Synthesis and biological activity of phospholipase C-resistant analogues of phosphatidylinositol 4,5-bisphosphate

Article abstract of DOI:10.1021/ja060621d

The membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) is an important regulator in cell physiology. Hydrolysis of PtdIns(4,5)P₂ by phospholipase C (PLC) releases two second messengers, Ins(1,4,5)P₃

Full text of DOI:10.1021/ja060621d

Published on Web 04/06/2006
Synthesis and Biological Activity of Phospholipase C-Resistant Analogues of
Phosphatidylinositol 4,5-bisphosphate
Honglu Zhang,† Yong Xu,† Zheng Zhang,‡ Emily R. Liman,‡ and Glenn D. Prestwich*,†
Department of Medicinal Chemistry, UniVersity of Utah, 419 Wakara Way, Suite 205, Salt Lake City, Utah
84108-1257, and Department of Biological Sciences and Program in Neuroscience, UniVersity of Southern
California, 3641 Watt Way, Los Angeles, California 90089-2520
Received February 6, 2006; E-mail: gprestwich@pharm.utah.edu
Scheme 1. Phospholipase C Catalyzes Hydrolysis of
PtdIns(4,5)P2 to Two Second Messengers, Ins(1,4,5)P3 and
Diacylglycerol
The membrane phospholipid phosphatidylinositol 4,5-bisphos-
phate (PtdIns(4,5)P2) is an important regulator of cytoskeletal
organization during a plethora of cellular functions, such as vesicle
trafficking, endocytosis, phagocytosis, focal adhesion formation,
and cell migration.1 PtdIns(4,5)P2 binds to and affects the function
of many actin-binding and actin-remodeling proteins2-4 and is a
cofactor in enzyme activation.5 In addition, PtdIns(4,5)P2 regulates
the activity of many ion channels and transporters.6,7 PtdIns(4,5)P2
is also the source of three second messengers: Ins(1,4,5)P3,
diacylglycerol (DAG),8,9 and PtdIns(3,4,5)P3.10 In many cases, it
is the decrease in PtdIns(4,5)P2, resulting from hydrolysis by
phospholipase C (PLC) (Scheme 1), and not the increase in Ins-
(1,4,5)P3 and DAG that constitutes the physiologically relevant
signal.11,12 Hydrolysis of PtdIns(4,5)P2 causes TRP channels to lose
some activity.13-19 Moreover, addition of PtdIns(4,5)P2 restores
sensitivity of TRPM4 and TRPM5 to activation by Ca2+ and restores
the sensitivity of TRPM8 and TRPV1 to thermal and chemical
stimuli.15,16,18,19
Scheme 2. Synthesis of Phosphonates 1-5a
The availability of a metabolically stabilized analogue of PtdIns-
(4,5)P2, that is, one that lacks the scissile P-O bond and thus could
not be hydrolyzed by PLC activity, would have many applications
in understanding the role of PtdIns(4,5)P2 in cell physiology.
R-Fluoroalkylphosphonates have emerged as important nonhydro-
lyzable mimics for phosphoesters in the synthesis of biologically
active “unnatural products”.20-23 Herein we describe the first
asymmetric total synthesis of isosteric and isoelectronic phosphonate
analogues 1-5 of PtdIns(4,5)P2 that cannot be hydrolyzed by PLC.
The synthesis employs a Pd(0) coupling not previously exploited
in phospholipid or phosphoinositide synthesis. Furthermore, we
demonstrate that both saturated and unsaturated R-fluorophospho-
nate analogues can substitute for exogenous PtdIns(4,5)P2 in
restoring the sensitivity of the TRPM4 channel to Ca2+
.
The synthetic sequence to the stabilized analogues 1-5 of PtdIns-
(4,5)P2 is illustrated in Scheme 2. A variety of attempts to connect
the intermediate 924 with a fluoromethylenephosphonic acid syn-
thon21 failed. Eventually, we turned to the Pd(0)-catalyzed coupling
of an H-phosphite with a vinyl bromide in order to form the desired
C-P linkage. Thus, coupling the protected inositol 9 with dibenzyl
N,N-diisopropylphosphoramidite gave the phosphoramidite inter-
mediate 10, which was converted to H-phosphonate 11 in 76%
isolated yield in two steps.25 The 1-bromo-1-fluoroolefin 7 (∼1:1
E/Z) was separately prepared via a Et2Zn-promoted olefination
reaction26 of CBr3F/PPh3 with glyceraldehyde 6 in excellent yield.
Few examples exist of Pd(0)-catalyzed formation of P-CF
bonds, and in our hands, only traces of coupled compound 12 with
a majority of the P-O cleaved compound 9 were obtained under
standard conditions using Et3N or K2CO3 as base. It appeared that
a Conditions: (a) CFBr3, PPh3, Et2Zn, THF, 76%; (b) (BnO)2P(NPr2-
i)2, N,N-diisopropylethylammonium •1H-tetrazole, CH2Cl2, rt; (c) H2O, 1H-
tetrazole, rt, 1 h, CH2Cl2, 76% for two steps; (d) Pd(OAc)2, dppf, propylene
oxide, THF, 70 °C, 62%; (e) 60% aqueous TFA, THF, 0 °C, 1 h, 86%; (f)
EDCI, DMAP, fatty acid, CH2Cl2, rt; (g) H2, Pd/C, MeOH, 6 h, EtSH; (h)
TMBr/TMSI (5:1), rt, 1.5 h; MeOH, 1 h.
the rate of decomposition was faster than the rate of coupling for
the more hindered H-phosphonate 11. To overcome this problem,
we selected propylene oxide as a weak Lewis base and an effective
scavenger of HBr.27 Using this modification, treatment of the
H-phosphonate 11 with Pd(OAc)2/dppf/propylene oxide in THF at
70 °C led to the formation of R-fluorovinylphosphonate 12 in 62%
† University of Utah.
‡ University of Southern California.
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J. AM. CHEM. SOC. 2006, 128, 5642-5643
10.1021/ja060621d CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Dose-response for recovery of TRPM4 currents by 2 and 4. After
TRPM4 desensitization, recovery was assessed. Data were normalized to
the response to 10 µM of each analogue in the same patch. (A) Averaged
data (n ) 5) for recovery of TRPM4 currents by 2 (EC50 ) 2.7 ( 0.6 µM
and nH ) 2.5 ( 1.2). (B) Averaged data (n ) 6) for 4 (EC50 ) 1.8 ( 0.1
µM and nH ) 3.2 ( 0.5).
Figure 1. PtdIns(4,5)P2 and analogues 2 and 4 restore TRPM4 currents
following desensitization. (A) An excised inside-out patch from Chok1 cell
expressing mouse TRPM4 (mTRPM4) shows activation and fast rundown
of an inward current in the presence of 100 µM Ca2+ and recovery by
dioctanoyl-PtdIns(4,5)P2 and analogues 2 and 4 (Vm ) 80 mV). (B) Initial
magnitudes of the mTRPM4 currents, currents after rundown, and currents
after recovery in response to 10 µM each of PtdIns(4,5)P2, 2, and 4
(averages, n ) 8).
TRPM4 currents. These results suggest that metabolically stabilized
analogues of PtdIns(4,5)P2 will have a wide variety of applications
in separating the role of the phosphoinositide per se from activities
that result when Ins(1,4,5)P3, DAG, Ca2+, or other downstream
signals are generated from the hydrolysis of PtdIns(4,5)P2 by PLC.
yield. Acetal 12 was selectively deprotected by treatment with 60%
aqueous trifluoroacetic acid in tetrahydrofuran at 0 °C to give diol
13. Next, acylation of 13 with either octanoic acid, palmitic acid,
or oleic acid provided the fully protected phosphonates 14a, 14b,
and 14c in 80, 73, and 82% yields, respectively. Hydrogenolysis
of 14a and 14b removed the benzyl groups, and then reaction with
ethanethiol removed the MOM groups to give the R-fluorometh-
ylenephosphonate analogues 1 and 2.28 The R-fluorovinylphospho-
nates 3-528 were obtained by deprotection of benzyl and MOM
groups simultaneously with TMSBr/TMSI (5:1).
Acknowledgment. We thank the NIH (Grant NS 29632 to
G.D.P. and DC 004564 to E.R.L.) for financial support of this work.
Supporting Information Available: Experimental details for
synthesis and characterization of new compounds, and protocols for
TRPM4 channel activity measurement. This material is available free
of charge via the Internet at http://pubs.acs.org.
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