Synthesis and biological activity of phosphatidylinositol-3,4,5-trisphosphorothioate

Article abstract of DOI:10.1016/j.bmcl.2007.11.041

Metabolically-stabilized analogs of PtdIns(3,4,5)P₃ have shown long-lived agonist activity for cellular events mediated by this phosphoinositide. We describe an efficient method for the total asymmetric synthesis of the trisphosphorothioate (PT

Full text of DOI:10.1016/j.bmcl.2007.11.041

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 762–766
Synthesis and biological activity of
phosphatidylinositol-3,4,5-trisphosphorothioate
Honglu Zhang,a Yong Xu,a Nicolas Markadieu,b Renaud Beauwens,b
Christophe Erneuxc and Glenn D. Prestwicha,*
aDepartment of Medicinal Chemistry, The University of Utah, 419 Wakara Way, Suite 205, Salt Lake City, UT 84108-1257, USA
bDepartment of Cell Physiology, Universite´ Libre de Bruxelles, Campus Erasme, Route de Lennik 808, 1070 Bruxelles, Belgium
cInstitut de Recherche Interdisciplinaire (IRIBHM), Universite´ Libre de Bruxelles, Campus Erasme, Route de Lennik 808,
1070 Bruxelles, Belgium
Received 28 September 2007; revised 8 November 2007; accepted 12 November 2007
Available online 17 November 2007
Abstract—Metabolically-stabilized analogs of PtdIns(3,4,5)P3 have shown long-lived agonist activity for cellular events mediated by
this phosphoinositide. We describe an efficient method for the total asymmetric synthesis of the trisphosphorothioate (PT) analog of
PtdIns(3,4,5)P3. Intracellular delivery of dipalmitoyl PtdIns(3,4,5)PT3-mimicked insulin in activating sodium transport in A6 cells.
Ó 2007 Elsevier Ltd. All rights reserved.
Phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3)
is a crucially important second messenger found ubiqui-
tously in higher eukaryotic cells.1 The activation of
affinity probes for isolation and characterization of
PtdInsPn binding proteins, and the availability of tools
for real-time localization of PtdInsPns in cells are pro-
viding a continuous influx of new information on the
roles of phosphoinositides in cellular processes10 How-
ever, many questions regarding the specific effects of
PtdIns(3,4,5)P3 remain unanswered due to its transient
nature in the cell.
Class
I PI 3-K and subsequent production of
PtdIns(3,4,5)P3 is an important cell signaling event that
has been causally linked to the activation of variety of
downstream cellular processes.2 These signaling events
include cell proliferation and transformation,3 cell shape
and motility,4 and the action of insulin in altering glu-
cose transport5 PtdIns(3,4,5)P3-regulated signaling is
governed both by its formation by PI 3-K and its
conversion into phosphatidylinositol 4,5- and 3,4-bis-
phosphates by the 3-phosphatase PTEN6 or the 5-phos-
phatase SHIP,7 respectively. Control of PI 3-K and
PtdIns(3,4,5)P3 levels have emerged as important tar-
gets8 in cardiovascular disease, allergy and autoimmune
disease,9 cancer, and diabetes.8
We recently described the preparation and activity of 3-
phosphatase-resistant, metabolically-stabilized (ms)
analogs of PtdIns(3,4,5)P3 that was both stable to degra-
dation by PTEN and acted as an inhibitor of PTEN11
One of these analogs incorporated a single phosphoro-
thioate (PT) substituent. Phosphorothioates are impor-
tant phosphomimetics that show greatly reduced rates
of enzyme-mediated hydrolysis12 However, the replace-
ment of P@O by P@S also affects the pKa of the phos-
13,14
In addition to regulation of PtdIns(3,4,5)P3 production,
catabolism by the phosphatases PTEN and SHIP have
emerged as crucial processes to control PtdIns(3,4,5)
P3-dependent signaling (Scheme 1). The availability of
phate and removes a H-bond acceptor.
Indeed,
we demonstrated that PtdIns(3)PT had reduced binding
activity for cognate PtdIns(3)P-selective FYVE and PX
domain binding proteins, which was attributable to re-
duced H-bonding.15 We hypothesized that a 3,4,5-
tris(PT) analog of PtdIns(3,4,5)P3 could be either an
antagonist or a long-lived agonist in the related signal-
ing pathway, because it would be more slowly dephos-
phorylated by both PTEN and SHIP, and could
potentially block the normal receptor-mediated signal-
ing involving PtdIns(3,4,5)P3. We also observed that a
Keywords: Metabolically-stabilized; PtdIns(3,4,5)P3; Asymmetric syn-
thesis; Activating sodium transport.
*
Corresponding author. Tel.: +1 801 585 9051; fax: +1 801 585
9053; e-mail: gprestwich@pharm.utah.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.041

H. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 762–766
763
O
OH
HO
=O3PO
=O3PO
O
O
O
P
HO
O-
O
O
O
PtdIns(4,5)P2
PI 3-K
PTEN
O
OH
O
=O3PO
=O3PO
=O3PO
O
O
P
HO
O-
O
O
O
SHIP
PtdIns(3,4,5)P3
O
O
OH
O
=O3PO
=O3PO
OH
O
P
HO
O-
O
O
O
PtdIns(3,4)P2
Scheme 1. Pathways for PtdIns(3,4,5)P3 biosynthesis and catabolism.
PT analog of PtdIns(5)P16 and a tris(PT) cyclopentyl
analog17 of Ins(1,4,5)P3 had agonistic activities and long
biological half-lives. We now describe the first asymmet-
ric total synthesis of a PtdIns(3,4,5)PT3, a trisphosp-
horothioate analog of PtdIns(3,4,5)P3 that is resistant
to hydrolysis by both PTEN and SHIP. In addition,
we document the ability of these analogs to effect in so-
dium transport in A6 cell monolayers, an amphibian re-
nal epithelium model expressing the epithelial sodium
channels.18
using ethanethiol and BF3ÆEt2O at rt to give the final
product 8a.15,21
Although this synthetic route afforded the desired
tris(PT) 8a, a more efficient synthetic approach was
deemed necessary. The disadvantages included the low
yield of oxidation with elemental sulfur, and the low
yield resulting from a TBDPS deprotection protocol
that required several weeks. The sulfur–pyridine reac-
tion conditions to convert the phosphite esters to the
phosphorthioates15 resulted in decomposition of the
six cyanoethyl groups (CE) under basic conditions. We
thus chose to replace the TBDPS group with TES, be-
cause TES is still sterically bulky but can be easily re-
moved under milder conditions. Also, phenylacetyl
disulfide11,22 was selected as an alternative to elemental
sulfur, and this reagent can be employed under neutral
conditions to minimize destruction of the CE protecting
groups.
Starting from 3,4,5-benzoyl inositol 1,19,20 the fully pro-
tected inositol intermediate 2 was obtained by reaction
of diol 1 with MOMCl and DIPEA at 50 °C (Scheme
2). Treatment of compound 2 with 1.0 M NaOMe in
methanol–THF gave the desired triol 3 in 85% yield,
which was then phosphorylated and oxidized with ele-
mental sulfur to yield the corresponding phosphorothio-
ate 4 in 28% yield.15 Use of the HFÆPy complex in THF–
pyridine solution selectively removed the TBDPS ether.
This deprotection reaction was very slow, and required
supplemental addition of HFÆPy after 3 days reaction,
and 3 weeks to give key intermediate 5 in low yield.15
The TES ether 9 was thus obtained in high yield in two
steps: first, deprotection of the TBDPS ether 2 with solid
TBAF in DMF for 3 h, followed by reaction with TESCl
and imidazole at room temperature.11 Next, removal of
the three benzoyl groups of TES–ether 9 sodium methox-
ide gave only a low yield of triol 10, accompanied by par-
tial TES cleavage. As an alternative, DIBAL-H/ꢀ78 °C
was employed for benzoyl removal,11 because the TES
and MOM groups were stable to these conditions; com-
pound 10 was obtained in 81% yield. Then, triol 10 was
converted to the protected tris(PT) in a two-step, one-
pot reaction. Phosphitylation with 1H-tetrazole and
bis(2-cyanoethoxy) (diisopropylamino)phosphine was
followed by oxidation of the resulting phosphite ester
with phenylacetyl disulfide in 30 min, giving the desired
phosphorothioate 11 in 72% yield.11 Since the cyanoethyl
The phosphoramidite 6a15 was then coupled with inositol
5 in the presence of 1H-tetrazole to yield the phosphite
intermediate, which was oxidized utilizing t-BuOOH to
give protected phosphorothioate 7a as shown in Scheme
2. The cyanoethyl groups (CE) on the phosphorothioate
were removed by using triethylamine (TEA) plus bis(tri-
methylsilyl)trifluoroacetamide (BSTFA) in anhydrous
acetonitrile. BSTFA was added to prevent the phosp-
horothioate anion from undergoing re-alkylation. The
cleavage of the O-silyl derivatives was achieved by aque-
ous hydrolysis at neutral pH to give the MOM ether-pro-
tected intermediate. The MOM groups were removed

764
H. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 762–766
S
CEO
CEO
OMOM
OMOM
OH
OMOM
OTBDPS
P
BzO
BzO
OTBDPS
BzO
BzO
OTBDPS
OH
O
HO
OTBDPS
a
b
c
S
OMOM
O
OMOM
OCE
HO
OMOM
P
CEO
OBz
OBz
O
S
OH
OCE
P
2
3
1
OCE
4
d
O
O
S
CEO
O
S
OMOM
O
P
O
C17H33
OCE
CEO
CEO
CEO
OMOM
OH
P
O
O
P
O
C17H33
O
e,f
O
CEO
O
C17H33
S
+
P
N
O
OMOM
S
P
CEO
O
OMOM
OCE
O
C17H33
O
S
O
P
CEO
OCE
OCE
O
S
P
OCE
P
O
OCE
OCE
6a
7a
5
g,h,i
O
O
S
H4NO
OH
O
P
O
C17H33
ONH4
H4NO
O
O
O
P
O
OH
C17H33
S
O
P
O
ONH4
H4NO
ONH4
P
S
ONH4
8a
Scheme 2. Reagents and conditions: (a) MOMCl, DIPEA, DMF, 50 °C, 75%; (b) NaOMe, CH3OH/THF, 85%; (c) Bis(2-cyanoethoxy)(diisopro-
pylamino)phosphine, 1H-tetrazole; S, CS2/Py, 28%; (d) HFÆPy, THF/Py, 3 weeks, 53%; (e) 1H-tetrazole, CH2Cl2; (f) t-BuOOH, 48%; (g) TEA,
BSTFA, CH3CN; (h) NH4OAc, H2O; (i) EtSH, BF3ÆEt2O, 88%.
groups were labile under basic conditions, we chose the
weakly acidic reagent NH4F for deprotection of the
TES. After reaction with NH4F in MeOH for 3 h the
key intermediate 5 was obtained in 90% yield as a single
product (Scheme 3)11 The modifications introduced here-
in gave the protected inositol intermediate more effi-
ciently and in higher overall yield.
exposure of the TMS-esters 12 to TMSBr in
CH2Cl2.23,24 This is very convenient for the purification
of the final product, because all the reaction reagents
are readily removed under vacuum. Finally, methanol
hydrolysis of the fully TMS protected intermediates
13b–d gave the final PtdIns(3,4,5)PT3 analogs 8b–d in
their acid forms (Scheme 3).
Condensation of 5 with each of the freshly prepared
phosphoramidites 6b–d in the presence of 1H-tetrazole
and t-BuOOH oxidation gave rise to the fully protected
phosphatidylinositide analogs 7b–d.11 The cyanoethyl
groups in intermediates 7b–d were removed by using
triethylamine and BSTFA in anhydrous acetonitrile.
After 24 h, we removed the solvents to afford a residue,
of which the 1H NMR showed that the cyanoethyl
groups were eliminated and the hydroxy groups were
protected with TMS. Thus, BSTFA effectively prevents
the phosphorothioate anions from undergoing realkyla-
tion. The TMS protected intermediates also provided
us a method to remove the methyl and MOM group;
that is, TMSBr could be added directly to the residue,
and this led to complete removal of the methyl and
MOM groups to give products 13b–d.11 In our previ-
ous synthesis15 and in work by the Bruzik laboratory,20
the TMS-esters 12 were generally converted to the
ammonium salts. Then, reaction of the ammonium
salts with TMSBr frequently gave only partial cleavage
of the MOM ethers. However, we observed that
the MOM groups were completely removed by direct
To test the function of these analogs, we used A6 cell mon-
olayers, a renal epithelium model that expresses epithelial
sodium channels (ENaC), in which carrier-mediated
intracellular delivery25 of PtdIns(3,4,5)P3 activates
GLUT4 translocation to the plasma membrane5 and so-
dium transport26 ENaC activity is the rate-limiting step
of the sodium transport and is stimulated by insulin27
DiC16-PtdIns(3,4,5)P3 is an early mediator of the insu-
lin-stimulated sodium transport in A6 cells. Thus, we
compared the effect of the unmodified diC16-PtdIns
(3,4,5)P3 with diC16-PtdIns(3,4,5)PT3 8d on sodium
transport across confluent monolayers of A6 cells. As
shown in Fig. 1, apical addition of analog 8d increased
sodium transport.28 The 3,4,5-tris(PT) analog 8d also
mimicked the activity of unstabilized PtdIns(3,4,5)P3.
The lag time observed between PtdIns(3,4,5)PT3 addition
and the final effect on sodium transport revealed that the
rate of intracellular delivery of this analog is faster than
that of unstabilized PtdIns(3,4,5)P3. We propose that
these PtdIns(3,4,5)PT3 analogs may have other applica-
tions as pharmacological tools to probe role of
PtdIns(3,4,5)P3 in a cellular context.

H. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 762–766
765
OCE
CEO
OMOM
P
OMOM
O
OMOM
O Si
O
O Si
S
BzO
BzO
Si
HO
HO
a, b
c
e
d
2
5
O
S
OMOM
OCE
CEO
CEO
P
OMOM
OMOM
O
OBz
OH
10
P
S
OCE
9
11
+
O
O
O
O
O
O
O
R
OTMS
OCE
O
O
R
O
R
TMSO
S
OMOM
O
CEO
OMOM
N
O
OTMS
OCE
O
P
P
f
P
O
O
O
g
S
P
O
P
OCE
R
O
R
R
O
S
OMOM
O
OMOM
TMSO
TMSO
CEO
CEO
O
P
P
O
O
O
P
O
OTMS
OCE
S
P
6b R =C3H7
6c R = C7H15
S
S
OTMS
OCE
6d R = C15H31
12b R =C3H7
12c R = C7H15
12d R = C15H31
7b R =C3H7
7c R = C7H15
7d R = C15H31
g, h, i
h
O
OH
OH
O
O
R
HO
OH
P
OTMS
O
O
O
O
R
S
TMSO
S
OTMS
P
OTMS
P
O
O
O
O
O
R
P
O
S
OH
HO
HO
i
O
OTMS
P
O
R
O
O
O
S
OH
TMSO
TMSO
P
P
O
S
O
OH
OTMS
P
8b R =C3H7
S
OTMS
8c R = C7H15
8d R = C15H31
13b R =C3H7
13c R = C7H15
13d R = C15H31
Scheme 3. Reagents and conditions: (a) TBAFÆ3H2O, DMF, 86%; (b) TESCl, imidazole, CH2Cl2, 87%; (c) DIBAL-H, CH2Cl2, ꢀ78 °C, 81%; (d)
Bis(2-cyanoethoxy)(diisopropylamino)phosphine, 1H-tetrazole; phenylacetyl disulfide, 72%; (e) NH4F, MeOH, 90%; (f) 1H-tetrazole, CH2Cl2, rt; t-
BuOOH; (g) TEA, BSTFA, CH3CN; (h) TMSBr/CH2Cl2 (2:3), rt; (i) MeOH.
Acknowledgments
We thank the NIH (Grant NS 29632 to G.D.P.) for
financial support of work at Utah and the ‘Fonds de
´
la Recherche Scientifique Medicale’ for support of work
in Bruxelles. We thank P. Neilsen (Echelon Biosciences,
Salt Lake City) for providing PtdIns(3,4,5)P3 and
histone.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.11.041.
Figure 1. Stimulation of A6 cell monolayers. Experiment and evalu-
ation of sodium transport (INa
,
lA/cm2) was carried out as
þ
described26 Briefly, A6 cells were subcultured onto 24-mm Millicell
inserts (Millipore, Bedford, MA) for 10 days and the day before the
experiment, incubated overnight in a serum-free 260 mosmol/kg H2O
amphibian Ringer solution. DiC16-PtdIns(3,4,5)P3, diC16-PtdIns
(3,4,5)PT3 8d (50 lM) was complexed by histone H1 carrier (50 lM)
and then added to the apical side of the monolayer. Results were
compared with insulin basolateral stimulation (100 nM) and control
(histone H1 alone).
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tured onto 24-mm Millicell inserts (Millipore, Bedford,
MA) for 10 days and the day before the experiment,
incubated overnight in a serum-free 260 mosmol/kg H2O
amphibian Ringer solution. DiC16-PtdIns(3,4,5)P3, analog
8d was complexed by histone H1 carrier (50 lM) and then
added to the apical side of the monolayer. Results were
compared with insulin basolateral stimulation (100 nM)
and control (histone H1 alone). This experiment is
representative of three independent experiments.