A scalable synthesis of the IP7 isomer, 5-PP-Ins(1,2,3,4,6) P5

Article abstract of DOI:10.1021/ol900149x

The phosphorylated inositol diphosphates, including the diphosphoinositol pentakisphosphate regioisomers, play critical roles in signal transduction and cellular regulation. In particular, the IP₇ isomer 5-PP-lns (1,2,3,4,6)P₅ is imp

Full text of DOI:10.1021/ol900149x

ORGANIC
LETTERS
2009
Vol. 11, No. 7
1551-1554
A Scalable Synthesis of the IP7 Isomer,
5-PP-Ins(1,2,3,4,6)P5
Honglu Zhang,† James Thompson,‡ and Glenn D. Prestwich*,†
Department of Medicinal Chemistry, The UniVersity of Utah, 419 Wakara Way, Suite
205, Salt Lake City, Utah 84108-1257, and Echelon Biosciences, 675 Arapeen DriVe,
Suite 302, Salt Lake City, Utah 84108
gprestwich@pharm.utah.edu
Received January 24, 2009
ABSTRACT
The phosphorylated inositol diphosphates, including the diphosphoinositol pentakisphosphate regioisomers, play critical roles in signal
transduction and cellular regulation. In particular, the IP7 isomer 5-PP-Ins(1,2,3,4,6)P5 is implicated in a nonenzymatic phosphate transfer
converting a protein serine phosphate residue to a serine diphosphate. A scalable, practical new synthesis of 5-PP-Ins(1,2,3,4,6)P5 is described
that also allows access to a variety of IP7 and IP8 regioisomers. The identity of the synthetic 5-PP-Ins(1,2,3,4,6)P5 was validated using IP6K1
to catalyze the conversion of IP7 + ADP to ATP + IP6.
Inositides, particulary inositol phosphates and phosphati-
dylinositol lipids, are crucially important cell signaling
molecules that are linked to a series of signaling events.1-3
These signaling events include ion-channel function,4,5
vesicle trafficking,6 apoptosis,7,8 transcriptional regulation,9
motility,10 cell proliferation, and transformation.11 Inositol
1,4,5-trisphosphate (IP3) is a ubiquitous second messenger,
which couples agonist stimulation of a wide variety of
receptors to the mobilization of intracellular calcium.12 The
more highly phosphorylated inositides, particularly the
“omnipotent” inositol hexakisphosphate13 and phosphory-
lated inositol diphosphates (commonly called pyrophosphates
in the biological literature), play critical roles in signal
transduction and cellular regulation.2,6
† The University of Utah.
‡ Echelon Biosciences.
In 1993, two inositol diphosphates, diphosphoinositol
pentakisphosphate (5-PP-Ins(1,2,3,4,6)P5, or IP7) and bis-
diphosphoinositol tetrakisphosphate (PP2-InsP4, or IP8), were
purified from Dictyostelium.6,14 The diphosphate bond of the
5-PP-Ins(1,2,3,4,6)P5 has a calculated phosphorylation po-
tential that equals or exceeds that of ATP,6,15 suggesting that
it could serve a similar function. 5-PP-Ins(1,2,3,4,6)P5 is
biosynthesized from inositol hexakisphosphate (IP6) by a
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10.1021/ol900149x CCC: $40.75
Published on Web 03/02/2009
2009 American Chemical Society

family of three inositol hexakisphosphate kinases
(InsP6Ks).16-18 Inositol hexakisphosphate kinase-2 (InsP6K2),
one of the InsP6Ks, was found to be an important physiologic
mediator of cell death. More recent studies revealed that IP7
plays an important role in regulation of insulin secretion.19-22
Recently, fluorometric detection of IP7 in the presence of
IP6 and lower IPn congeners was achieved by the Matile
laboratory using a synthetic multifunctional pore.23 A dif-
ferent regioisomer of IP7 is produced by yeast Vip1 and is
necessary for Pho81 cyclin-dependent kinase (CDK) inhibi-
tion of the cyclin-CDK complex Pho80-Pho85, thereby
regulating an important metabolic network.24,25 Although first
identified24 as 4(6)-PP-InsP5, this was recently revised26 to
1(3)-PP-InsP5, a result that has been independently validated
and extended by synthesis and assay of the enantiopure 1-PP
and 3-PP-InsP5 isomers.27
The reversible phosphorylation of proteins regulates nearly
every aspect of cell physiology.28 Phosphorylation and
dephosphorylation, catalyzed by protein kinases29 and protein
phosphatases, are depicted in Figure 1. Some 30% of the
proteins encoded by the human genome are phosphorylated,
and abnormal phosphorylation is now recognized as a cause
or a consequence of many human pathologies. As a result,
protein kinases are already the second largest group of drug
targets after G-protein-coupled receptors, and they account
for 20-30% of the drug discovery programs of many
companies.30
Unlike ATP, however, the inositol diphosphate IP7 appears
to phosphorylate serine phosphate residues nonenzymatically
(Figure 1).31 5-PP-Ins(1,2,3,4,6)P5 has been demonstrated
to phosphorylate a variety of Ser-rich protein targets in yeast
and mammals.15 The resulting 5-PP-Ins(1,2,3,4,6)P5-phos-
phorylated peptides are more acid-labile and more resistant
to phosphatases, suggesting that a protein diphosphate bond
had been formed (Figure 1).31 Moreover, only the Ser-rich
Figure 1. Reversible protein phosphorylation and pyrophosphory-
lation mechanisms.
regions of target proteins that had been previously phospho-
rylated by a protein kinase were substrates, strongly impli-
cating Ser-PP, a serine diphosphate (pyrophosphate), as the
product of the nonenzymatic diphosphorylation. This diphos-
phorylation may represent a novel mode of signaling,31 but
its study in many cases is limited by the availability of 5-PP-
Ins(1,2,3,4,6)P5 and a family of chemical and biological tools
to probe the structure and function of diphosphorylated
proteins. To address this unmet need, we describe herein a
scalable and efficient new method for the synthesis of 5-PP-
Ins(1,2,3,4,6)P5 based on modifications of previous inositide
syntheses by the laboratories of Prestwich31 and Falck.32,33
The three key problems in the synthesis of any IP7
stereoisomer are (i) the method of stereoselective introduction
of the protected 5-diphosphate, (ii) the stability of the
protected diphosphorylated intermediate(s), and (iii) the
complete removal of protecting groups under mild conditions
with minimal degradation of the desired IP7. We selected
the synthetic route as shown in Scheme 1 to prepare the key
intermediate 8. It is important to note that the starting
materials, intermediates, and final products are all formally
meso compounds, since the 5-diphosphate will be positioned
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Scheme 1. Synthesis of Advanced Intermediate 8
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1552
Org. Lett., Vol. 11, No. 7, 2009

on the C-2-C-5 plane of symmetry. Following known
procedures, myo-inositol 1,3,5-orthoformate 2, available in
one step from myo-inositol,34 was selectively benzoylated
at 2-OH with benzoyl chloride.35 Hydrolysis of orthoformate
2 in 4 M HCl/MeOH afforded benzoate 4,35 which was
converted to the acetonide 5 as a mixture with other
isomers.36 Compound 5 was easily separated from other
isomers by flash chromatography. Introduction of the 4-meth-
oxybenzyl ether was unexpectedly problematic. Under
standard benzylation conditions (NaH or nBuLi, 4-MeOBnCl
or 4-MeOBnBr, THF or DMF) or phase-transfer catalysis
conditions (4-MeOBnCl, Bu4NHSO4, aqueous NaOH), start-
ing material was converted to complex product mixtures.
Nonetheless, reaction of 5 with 4-methoxybenzyl bromide
in the presence of the hindered organic base, 2-tert-
butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-di-
azaphosphorine (BEMP),37 in CH3CN afforded the fully
protected inositol 6 in 84% yield. Treatment of benzoate 6
with NaOMe gave 2-OH inositol 7 in 82% yield, and
hydrolysis of the acetonides with pTsOH in acetone/H2O
gave the 5-PMB-protected inositol 8 in 79% yield. Since 8
was poorly soluble in EtOAc and CH2Cl2, purification was
accomplished simply by trituration of the white solid with
these two solvents.
With the advanced intermediate 5-PMB inositol in hand,
different phosphoramidites coupling with compound 8 would
generate different phosphates. Considering the stability of
the IP7, dimethyl phosphate and diethyl phosphate could not
be used as protecting groups because the use of TMSBr to
remove these protecting groups could cause decomposition
of the diphosphate moiety. The dibenzyl phosphate32,33 and
o-xylylene phosphate are the best candidates because they
are readily removed by hydrogenolysis. In addition, since
the o-xylylene phosphate is sterically smaller than dibenzyl
phosphate, we expected that the o-xylylene phosphate
intermediate 13 would be more stable in this highly sterically
congested intermediate. Thus, the pentakis(o-xylylene phos-
phate) 9 was prepared in 99% yield by global phosphory-
lation of pentaol 8 with o-xylylene N,N-diethylphosphora-
midite and 1H-tetrazole in CH2Cl2. The 4-methoxybenzyl
functional group was removed with TFA/CH2Cl2/H2O
(5:1:1, v/v) to furnish alcohol 10.
generated the differentially protected hexakisphosphate 11.
Selective removal of the cyanoethyl group under mild
conditions (TEA/BSTFA/CH3CN)38-40 and purification by
Dowex-H+ chromatography afforded pure monophosphoric
acid 12 in 92% yield (Scheme 2). After treatment of 12 (1
Scheme 2
.
Global Phosphorylation and Final Conversion to
5-PP-Ins(1,2,3,4,6)P5
equiv) with triethylamine (2 equiv) in anhydrous CH2Cl2 at
0 °C followed by dibenzylphosphoryl chloride (2 equiv) for
2 h at rt, solvents were removed to stop the reaction and
give a crude product. The crude residue 13 was then
deprotected by stirring an aqueous t-butanol solution under
H2 (60 psi) with PtO2 for 4 h. After removal of the catalyst
and concentration, the residue was dissolved in water and
washed with EtOAc and CH2Cl2. The aqueous solution was
concentrated and purified through ion exchange chromatog-
raphy (Dowex 50W × 8 - 200 Na+ exchange resin) by
elution with water to afford the 5-PP-Ins(1,2,3,4,6)P5 sodium
form in 81% yield as white solid. The 31P NMR spectrum
of 5-PP-Ins(1,2,3,4,6)P5 in D2O displayed a characteristic
pyrophosphate peak, at -9.40 to -10.20 ppm, integrating
at a 2.0:5.04 ratio relative to the monophosphate resonances
between 0.2 and 1.2 ppm (Figure 2). The complexity of the
signals reflects a distribution of sodium salt and protonated
species each with characteristic chemical shifts. Similarly,
the key feature of the complex proton spectrum is the 1.0:
1
4.88 ratio of peaks for H-5:H1-4, H6. Both H NMR and
Here again, our route diverges from previous methods.
The unsymmetrical phosphoramidite, benzyl-2-cyanoethyl
N,N-diisopropylphosphoramidite, was prepared in 86% yield
from commercial 2-cyanoethyl N,N-diisopropyl-chlorophos-
phoramidite and benzyl alcohol in the presence of Hunig’s
base. Reaction of 10 with this phosphoramidite in the
presence of 1H-tetrazole, followed by m-CPBA oxidation,
31P NMR were consistent with the 5-PP-Ins(1,2,3,4,6)P5
isolated from Polysphondylium.41
In this new synthetic route, intermediate 5 could be readily
prepared in 10 g amounts in four steps, including removal
of stereoisomers, in 18% yield from inexpensive com-
mercially available materials. With this known material, the
final 5-PP-InsP5 was prepared in 33% overall yield for seven
steps. Importantly, the o-xylylene phosphate 13 was more
stable than the globally benzyl-protected 5-PP-InsP5 inter-
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Org. Lett., Vol. 11, No. 7, 2009
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Figure 3. Effects of the concentration of ADP and synthetic 5-PP-
Ins(1,2,3,4,6)P5 on ATP production by IP6K1.
Figure 2.
1H NMR and 31P NMR of 5-PP-InsP5.
case, no increase in luciferase activity was observed, indicat-
ing IP6K1 is indeed required for ATP synthase activity
(Supporting Information, Figures 1 and 2). Additionally,
incubation of 5-PP-Ins(1,2,3,4,6)P5 with ATP and the enzyme
luciferase results in decreased conversion of luciferin to
oxyluciferin indicating 5-PP-Ins(1,2,3,4,6)P5 may inhibit the
luciferase enzyme (Supporting Information, Figure 1).
In conclusion, we have developed an efficient and scalable
method for the synthesis of the important IP7 regioisomer
5-PP-Ins(1,2,3,4,6)P5, which will meet the demand of cell
signaling scientists in the studies of protein pyrophospho-
rylation, exocytosis in pancreatic beta cells, and other key
cellular processes. In addition, this new synthetic approach
method can be adapted to the chemical synthesis of a variety
of regioisomers of IP8, the bisdiphosphoinositol tetrakispho-
sphates (PP2-InsP4), which have recognized importance in
cell signaling research.
mediate, which only gave a 25% yield for the final three
steps described by us previously.31
To validate the biological activity of the synthetic 5-PP-
Ins(1,2,3,4,6)P5, we used it as a substrate to phosphorylate
ADP. Inositol hexakisphosphate kinase 1 (IP6K1) not only
catalyzes the transfer of a phosphate from ATP to IP6
yielding 5-PP-Ins(1,2,3,4,6)P5 plus ADP but also readily
catalyzes the reverse reaction in which a phosphate is
transferred from 5-PP-Ins(1,2,3,4,6)P5 to ADP yielding ATP
and IP6.42 Thus, synthetic 5-PP-Ins(1,2,3,4,6)P5 was incu-
bated with ADP and recombinant IP6K1, and ATP synthase
activity was monitored using the activity of the enzyme
luciferase. An increase in ATP, resulting from its production
from 5-PP-Ins(1,2,3,4,6)P5 plus ADP, was quantified by
measuring the luciferase-catalyzed conversion of luciferol
to oxyluciferol (Figure 3). Synthetic 5-PP-Ins(1,2,3,4,6)P5
may be used as a substrate for ATP synthesis in a reaction
catalyzed by IP6K1.
Acknowledgment. We thank the NIH for financial support
(NS 29632 to GDP).
Supporting Information Available: Experimental details
for synthesis, characterization of new compounds, and
experimental methods for IP6K1-catalyzed reaction figures
for enzyme-free experiment. This material is available free
of charge via the Internet at http://pubs.acs.org.
To ensure IP7 was not directly phosphorylating ADP to
ATP without the enzyme IP6K1, the luciferase reaction was
repeated without the addition of the enzyme IP6K1. In this
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Olszewski, J. D.; Prestwich, G. D.; Snyder, S. H. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 4305–4310.
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Org. Lett., Vol. 11, No. 7, 2009