Synthesis and biological activity of PTEN-resistant analogues of phosphatidylinositol 3,4,5-trisphosphate

Article abstract of DOI:10.1021/ja065002j

The activation of phosphatidylinositol 3-kinase (PI 3-K) and subsequent production of PtdIns(3,4,5)P₃ launches a signal transduction cascade that impinges on a plethora of downstream effects on cell physiology. Control of PI 3-K and PtdIns(3,4,

Full text of DOI:10.1021/ja065002j

Published on Web 12/06/2006
Synthesis and Biological Activity of PTEN-Resistant Analogues of
Phosphatidylinositol 3,4,5-Trisphosphate
Honglu Zhang,† Nicolas Markadieu,‡ Renaud Beauwens,‡ Christophe Erneux,§ and
Glenn D. Prestwich*,†
Department of Medicinal Chemistry, The UniVersity of Utah, 419 Wakara Way, Suite 205, Salt Lake City, Utah
84108-1257, Department of Cell Physiology and Institut de Recherche Interdisciplinaire (IRIBHM), UniVersite´ Libre
de Bruxelles, Campus Erasme, Route de Lennik 808, 1070 Bruxelles, Belgium
Received July 24, 2006; E-mail: gprestwich@pharm.utah.edu
Scheme 1. Synthesis of Phosphorothioates 1a
The phosphoinositide 3-kinase (PI 3-K) signaling pathway
contains important therapeutic targets in human pathophysiology.1,2
Phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is a ubiq-
uitous signaling lipid found in higher eukaryotic cells3 and activates
a plethora of downstream cellular processes.4 These signaling events
include cell proliferation and transformation,5 cell shape and
motility,6 and insulin action and alteration of glucose transport.7
PtdIns(3,4,5)P3-regulated signaling is modulated by the lipid
3-phosphatase PTEN8 and SH2 domain-containing inositol 5-phos-
phatase SHIP.9
A metabolically stabilized (ms) analogue of PtdIns(3,4,5)P3 that
resists lipid 3- and 5-phosphatases would have numerous applica-
tions in understanding the role of PtdIns(3,4,5)P3 in cell physiology.
The ms-PtdIns(3,4,5)P3 analogues could separate the activation
of signal transduction from the degradation of the signal by
phosphatase action in cells. This chemical biology approach to
a Conditions: (a) TIPDSCl2, imidazole, Py, 88%; (b) MOMCl, DIPEA,
dissection of the PI 3-K pathway is complementary to the use of
siRNA knockdowns or genetic knockouts for PTEN and SHIP. We
focused first on a 3-stabilized PtdIns(3,4,5)P3 analogue, that is, one
resistant to hydrolysis by PTEN, and we selected two stabilized
phosphomimetic isosteres to replace the 3-phosphate of PtdIns-
(3,4,5)P3.
DMF, 65 °C, 63%; (c) TBAF, THF, 77%; (d) N,N-dimethylphosphoramidite,
1H-tetrazole, m-CPBA, 81%; (e) TBAF‚3H2O, DMF, 91%; (f) TESCl,
imidazole, CH2Cl2, 88%; (g) DIBAL-H, CH2Cl2, -78 °C, 84%; (h) bis(2-
cyanoethoxy)(diisopropylamino)phosphine, 1H-tetrazole, phenylacetyl dis-
ulfide, 72%; (i) NH4F, MeOH, 85%; (j) 1H-tetrazole, CH2Cl2, rt, t-BuOOH;
(k) TEA, BSTFA, CH3CN; (l) TMSBr/CH2Cl2 (2:3), rt; (m) MeOH.
Phosphorothioates are phosphomimetics that show reduced rates
of enzyme-mediated hydrolysis.10 However, the replacement of Pd
O by PdS also affects the pKa of the phosphate and removes a
H-bond acceptor.11,12 For example, the phosphorothioate analogue
of PtdIns(3)P had reduced binding activity for cognate binding
proteins, due in part to reduced H-bonding.13 We hypothesized that
a 3-phosphorothioate of PtdIns(3,4,5)P3 could be either an antagonist
or a long-lived agonist in the PI 3-K signaling pathway because of
reduced dephosphorylation by PTEN. Moreover, the methylene-
phosphonate analogue of PtdIns(3)P bound selectively to one of
two cognate binding proteins.14 We now describe the first asym-
metric total syntheses of two PtdIns(3,4,5)P3 analogues that are
resistant to the 3-phosphatase PTEN: 3-PT-PtdIns(3,4,5)P3 and
3-MP-PtdIns(3,4,5)P3. Further, we show both selective binding
to a PtdIns(3,4,5)P3-binding protein and the ability of these
analogues to increase sodium transport in A6 cell monolayers.
The synthetic sequence to 3-phosphorothioate-PtdIns(3,4,5)P3
(3-PT-PtdIns(3,4,5)P3) is illustrated in Scheme 1. Treatment of
TBDPS ether 315,16 with the bulky bifunctional reagent TBDPSCl2
in the presence of imidazole selectively afforded the diol 4,5-bis-
silyl ether in 88% yield as a single product; the diols were then
protected to give compound 4. Next, TIPDS deprotection, bisphos-
phorylation with dimethyl N,N-diisopropylphosphoramidite, and
subsequent m-CPBA oxidation generated the protected 4,5-bis-
phosphate 5 in good yield. Since attempts to remove TBDPS in
the presence of the cyanoethyl phosphate protecting groups failed
to give a satisfactory result, the TBDPS was replaced with TES at
this stage. Reduction of the benzoyl ester 6 with DIBAL-H at -78
°C followed by thiophosphorylation with bis(2-cyanoethoxy)-
(diisopropylamino)phosphine in the presence of 1H-tetrazole and
phenylacetyl disulfide provided the desired TES ether.17 Depro-
tection of TES with the weakly acidic reagent NH4F in methanol
gave the key advanced intermediate 7 in 80% yield. Condensation
of 7 with each of four different freshly prepared 1,2-di-O-acyl-sn-
glycero cyanoethyl (N,N-diisopropylamino) phosphoramidites 8a-d
in the presence of 1H-tetrazole, followed by t-BuOOH oxidation,
gave the fully protected lipids 9a-d.13 Removal of the cyanoethyl
groups with triethylamine and bis(trimethylsilyl)trifluoroacetamide
(BSTFA) followed by removal of the MOM and methyl ester groups
with TMSBr afforded the 3-PT-PtdIns(3,4,5)P3 analogues 1a-d.
Scheme 2 summarizes the preparation of the 3-methylenephos-
phonate-PtdIns(3,4,5)P3 (3-MP-PtdIns(3,4,5)P3, 2), in which
reduction of 4 with DIBAL-H was followed by alkylation with
dimethyl phosphonomethyltriflate (n-BuLi/HMPA) to give meth-
ylenephosphonate 10 in 80% yield. Use of excess HMPA to chelate
the Li+ cation and enhance the nucleophilicity of the alkoxide was
the key to obtaining a high yield. Selective desilylation of 10 with
1 M TBAF in THF provided the 4,5-diol, which was bisphospho-
rylated to give TBDPS ether 11. Removal of the TBDPS group
† The University of Utah.
‡ Department of Cell Physiology, Universite´ Libre de Bruxelles.
§ IRIBHM, Universite´ Libre de Bruxelles.
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16464
J. AM. CHEM. SOC. 2006, 128, 16464-16465
10.1021/ja065002j CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2. Synthesis of Methylenephosphonates 2a
poral coordination of lipid production and removal are likely
required for normal physiology, and thus PtdIns(3,4,5)P3 is neces-
sary but not sufficient to fully mimic the action of insulin.
We tested the binding of the 3-PT and 3-MP analogues to the
specific PtdIns(3,4,5)P3-binding protein Grp1 (Supporting Informa-
tion Figure 2). DiC8-3-PT-PtdIns(3,4,5)P3 1b bound to Grp1 with
5-fold reduced affinity relative to that of diC8-PtdIns(3,4,5)P3, but
the diC8-3-MP analogue 2b showed no binding at all. Moreover,
while PTEN rapidly hydrolyzed diC8-PtdIns(3,4,5)P3, no hydrolysis
was observed with either 1b or 2b (Supporting Information Figure
3). Interestingly, diC8-3-PT analogue 1b showed >90% inhibition
of PTEN activity at 0.4 µM, while the diC8-3-MP analogue 2b
required 40 µM for >90% inhibition (A. Branch, P. Neilsen,
personal communication). Thus, analogues 1 and 2 have potential
as protein-selective biological tools in the PI 3-K signaling pathway.
Additional functional assays and interactions with PTEN will be
reported in due course.
a Conditions: (a) DIBAL-H, CH2Cl2, -78 °C, 88%; (b) n-BuLi, HMPA,
dimethyl phosphonomethyltriflate, THF, -78 °C to rt, 80%; (c) TBAF,
THF, 90%; (d) N,N-dimethylphosphoramidite, 1H-tetrazole, m-CPBA, 95%;
(e) TBAF‚3H2O, DMF, 75%; (f) 1H-tetrazole, 8a-d, CH2Cl2, rt, t-BuOOH;
(g) TEA, BSTFA, CH3CN; (h) TMSBr/CH2Cl2 (2:3), rt; (i) MeOH.
Acknowledgment. We thank the NIH (NS 29632 to GDP) and
the “Fonds de la Recherche Scientifique Me´dicale” for support,
Dr. C. Ferguson (Echelon Biosciences, Inc.) for Grp1 binding data,
and Dr. P. Neilsen and Ms. A. Branch (Echelon) for PtdIns(3,4,5)-
P3, histone, PTEN, and assistance with the PTEN assays.
Supporting Information Available: Experimental details for
synthesis, characterization of new compounds, binding data, and PTEN
assays. This material is available free of charge via the Internet at http://
pubs.acs.org.
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