Dynabeads (M-270 carboxylic acid) were
found to couple with the amino-IP5 efficiently and facili-
tate quick and effective washing (see the Supporting Infor-
mation for further details).
Before affinity pull-downs from complex mixtures of
proteins, such as cell extracts, it was important to confirm
that the beads were working as expected. It is well known
that IP5 competes with a number of PIPs for the binding of
the Pleckstrin homology domain (PH domain) of GRP (gen-
eral receptor for phosphoinositides 1),26 and is known to
bind to protein kinase B (PKB or AKT). The PH domains of
Phosphorylation of tetraol 7 with phosphoramidite 13
in the presence of 1H-tetrazole gave the tetraphosphorylat-
ed inositol 14 in 80% yield (Scheme 3). Subsequent deallyla-
tion using palladium(II) chloride gave alcohol 15 that
demonstrated downfield shifts of the 31P NMR resonances.
Coupling of the phosphoramidite 12 with alcohol 15 gave
the fully protected IP5 precursor 16 in good yield. Tradi-
1
tional analysis by H NMR spectroscopy of these materials
was difficult owing to the intense signals of the benzyl and
xylyl protons, however, 31P NMR spectroscopy revealed five
signals at δ –1.45, δ –1.53, δ –1.60, δ –2.14 and δ –2.64 rep-
resenting the five now non-equivalent phosphorus atoms,
due to the chiral phosphorus at C-5. A peak in the HRMS of
1412.2718 (1412.2712 calculated for C64H68NO24P5Na [M +
Na]+) was also observed, further supporting the identity of
the product 16.
cytohesin-3/general receptor of phosphoinositides
1
(GRP1)(O43739: 264–381) and RAC-alpha serine/thre-
onine-protein kinase (AKT1)(P31749: 5–108) were pro-
duced as glutathione S-transferase (GST) fusion proteins.
Transformations were carried out using BL21 competent
cells and the PH domains in pGEXT2TK as previously de-
scribed.16a GST fusion proteins were purified using glutathi-
one agarose beads followed by micropreparative size-exclu-
sion chromatography with
a
Superose 12 column
(HR10/30) equilibrated in phosphate-buffered saline (PBS)
and connected to an AKTA purifier system. Protein purity
was analysed using Coomassie-stained sodium dodecyl sul-
fate polyacrylamide gel electrophoresis (SDS-PAGE) and
protein identity was confirmed using LC–MS/MS analysis.
The GST-GRP PH domain was diluted in PBS and was sepa-
rately incubated with blank beads and IP5 beads. The beads
were then washed three times with PBS and heated in SDS-
PAGE reduction buffer at 95 °C for 10 minutes to extract any
protein from the beads; the protein was then separated by
SDS-PAGE (Figure 2,A).
The IP5 beads show a distinct band corresponding to
the GRP protein and the blank beads show very little pres-
ence of the same band. This indicates that the function-
alised IP5 beads are distinctly different from the blank
beads and are specifically binding to and pulling down GRP.
This not only acts as further validation that the amino-IP5
compound is indeed tethered to the beads, but also sug-
gests that the inositol ring is on the surface of the bead and
enables binding to the protein. Another possible purifica-
tion procedure would be to elute the proteins from the IP5-
affinity beads with free IP5. In the absence of ligand elution,
we have validated the IP5 binding using an IP5 biosensor
chip (Figure 2,B).
Scheme 3 Synthesis of tethered IP5 derivative 1
Finally, the fully protected precursor 16 was then glob-
ally deprotected by hydrogenolysis (at a pressure of 10 bar)
in the presence of palladium hydroxide to give the desired
product 1. All phosphate benzyl esters, the C-2 benzyl ether
and the benzyloxycarbonyl protecting groups had been re-
moved to reveal the IP5 structure with a C-5 position linker
group, culminating in a free amine to attach to reporter
molecules, beads and surfaces. All the resonances in the
31P NMR spectrum were shifted upfield [δ 4.22 (2 P), δ 3.01
(2 P) and δ 0.44 (1 P)], consistent with deprotection of the
phosphate groups. HRMS identification of m/z 665.9587
(665.9562 calculated for C10H25NO22P5 [M – H]–) and other
characterisations confirmed that the desired structure 1
was obtained.25
We have previously immobilised the ω-amino PIP deriv-
atives onto affinity beads and surfaces.15 It was hoped that
this technique could be applied in the field of IPs, firstly us-
ing the terminal amino IP5 derivative 1 to couple onto
beads for pull-down experiments. In addition, the com-
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including regioselective protection and deprotection, phos-
phite coupling (and oxidation), followed by hydrogenolytic
global deprotection chemistry. The use of an orthoformate
group in the key intermediate 4 provides an opportunity to
differentiate the six positions on the inositol ring and can
also be used in a similar fashion for the synthesis of other
members of the inositol phosphate family.
The amino-IP5 compound was successfully conjugated
to magnetic Dynabeads to form a probe for pull-down ex-
periments. Its ability to bind to proteins of interest was val-
idated with a known binding protein. The specific binding
of purified GRP and AKT with IP5 was confirmed using a bio-
sensor technique which also validated the capacity of the
IP5 probe. These initial results provide an excellent plat-
form for further biological study.
Acknowledgment
Figure 2 (A) Gel electrophoresis (SDS-PAGE) display of the products
from the pull-down of GST-GRP-PH domain to validate IP5 beads using
MES buffer. Lanes: (1) marker, (2) crude GST-GRP-PH domain, (3) pull-
down with blank beads, (4) pull-down with IP5 beads. (B) Biosensor
analysis of an IP5-derivatised biosensor chip with GRP-PH domain at
varying concentrations (2.5 μM, 1.25 μM, 625 nM, 312 nM and 156
nm); 1:1 Langmuir analysis gives an approximate KD of 520 nM. (C) Bio-
sensor analysis of an IP5-derivatised biosensor chip with AKT-2-PH do-
main at varying concentrations (2.5 μM, 1.25 μM, 625 nM, 312 nM and
156 nm); 1:1 Langmuir analysis gives an approximate KD of 80 nM
This work was supported by the Australian Research Council, Discov-
ery Project DP1094497 and the University of Melbourne (MIRS and
MIFRS scholarships to M.G.). We thank Dr. N. Aberle (University of
Melbourne) for his interest in this work. An NHMRC Program grant
487922 provided access to the facilities in the Walter and Eliza Hall
Institute.
Supporting Information
Supporting information for this article is available online at
To further validate the amino-IP5 compound, and to de-
velop a method for analysing the specificity and affinity of
IP5 for any proteins in proteomics experiments, the IP5
compound was attached to a chip for biosensor studies. The
amino-IP5 compound was coupled with an N-hydroxysuc-
cinimide (NHS) activated biotin reporter group and purified
by anion exchange column chromatography using a linear
gradient of ammonium bicarbonate (0–2 M). The purified
biotinylated IP5 was then immobilised directly onto a neu-
travidin-derivatised sensor surface.27 Injection of varying
concentrations of GRP (2.5 μM, 1.25 μM, 625 nM, 312 nM
and 156 nM) across the immobilised IP5 chip, with a neu-
travidin channel as a control, provided the binding curves
shown in Figure 2,B.
This kinetic analysis determined that the dissociation
constant (KD) of GRP for the IP5 compound was approxi-
mately 520 nM. In addition the same studies for another
known IP5 binding protein, AKT, were performed and gave
a dissociation constant of around 80 nM (Figure 2,C), being
comparable to literature values.28
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To a solution of the fully protected derivative 16 (10 mg, 72
μmol) dissolved in MeOH–THF (2:3, 2 mL) was added Pd(OH)2
(10 mg). The mixture was reacted under H2 (10 bar pressure, in
a Buchi hydrogenation vessel) and was allowed to stir vigor-
ously at r.t. for 48 h. The mixture was filtered twice through
Celite and the filtrate was lyophilised to give the desired
product 1 (5 mg, quant.) as a white powder.
IR (neat): 3500–3000 (br), 1579, 1409, 1353, 1087, 973 cm-1.
1H NMR (500 MHz, D2O): δ = 4.60–4.40 (m, 5 H), 4.25 (br s, 1 H),
4.14 (br s, 2 H), 3.88–3.81 (m, 4 H), 3.76–3.64 (m, 2 H), 3.14 (br
s, 2 H). 13C NMR (125 MHz, CDCl3): δ = 76.7, 76.2, 75.5, 73.0,
71.6, 69.9, 66.3, 66.0, 60.4, 39.2. 31P NMR (202 MHz, CDCl3):
δ = 4.22 (2 P), 3.01 (2 P), 0.44 (1 P). HRMS (ESI): m/z [M – H]–
calcd for C10H25NO22P5: 665.9562; found: 665.9587; m/z [M – 2
H]2– calcd for C10H24NO22P5: 332.4745; found: 332.4755.
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