Synthesis of dipalmitoyl-phosphatidylinositol 5-phosphate and its modified biological tools

Article abstract of DOI:10.1016/S0040-4039(00)01541-0

Synthesis of a dipalmitoyl analog of phosphatidylinositol 5-phosphate with the racemic inositol skeleton was achieved via a key intermediate, 1,2-cyclohexylidene-3,4-tetraisopropyldisiloxanyl-myo-inositol. Probes bearing a fluorophore, NBD on a fatty acid chain and a resin for affinity chromatography were also prepared due to biological interest in cell division. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01541-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 8509–8512
Synthesis of dipalmitoyl–phosphatidylinositol 5-phosphate
and its modified biological tools
Yutaka Watanabe* and Hideki Ishikawa
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan
Received 21 July 2000; revised 4 September 2000; accepted 8 September 2000
Abstract
Synthesis of a dipalmitoyl analog of phosphatidylinositol 5-phosphate with the racemic inositol skeleton
was achieved via a key intermediate, 1,2-cyclohexylidene-3,4-tetraisopropyldisiloxanyl-myo-inositol.
Probes bearing a fluorophore, NBD on a fatty acid chain and a resin for affinity chromatography were
also prepared due to biological interest in cell division. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: phosphatidylinositol 5-phosphate; fluorescent analog; affinity chromatography.
Phosphatidylinositol 4,5-bisphosphate (PI4,5ꢀP2) is a key substance in the phosphatidylinosi-
tol cell signaling pathway. It is converted to two second messengers, myo-inositol 1,4,5-trispho-
sphate and diacylglycerol, and is also transformed to 3-O-phosphorylated PI3,4,5ꢀP3 that
engages in a variety of signal cascades.1 In addition, PI4,5ꢀP2 itself acts as a modulator for
regulatory proteins. PI4,5ꢀP2 is known to be formed generally from PI4ꢀP by phosphatidylinos-
itol 4-phosphate specific 5-kinase (PI4Pꢀ5K) catalyzed phosphorylation. Recently, PI5ꢀP, the
content of which was 1/100 of that of PI4ꢀP, was discovered in mammalian fibroblasts and
shown to be transformed into PI4,5ꢀP2 by phosphorylation with PI5Pꢀ4K.2 Although the
physiological role of the new pathway is still unclear, its significance is anticipated due to the
low concentration of PI4,5ꢀP2. Chemical synthesis of PI5ꢀP with dipalmitoyl groups was
reported by the Prestwich3a and Falck3b groups. In this communication, we describe a conve-
nient alternative synthesis of a dipalmitoyl analog4 of natural PI5ꢀP and an analog bearing a
fluorescent group, NBD. Preparation of a PI5ꢀP attached resin for affinity chromatography is
also reported.
* Corresponding author.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01541-0

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A pivotal intermediate, diol 1 with DL-form, derived regioselectively from myo-inositol in two
steps,5 was acylated exclusively at the 6 position with levulinic acid in the presence of DCC and
DMAP, followed by phosphorylation via a phosphoramidite method to give 3 in good yield.
Removal of the cyclohexylidene group in 3 was achieved by the action of pyridinium poly(hy-
drogen fluoride)6 (PPF) in an anhydrous dichloromethane solution, affording 1,2-diol 4 in 89%
yield. The TIPDS group was intact in this reaction whereas PPF was reported to decompose
TIPDS ethers in sugar derivatives.7 The scope and limitation of the PPF-promoted deacetaliza-
tion will be described elsewhere. A conventional decyclohexylidenation using ethylene glycol and
p-toluenesulfonic acid resulted in the formation of ethylene ketals on the levulinoyl groups of
the starting material and the desired product (14% each) along with the diol 4 (48%).
The diol 4 thus derived was regioselectively phosphorylated by reaction with sn-3-glyceryl
phosphite 6 in the presence of pyridinium tribromide to give 1-O-phosphate 5 without formation
of the 2-O-phosphoric regioisomer. The phosphorylation site was confirmed by derivatization of
5 to 2-O-chloracetate and its NMR analysis. The phosphate 5 was deprotected by two
sequential procedures as shown in Scheme 1 to give the final product as a white solid.8 Removal
of the levulinoyl group before that of the benzyl groups in the phosphates was performed
successfully (Method 2) while, in general, deprotection of the phosphate functions was carried
out prior to removal of other protecting groups to avoid migration of the phosphates as done
in Method 1. Since the chiral 1,2-cyclohexylidene derivative 1 is available,9 an optically active
PI5ꢀP analog with saturated fatty acid chains has now also been formally prepared.
Scheme 1. Synthesis of dipalmitoylꢀPI5ꢀP. (i) CH3C(O)(CH2)2CO2H, DCC, DMAP; (ii) (BnO)2Pni-Pr2, tetrazole,
then mCPBA; (iii) Py(HF)x; (iv) Lutidine; (v) H2, Pd black, NaHCO3, t-BuOH/H2O (6:1 for Method 1, 4:1 for
Method 2); (vi) TBAF·3H2O, AcOH; (vii) NH2NH2·H2O, Py/AcOH (4:1). Abbreviations: DCC=dicyclohexylcar-
bodiimide, DMAP=4-(dimethylamino)pyridine, Bn=benzyl, mCPBA=m-chloroperbenzoic acid, Py=pyridine,
TBAF=tetrabutyl-ammonium fluoride
DipalmitoylꢀPI5ꢀP thus synthesized, was assumed to be concerned with cell division due to an
experiment using PI5Pꢀ4K over-expressed cells. To explore its physiological function in the
system, a fluorescent probe for detection of the localized site and resin for isolation of a PI5ꢀP

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recognition protein by affinity chromatography were designed and prepared as biological tools.
Thus, 1,2-diol 4 was phosphorylated with aminohexanoylglycerol phosphite 8 by a similar
procedure to that used above to furnish 7 in 89% yield. A sequential procedure for deprotection
of 7 involving hydrazinolysis, hydrogenolysis, and desilylation afforded 9. On the other hand,
when hydrogenolysis was first done according to Method 1 shown in Scheme 1, the reaction did
not proceed smoothly under various reaction conditions because NaHCO3, added to avoid
reduction of the levulinoyl ketone function, retarded the reaction. Aminoacyl PI5ꢀP 9 thus
obtained was converted to fluorophore-attached PI5ꢀP 10 by the reaction with N-NBD-amino-
hexanoic acid N-succinimidyl ester 11 in a 1:1 solution of DMF and aqueous triethylammonium
bicarbonate (TEAB) solution (Scheme 2). The structure of 10 was principally confirmed by 31P
NMR and FABMS data.10 Analogous NBD derivatives of PI4,5ꢀP211 and PI12 were reported,
and similar fluorescent probes for other inositol phospholipids are now commercially available
from Echeron Laboratories Inc.13
Scheme 2. Preparation of PI5ꢀP based biological tools. (i) NH2NH2·H2O, Py/AcOH, (4:1), 86%; (ii) H2, Pd black,
t-BuOH/H2O (6:1), 97%; (iii) TBAF·3H2O, AcOH, 77%. Abbreviations: Z=benzyloxycarbonyl, TEAB=triethylam-
monium bicarbonate
On the other hand, 9 was treated with Affigel10™ involving succinimide active esters
(Bio-Rad Laboratories) in a solution similar to that used above for the preparation of 10. The
reaction took place smoothly, and it was concluded the reactive sites of the resin were
quantitatively converted to amides, based on the quantity of recovered 9, giving 12,
quantitatively.

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