Asymmetric Synthesis of Water-Soluble, Nonhydrolyzable Phosphonate Analogue of Phosphatidylinositol 4,5-Bisphosphate

Full text of DOI:10.1021/jo972046p

430
J . Org. Chem. 1998, 63, 430-431
tives and analogues of the cyclic 1,2-phosphate intermedi-
ates18 can be employed. Most recently, a number of phos-
phonate analogues of PtdIns have been prepared to study
the structure and mechanism of bacterial PtdIns-specific
PLC.19,20 To date, however, no phosphonate analogues of
PtdIns(4,5)P2 have been reported as nonhydrolyzable sub-
strates for the mammalian enzyme. The difficulty in
synthesizing such analogues appeared to be the absence of
an appropriate method to form the phosphonate C-P bond.
We report herein an efficient route for the asymmetric
synthesis of nonhydrolyzable phosphoinositide polyphos-
phates, and we show the application of this route for the
preparation of a dibutyryl derivative of the PtdIns(4,5)P2
phosphonate analogue.
Asym m etr ic Syn th esis of Wa ter -Solu ble,
Non h yd r olyza ble P h osp h on a te An a logu e of
P h osp h a tid ylin ositol 4,5-Bisp h osp h a te
J ian Chen and Glenn D. Prestwich*
Department of Medicinal Chemistry, The University of Utah,
30 South 2000 East, Room 201,
Salt Lake City, Utah 84112-5820
Received November 7, 1997
The hydrolysis of L-R-phosphatidyl-D-myo-inositol 4,5-bis-
phosphate (PtdIns(4,5)P2) by phospholipase C (PLC) yields
inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol,
two key second messengers in cell signaling.1 Four PLC
isozymes (R, â, γ, and δ) are recognized on the basis of their
modular structures and the mechanisms of their regulation.2
The vertebrate δ1 isoform contains two binding sites for
PtdIns(4,5)P2, the catalytic domain and the pleckstrin
homology (PH) domain, which are important for recruitment
of PLC to the membrane3 and for activation of the enzymatic
activity.4 Three-dimensional structures of this PH domain
complexed to Ins(1,4,5)P3 have been solved by NMR and
X-ray crystallographic methods.5-7 Moreover, X-ray struc-
tures of the combined C2, EF-hand, and catalytic domains
have revealed many intimate details germane to the cata-
lytic mechanism.8-12 In biophysical studies, inhibition of
PLC by basic peptides occurs by sequestration of substrates
in two-dimensional domains.13 Despite considerable effort,
however, the precise mode of binding of the actual PtdIns-
(4,5)P2 substrate in the active site or in the PH domain
remains elusive. The two principal difficulties are chemical
in nature: (a) the natural substrate, with sn-1-O-stearoyl
and sn-2-O-arachidonyl side diacylglyceryl chains, is micellar
and cannot be readily employed in structural biological
studies, and (b) the hydrolysis of the P-O bond occurs too
rapidly for collection of NMR or crystallographic data.
Both problems also have chemical solutions. First, shorter
chain analogues may be synthesized and employed for either
NMR or cocrystallization experiments. The usefulness of
short chain analogues as substrates for PLC isozymes is
already recognized; that is, a micellar substrate is not
required for PLC activity.14 Second, analogues with slower
hydrolytic rates, such as phosphorothioates,15-17 or deriva-
Trivalent phosphorus chemistry is an essential component
in the preparation of intermediates in the synthesis of
inositol polyphosphates and polyphosphoinositides.16,21,22
The
tautomeric equilibrium between the trivalent hydrogen
dialkyl phosphite and the pentavalent H-phosphonates has
been employed to form new C-P bonds during the prepara-
tion of phosphonates. However, hydrogen dialkyl phos-
phites, often prepared by esterification of PCl3 followed by
hydrolysis, have not been extensively utilized in inositol
chemistry. In the work described below, a hydrogen benzyl
inosityl phosphite was prepared by addition of water to a
phosphoramidite. This intermediate could be used to form
the phosphonate analogue of the phosphodiester between a
phosphorylated inositol headgroup and the sn-3-O-linked
diacylglyceryl moiety.
The D-myo-inositol intermediate 1 was synthesized from
methyl R-D-glucopyranoside as previously described23-26 via
a Ferrier rearrangement route.27 Coupling the inositol 1
with a phosphoramidite in the presence of N,N-diisopropyl-
ethylammonium 1H-tetrazole at rt gave the phosphoramid-
ite intermediate 2 (Scheme 1). Because of the steric
* To whom correspondence should be addressed. Tele.: (801) 585-9051.
Fax: (801) 585-9053. E-mail: gprestwich@deans.pharm.utah.edu.
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S0022-3263(97)02046-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/22/1998

Communications
J . Org. Chem., Vol. 63, No. 3, 1998 431
Sch em e 1a
Sch em e 3a
a
Reaction conditions: (a) (BnO)P(NPr2-i)2, N,N-isopropylethylam-
monium 1H-tetrazole, CH2Cl2, rt, 2 d. (b) H2O, 1H-tetrazole, rt, 1 h,
CH2Cl2.
Sch em e 2
a
Reaction conditions: (a) NaH, DMF, 8, rt, 30 min; (b) DDQ,
CH2Cl2-H2O (100/1, v/v), rt, 2 h; (c) (BnO)2P(NPr2-i), 1H-tetrazole,
CH2Cl2, rt, 1 h, then m-CPBA, 30 min; (d) p-TsOH‚H2O, MeOH, rt, 1
h; (e) butyric acid, DCC, DMAP, CH2Cl2, rt, overnight; (f) Pd/C (10%),
95% EtOH, H2, 50 psi; then ion exchange.
hindrance of the secondary hydroxyl in precursor 1, the
sluggish reaction required more than 48 h for completion.
Heating or using 1H-tetrazole accelerated the reaction but
at the expense of increased byproduct formation. In the
optimized reaction, 2 equiv of phosphoramidite and 1 equiv
of N,N-diisopropylethylammonium 1H-tetrazole were added
at t ) 0 and again at t ) 24 h; after 2 d, TLC showed >95%
completion. At this point, 1H-tetrazole (2 equiv) was added
followed by a small amount of water, and the reaction was
stirred for an additional 1 h. The hydrogen phosphite 3 was
obtained in 86% isolated yield (for two steps) based on the
consumed starting inositol 1. Two compounds, separable on
SiO2 column, corresponded to the diastereomers arising from
the newly introduced chiral phosphorus. Moreover, the
diastereomeric phosphites were in equilibrium with the
H-phosphonate forms 3a and 3b, as recognized in the 31P
NMR spectrum of the mixture. The mixture was employed
without separation, as the final product would not possess
P-1 chirality.
The glyceryl synthon was prepared from (S)-(-)-1,2,4-
butanetriol 4. Thus, protection of the vicinal hydroxyls as
an acetone acetal gave alcohol 5, which was converted
sequentially to the tosylate 6, bromide28 7, and finally to
iodide 8 (Scheme 2). Reaction of the hydrogen phosphite 3
with sodium hydride in DMF generated an anion that was
expected to react with tosylate 6 or with bromide 7. In the
event, the tosylate 6 and bromide 7 were not sufficiently
reactive and debenzylation was observed as the predominant
reaction. In contrast, iodide 8 reacted smoothly with the
anion derived from 3 and gave the desired product 9 in 96%
isolated yield (Scheme 3). Product 9 and compounds pre-
pared from 9 were obtained as mixtures of two diastereomers
(31P, 34 and 35 ppm, in a 2:1 ratio) resulting from chirality
of the tetrahedral phosphorus.29 To avoid competing de-
benzylation, a reaction time of 30 min and only a 2-fold
excess of sodium hydride is recommended. Oxidative re-
moval of the PMB ethers from compound 9 with DDQ gave
diol 10 in 93% yield, and reaction of diol 10 with dibenzyl
N,N-diisopropylphosphoramidite followed by m-CPBA oxida-
tion gave the protected bisphosphate 11. The acetal in crude
phosphonate 11 was then hydrolyzed; homogeneous 12 was
obtained in 95% yield for two steps. The diol 12 could be
acylated with any fatty acid; in this case, for the purpose of
obtaining a water-soluble PtdIns(4,5)P2, the dibutyrate was
synthesized. Thus, carbodiimide-mediated esterification
with butyric acid gave the fully protected diester 13 in 92%
yield with minor contamination by the dicyclohexylurea
(DCU) byproduct. The target phosphonate analogue 14 was
obtained in essentially quantitative yield, free from DCU,
by hydrogenolysis of all protecting groups at 50 psi H2
followed by ion-exchange purification. Removal of the
protecting groups converted the diastereomeric mixtures for
compounds 9-13 into a single stereoisomer with the D-myo
inosityl headgroup and the methylene analogue of the sn-
1,2-O-diacyl-3-phosphoglyceryl moiety.29
In conclusion, an efficient route for preparation of the
nonhydrolyzable phosphoinositide polyphosphates is de-
scribed. Specifically, a phosphonate analogue of a short-
chain analogue of PtdIns(4,5)P2 was prepared in which the
sn-3 oxygen of the diacylglyceryl moiety is substituted by
CH2, rendering the product resistant to hydrolysis by PLC.
This phosphonate analogue was obtained in 67% yield for
seven steps from a protected inositol precursor. This route
offers a high-yield strategy for preparation of diacylglyceryl
analogues (with any chain length) of phosphoinositide phos-
phonates (with any inositol phosphorylation pattern) from
readily available homochiral inositol precursors.
Ack n ow led gm en t. We thank the NIH (Grant No. NS
29632) for financial support and Dr. R. L. Williams (MRC,
Cambridge, U.K.) for suggesting this molecular target and
implementing its use.
Su p p or tin g In for m a tion Ava ila ble: Experimental proce-
dures for the synthesis as well as spectral and analytical data of
compounds 3 and 8-14 (5 pages).
J O972046P
(29) Compound 9: 31P NMR (81 MHz, CDCl3) δ 34.88, 33.91 (2s, 1: 2);
HRMS (FAB) calcd for
C59H70O14P
(MH+) 1033.4503, found 1033.4515.
Compound 13: 31P NMR (81 MHz, CDCl3) δ 34.41, 33.71 (2s, 1: 2, combined
as 1P), -0.49, -0.58 (combined as 1P), -0.81, -0.87 (combined as 1P);
HRMS (FAB) calcd for C68H76O18P3 (MH+) 1273.4244, found 1273.4294.
Compound 14: 1H NMR (500 MHz, D2O) δ 5.10-5.00 (m, 1H, sn-2), 4.06
(dd, J ) 2.5, 12 Hz, 1H), 4.15 (q, J ) 8.5 Hz, 1H), 4.10-4.05 (m, 2H), 3.95-
3.90 (m, 2H), 3.87 (t, J ) 9.0 Hz, 1H), 3.78 (t, J ) 9.5 Hz, 1H), 3.60 (dd, J
) 3, 10 Hz, 1H), 2.35-2.25 (m, 4H, CH2CO), 1.85-1.45 (m, 8H, 2CH2
+
CH2CH2P), 0.82 (2t, J ) 7.2 Hz, 6H, CH3) ppm; 31P NMR (1H-decoupled, 81
MHz, D2O) δ 30.22 (s, 1P), 6.45 (s, 1P); 5.35 (s, 1P). 31P NMR (1H-coupled,
2
3
3
81 MHz, D2O) δ 30.30 (dt, J HP ) 16.4 Hz, J HP ) 8.1 Hz, 1P), 6.58 (d, J HP
) 8.4 Hz, 1P), 5.30 (d, 3J HP ) 7.0 Hz, 1P); 13C NMR (50 MHz, D2O) δ 178.85,
178.80, 80.28 (t, J ) 5.0 Hz), 78.30 (q, J ) 2.9 Hz), 76.65 (d, J ) 6.6 Hz),
75.11, 74.72, 73.70, 73.45, 73.39, 79.09, 38.35, 37.93, 26.50 (t, J ) 3.1 Hz),
20.28, 20.17, 15.07.
(28) Hayashi, H.; Nakanishi, K.; Brandon, C.; Marmur, J . J . Am. Chem.
Soc. 1973, 95, 8749-8757.