Synthesis and molecular recognition of phosphatidylinositol-3- methylenephosphate

Article abstract of DOI:10.1021/ol060903i

Phosphatidylinositol-3-phosphate (Ptdlns(3)P) is a spatial regulator of vesicular trafficking and other vital cellular processes. We describe the asymmetric total synthesis of a metabolically stabilized analogue, phosphatidylinositol-3-methylenephosphate (Ptdlns(3)MP) from a differentially protected myo-inositol. NMR studies of Ptdlns(3)MP bound to the ¹⁵N-labeled FYVE domain showed significant ¹H and ¹⁵N chemical shift changes relative to the unliganded protein.

Full text of DOI:10.1021/ol060903i

ORGANIC
LETTERS
2006
Vol. 8, No. 13
2811-2813
Synthesis and Molecular Recognition of
Phosphatidylinositol-3-methylenephosphate
Joanna Gajewiak,† Yong Xu,† Stephanie A. Lee,‡ Tatiana G. Kutateladze,‡ and
Glenn D. Prestwich*,†
Department of Medicinal Chemistry, The UniVersity of Utah, 419 Wakara Way,
Suite 205, Salt Lake City, Utah 84108-1257, and Department of Pharmacology,
UniVersity of Colorado Health Sciences Center, Aurora, Colorado 80045
glenn.prestwich@hsc.utah.edu
Received April 13, 2006
ABSTRACT
Phosphatidylinositol-3-phosphate (PtdIns(3)P) is a spatial regulator of vesicular trafficking and other vital cellular processes. We describe the
asymmetric total synthesis of a metabolically stabilized analogue, phosphatidylinositol-3-methylenephosphate (PtdIns(3)MP) from a differentially
protected myo-inositol. NMR studies of PtdIns(3)MP bound to the 15N-labeled FYVE domain showed significant 1H and 15N chemical shift
changes relative to the unliganded protein.
Phosphoinositide (PtdInsPn) signaling networks are dynami-
cally modulated by proteins with lipid recognition motifs as
well as kinase, phosphatase, and phospholipase enzymatic
activities. In particular, the 3-phosphorylated PtdInsPn lipids
have been implicated as activators of protein kinase C
isoforms and are messengers in cellular signal cascades
pertinent to inflammation, cell proliferation, transformation,
protein kinesis, and cytoskeletal assembly.1-3 PtdIns(3)P is
produced by the action of phosphoinositide-3-kinase (PI
3-K)1,4 on PtdIns, and its interactions with cognate binding
proteins, kinase, and phosphatases are important in cell
physiology. PtdIns(3)P specifically binds FYVE domains5-7
and is involved in phagocytosis,8 membrane trafficking, and
protein sorting.9 PX domains also recognize PtdIns(3)P, and
spatiotemporal changes mediate important aspects of cell
respiration.10 The myotubularin-related (MTMR) protein
family11 is comprised of PtdIns(3)P phosphatases that
contribute to lipid remodeling and are mutated in genetic
diseases.12
To gain deeper insight into these biological pathways,
selective reagents that can interfere with ligand binding,
inhibit enzyme activity, and activate protein-mediated lipid
signaling are needed.13 We recently described a general
approach to the synthesis of methylphosphonate, (mono-
fluoromethyl)phosphonate, and phosphorothioate analogues
(5) Kutateladze, T.; Ogburn, K.; Watson, W.; de Beer, T.; Emr, S.; Burd,
C.; Overduin, M. Mol. Cell 1999, 3, 805-811.
* To whom correspondence should be addressed. Phone: +1-801-585-
9051. Fax: +1-801-585-9053.
(6) Gaullier, J. M.; Simonsen, A.; D’Arrigo, A.; Bremnes, B.; Stenmark,
H.; Aasland, R. Nature 1998, 394, 432-433.
† The University of Utah.
‡ The University of Colorado Health Sciences Center.
(1) Vanhaesebroeck, B.; Leevers, S. J.; Ahmadi, K.; Timms, J.; Katso,
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ReV. Biochem. 2001, 70, 535-602.
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(7) Kutateladze, T. BBA - Mol. Cell Biol. Lipids 2006, in press.
(8) Gillooly, D. J.; Simonsen, A.; Stenmark, H. J. Cell Biol. 2001, 155,
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2003, 12, R285-R292.
(13) Prestwich, G. D. Chem. Biol. 2004, 11, 619-637.
10.1021/ol060903i CCC: $33.50
© 2006 American Chemical Society
Published on Web 05/24/2006

of PtdIns(3)P.14 These metabolically stabilized ligands were
recognized by 15N-labeled FYVE and PX domains and were
also substrates for PIKfyve, a 5-kinase required for the
formation of multivesicular bodies. We now introduce a
modified synthetic route that provides access to a stabilized
methylenephosphonate analogue, PtdIns(3)MP, which retains
the inositol 3-oxygen as well as the dianionic headgroup. In
this modification, a methylene bridge was inserted between
the oxygen of the inositol moiety and the phosphate
headgroup. A similar approach was also used to generate
alkoxymethylene phosphonate-containing geranylgeranyl pro-
tein transferase inhibitors15 and antiviral drugs,16 including
anti-HIV phosphorylated nucleoside analogues.17,18 We have
also used this approach to synthesize potent analogues of
lysophosphatidic acid and phosphatidic acid, and these results
will be presented in due course. Herein, we describe the
asymmetric total synthesis of the methylenephosphonate
analogue of PtdIns(3)P, and we illustrate the binding of
PtdIns(3)MP to the FYVE domain.
The synthetic strategy employed the simple and elegant
protection scheme of Bruzik,19 in which the 1-position of
myo-inositol (1) was silylated with the TBDPS group, the
phosphomonoester 3-position was protected as a benzoate
group, and all remaining hydroxyl groups were protected as
methoxymethyl (MOM) ethers. Thus, 1-O-(tert-butyl-di-
phenylsilyl)-2,4,5,6-O-tetrakis(methoxymethylene)-myo-inositol
(2) was synthesized from myo-inositol in six steps. Instal-
lation of the methylenephosphonate moiety required the
preparation (Scheme 1) of dimethyl phosphonomethyltriflate
lutidine as the base and was employed without further
purification.
The alkoxide of protected inositide 2 (n-BuLi,15 -78 °C)
was alkylated with triflate 5 in 64% yield (Scheme 2). Use
of NaH or t-BuOK as the base did not significantly improve
the yield and resulted in greater decomposition of the starting
material. The TBDPS group was removed by treating
intermediate 6 with n-Bu4F. The resulting alcohol 7 was
coupled with the dibutanoylglyceryl phosphoramidite14 in the
presence of 1H-tetrazole, followed by mild oxidation with
14
n-BuNIO4 to give fully protected PtdIns(3)MP (8). The
removal of phosphate and hydroxyl protecting groups of 8
was accomplished under strictly anhydrous conditions with
20 equiv of fresh TMSBr (CH2Cl2, room temperature, 1 h).
After concentration in vacuo, the residue was dissolved in
90% aq CH3OH and stirred for 40 min to hydrolyze the silyl
phosphate esters. We found that under these conditions not
only were the phosphates deprotected but also all MOM
groups were removed. After complete evaporation of the
organic solvent in vacuo, the crude compound was dissolved
in water and passed through a short column of acidic Dowex
ion-exchange resin to yield the final product in >98% purity.
In endosomal membranes, PtdIns(3)P is specifically rec-
ognized by a number of protein binding partners including
FYVE and PX domains. We next investigated the interactions
of human EEA1 FYVE and yeast Vam7 PX domains with
PtdIns(3)MP by NMR spectroscopy. Significant changes
were observed in 1H and 15N resonances in the FYVE domain
when titrating in dibutanoyl PtdIns(3)MP (9) (Figure 1a).
These perturbations were of reduced magnitude but paralleled
the chemical shift changes apparent in the complex of the
FYVE domain with dibutanoyl-PtdIns(3)P (Figure 1b). Thus,
the PtdIns(3)MP analogue and native lipid are accommodated
by the same binding pocket consisting of four Arg and two
Scheme 1. Synthesis of Dimethyl Phosphonomethyltriflate (5)
1
His residues of the FYVE domain. On the basis of H and
15N chemical shift changes, the FYVE domain affinity for
PtdIns(3)MP was calculated to be 3.8 ( 0.5 mM (see
supplementary Figure 2 in Supporting Information). To put
this in perspective, the local concentration of PtdIns(3)MP
(5) following the literature route.20,21 Reaction of paraform-
aldehyde with trimethyl phosphite gave hydroxymethyl
phosphonate 4, which was converted to triflate 5 using 2,6-
Scheme 2. Synthesis of PtdIns(3)MP (9)
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Org. Lett., Vol. 8, No. 13, 2006

1
Figure 1. Binding of PtdIns(3)P and PtdIns(3)MP to the FYVE domain. H-15N heteronuclear single-quantum coherence (HSQC) NMR
spectra of the 0.2 mM EEA1 FYVE domain before and after addition of (a) dibutanoyl-PtdIns(3)MP (9) and (b) dibutanoyl-PtdIns(3)P.
in early endosomal membranes is quite high (∼200 µM)22
and the FYVE-dibutanoyl-PtdIns(3)P affinity is 135 µM
under similar experimental conditions.23 A similar experiment
with the PX domain showed a much weaker binding without
significant chemical shift changes. Addition of up to 11.3
mM (57-fold excess) PtdIns(3)MP to a 0.2 mM PX domain
sample induced no noticeable resonance changes. For
comparison, the Kd of the PX domain for dibutanoyl-
PtdIns(3)P under these conditions is approximately 300 µM.24
This result may be attributed to the difference in the binding
modes for the two complexes. PtdIns(3)P inserts its 3-phos-
phate between two loops and an R-helix of the PX domain.25
However, in the case of the FYVE domain, PtdIns(3)P
occupies a shallow pocket in a side-on orientation.26 Appar-
ently, the extended methylenephosphonate group is too bulky
to be accommodated in the PX domain binding pocket. Thus,
PtdIns(3)MP is the first analogue of PtdIns(3)P that shows
discrimination in its protein-ligand interactions, suggesting
that it could be useful in selectively perturbing distinct
PtdIns(3)P signaling pathways.
(14) Xu, Y.; Lee, S. A.; Kutateladze, T. G.; Sbrissa, D.; Shisheva, A.;
Prestwich, G. D. J. Am. Chem. Soc. 2006, 128, 885-897.
(15) Minutolo, F.; Antonello, M.; Barontini, S.; Bertini, S.; Betti, L.;
Danesi, R.; Gervasi, G.; Giannaccini, G.; Papi, C.; Placanic, G.; Rapposelli,
S.; Macchia, M. Il Farmaco 2004, 59, 887-892.
(16) De Clercq, E.; Holy, A. Nat. ReV. Drug. DiscoVery 2005, 4, 928-
940.
Acknowledgment. We thank the NIH (Grant NS 29632
to G.D.P.) and the American Cancer Society (T.G.K.) for
financial support of this work.
Supporting Information Available: Experimental details
(17) Jie, L.; Van Aerschot, A.; Balzarini, J.; Janssen, G.; Busson, R.;
Hoogmartens, J.; De Clercq, E.; Hardewijn, P. J. Med. Chem. 1990, 33,
241-245.
for the synthesis and characterization of new compounds and
1
protocols for the H and 15N NMR binding measurements.
(18) Van Aeroschot, A.; Jie, L.; Herdewijn, P. Tetrahedron Lett. 1991,
32, 1905-1908.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(19) Kubiak, R. J.; Bruzik, K. S. J. Org. Chem. 2003, 68, 960-968.
(20) Phillion, D. P.; Andrew, S. S. Tetrahedron Lett. 1986, 27, 1477-
1480.
OL060903I
(21) Hamilton, C. J.; Roberts, S. M. J. Chem. Soc., Perkin Trans. 1 1999,
1051-1056.
(22) Stenmark, H.; Gillooly, D. J. Semin. Cell DeV. Biol. 2001, 12, 193-
(24) Cheever, M.; Kutateladze, T.; Overduin, M. Unpublished results.
(25) Bravo, J.; Karathanassis, D.; Pacold, C. M.; Pacold, M.; Ellson, C.
D.; Anderson, K. E.; Butler, P.; Lavenir, I.; Perisic, O.; Hawkins, P. T.;
Stephens, L.; Williams, R. Mol. Cells 2001, 8, 829-839.
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(23) Lee, S. A.; Eyeson, R.; Cheever, M. L.; Geng, J.; Verkhusha, V.
V.; Burd, C.; Overduin, M.; Kutateladze, T. G. PNAS 2005, 102, 13052-
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