Synthesis of dipalmitoyl phosphatidylinositol 3,4-bis(phosphate) and 3,4,5-tris(phosphate) and their enantiomers

Article abstract of DOI:10.1039/a703045b

The dipalmitoyl derivatives 4 and 5 of 3-phosphorylated myo-inositol phospholipids 2 and 3 and their enantiomers are synthesised from homochiral myo-inositol precursors 6 and 11; they serve as biological probes for cell signal transduction.

Full text of DOI:10.1039/a703045b

View Article Online / Journal Homepage / Table of Contents for this issue
Synthesis of dipalmitoyl phosphatidylinositol 3,4-bis(phosphate) and
3,4,5-tris(phosphate) and their enantiomers
Simon J. A. Grove,a Andrew B. Holmes,*a,b Gavin F. Painter,a,b Phillip T. Hawkinsc and Leonard R. Stephensc
a Cambridge Centre for Molecular Recognition, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge,
UK CB2 1EW
b Melville Laboratory for Polymer Synthesis, Department of Chemistry, University of Cambridge, Pembroke Street, Cambridge,
UK CB2 3RA
c Babraham Institute, Department of Signalling, Inositide Laboratory, Cambridge, UK CB2 4AT
The dipalmitoyl derivatives 4 and 5 of 3-phosphorylated
myo-inositol phospholipids 2 and 3 and their enantiomers
are synthesised from homochiral myo-inositol precursors 6
and 11; they serve as biological probes for cell signal
transduction.
(Scheme 2). Similarly (2)-5† was synthesised from (+)-11 and
(2)-9 by the route used in Scheme 2.
An alternative synthesis of dipalmitoyl PtdIns(3,4)P2 (+)-4
was also carried out from the 3,4-protected alcohol (2)-12
(Scheme 3).6 The alcohol was coupled directly with the
The group of myo-inositol phospholipids PtdIns(3)P 1,
PtdIns(3,4)P2 2 and PtdIns(3,4,5)P3 3 are believed to have a role
in the mechanisms by which cell surface receptors for antigens,
BnO
HO
BnO
OPMB
OPMB
i
OBn
OBn
OBn
OBn
(BnO)2(O)PO
(–)-7
OH
(–)-6
OP(O)(OBn)2
OCOR2
OCOR1
O
O
P
ii
HO
O
OH
BnO
OH
OH
R3O
OR5
OBn
OBn
OR4
(BnO)2(O)PO
OP(O)(OBn)2
1 R1 = C19H31, R2 = C17H35, R3 = P(O)(OH)2, R4 = R5 = H
2 R1 = C19H31, R2 = C17H35, R3 = R4 = P(O)(OH)2, R5 = H
3 R1 = C19H31, R2 = C17H35, R3 = R4 = R5 = P(O)(OH)2
4 R1 = R2 = C15H31, R3 = R4 = P(O)(OH)2, R5 = H
5 R1 = R2 = C15H31, R3 = R4 = R5 = P(O)(OH)2
(–)-8
OCOC15H31
OCOC15H31
iii
O
O
P
BnO
O
OBn
OBn
OBn
(BnO)2(O)PO
OP(O)(OBn)2
OCOC15H31
OCOC15H31
(+)-10
BnO
O
inflammatory stimuli and growth factors control a variety of
cellular functions.1–3 These lipids appear to be resistant to
hydrolysis by phosphatidyl inositol-specific phospholipase C
(PI-PLC),4 implying an independent cell signalling pathway
from that already established for PtdIns(4,5)P2.5 In order to
probe the function of the phospholipids 2 and 3 in signal
transduction we proposed to synthesise the functionally similar
dipalmitoyl derivatives 4 and 5 and their enantiomers for use in
biological assays. The synthetic work presented here utilises
differentially protected homochiral myo-inositol derivatives 6
and 11 which were obtained efficiently from myo-inositol
orthoformate.6
Dipalmitoyl PtdIns(3,4)P2 4 was prepared from (2)-6 in four
synthetic steps with an overall yield of 72% (Scheme 1). The
3,4-bisphosphorylated derivative (2)-7 was prepared via phos-
phitylation of (2)-6 with bis(benzyloxy)(N,N-diisopropy-
lamino)phosphine7 and 1H-tetrazole followed by in situ oxida-
tion with MCPBA. After removal of the p-methoxybenzyl ether
with ceric ammonium nitrate the 1-hydroxy group was coupled
with the phosphoramidite (+)-9.8,9 In situ oxidation of the
phosphorus(iii) species afforded the fully protected phospho-
lipid (+)-10. Reductive debenzylation was readily effected
using the conditions reported10 by Kozikowski et al., furnishing
dipalmitoyl PtdIns(3,4)P2 (+)-4.†
P
iv
NPri2
(+)-4
(+)-9
Scheme 1 Reagents and conditions: (BnO)2PNPri2, 1H-tetrazole, CH2Cl2,
then MCPBA; ii, (NH4)2Ce(NO3)6, MeCN–H2O (4:1); iii, (+)-9,
1H-tetrazole, CH2Cl2, then MCPBA; iv, Pd(OH)2–C, H2 (60 psi), ButOH
OCOC15H31
O
O
OCOC15H31
P
BnO
HO
HO
i–iv
OPMB
O
OH
OBn
OH
OH
OP(O)(OH)2
(HO)2(O)PO
OH
(–)-11
OP(O)(OH)2
(+)-5
Scheme 2 Reagents and conditions: i, (BnO)2PNPri2, 1H-tetrazole, CH2Cl2,
then MCPBA; ii, (NH4)2Ce(NO3)6, MeCN–H2O (4:1); iii, (+)-9,
1H-tetrazole, CH2Cl2, then MCPBA; iv, Pd(OH)2–C, H2 (60 psi), ButOH
BnO
OCOC15H31
OH
O
OBn
OBn
O
OCOC15H31
iii
O
P
BnO
HO
i, ii
O
OBn
O
(+)-10
OBn
OBn
Elaboration of the antipodal diol (+)-6 via the same reaction
sequence as shown in Scheme 1 and coupling with (2)-9,‡
afforded (2)-4,† the enantiomer of dipalmitoyl PtdIns(3,4)P2.
The use of similar reagents and reaction conditions as
outlined for the synthesis of (+)-4 afforded dipalmitoyl
PtdIns(3,4,5)P3 (+)-5† in 70% yield from the 3,4,5-triol (2)-11
OH
13
(–)-12
Scheme 3 Reagents and conditions: i, (+)-9, 1H-tetrazole, CH2Cl2, then
MCPBA; ii, AcCl, MeOH–CH2Cl2 (1:1); iii, Pd(OH)2–C, H2 (60 psi),
ButOH
Chem. Commun., 1997
1635

View Article Online
For (2)-4: [a]2D2 20.75 [c 0.70 in CHCl3–MeOH (1:1)].
phosphoramidite (+)-9, and the product was then cleaved at the
3,4-acetal with acid (AcCl–MeOH) to afford the 3,4-diol 13.
Phosphitylation of the free hydroxy groups with bis(benzyl-
oxy)(N,N-diisopropylamino)phosphine and 1H-tetrazole, fol-
lowed by in situ oxidation with MCPBA, afforded the common
fully protected phospholipid (+)-10. This route required two
fewer steps than the original procedure, as the p-methoxybenzyl
protection and deprotection at the d-1-hydroxy are not required.
The overall yield is slightly better than that of the original
procedure.
For (+)-5, the dipalmitoyl analogue of PtdIns(3,4,5)P3: [a]2D2 +1.85 [c
0.60 in CHCl3–MeOH (1:1)]; dH (400 MHz, [2H6]DMSO) 5.19–5.11 (1 H,
m), 4.47 (1 H, m), 4.31–3.99 (8 H, m), 3.75 (1 H, t, J 9.4), 2.33–2.22 (4 H,
m), 1.54–1.45 (4 H, m), 1.29–1.19 (48 H, m), 0.85 (6 H, m); dP (101 MHz,
[2H6]DMSO) 1.22, 0.98, 0.59, 20.20; Found (FAB): (M + H)+, 1051.4350.
C41H83O22P4 requires (M + H)+, 1051.4326].
For (2)-5: [a]2D2 21.75 [c 0.75 in CHCl3–MeOH (1:1)].
‡ Compound (2)-9 was synthesised in a similar manner to (+)-9 starting
from (R)-(2)-2,2-dimethyl-1,3-dioxolane-4-methanol (Aldrich).
In summary we have demonstrated a general synthesis of
3-phosphorylated myo-inositol phospholipids 4 and 5 from the
homochiral precursors 6 and 11 which also allows the synthesis
of enantiomeric derivatives containing the unnatural stereo-
chemistry. These materials play an important roˆle in evaluating
the activation of protein kinase B.11
1 A. Toker, M. Meyer, K. K. Reddy, J. R. Falck, R. Aneja, S. Aneja,
A. Parra, D. J. Burns, L. M. Ballas and L. C. Cantley, J. Biol. Chem.,
1994, 269, 32 358.
2 M. J. Berridge, Nature, 1993, 361, 315.
3 L. R. Stephens, T. R. Jackson and P. T. Hawkins, Biochem. Biophys.
Acta, 1993, 1179, 27.
We thank the EPSRC and BBSRC for financial support and
provision of the Swansea Mass Spectrometry Service, Glaxo-
Wellcome for a CASE studentship (to S. J. A. G.), Drs D. R.
Marshall and M. L. Hill for their interest in this work and Dr A.
P. Kozikowski for experimental details of debenzylation
procedures.
4 L. A. Serunian, M. T. Haber, T. Fukui, J. W. Kim, S. G. Rhee,
J. M. Lowenstein and L. C. Cantley, J. Biol. Chem., 1989, 264,
17 809.
5 M. J. Berridge and R. F. Irvine, Nature, 1989, 341, 197; S. G. Rhee and
K. D. Choi, J. Biol. Chem., 1992, 267, 12 393.
6 S. J. A. Grove, I. H. Gilbert, A. B. Holmes, G. F. Painter and M. L. Hill,
preceding communication.
7 K.-L. Yu and B. Fraser-Reid, Tetrahedron Lett., 1988, 29, 979.
8 J. C. Snowden and H. O. L. Fischer, J. Am. Chem. Soc., 1941, 63,
3244.
9 C. E. Dreef, C. J. J. Elie, P. Hoogerhout, G. A. van der Marel and
J. H. van Boom, Tetrahedron Lett., 1988, 29, 6513.
10 A. P. Kozikowski, G. Powis and W. Tu¨ckmantel, Angew. Chem., Int.
Ed. Engl., 1992, 31, 1379.
11 D. Stokoe, L. R. Stephens, T. Copeland, P. Gaffney, C. B. Reese,
G. F. Painter, A. B. Holmes, F. McCormick and P. T. Hawkins, Science,
1997, in the press.
Footnotes and References
E-mail: abh1@cus.cam.ac.uk
† All new compounds exhibited spectroscopic and analytical data in accord
with the assigned structure (J values in Hz). Selected data for (+)-4, the
dipalmitoyl analogue of PtdIns(3,4)P2: [a]2D2 +0.65 [c 1.1 in CHCl3–MeOH
(1:1)]; dH (400 MHz, [2H6]DMSO) 5.18–5.11 (1 H, m), 4.35–4.28 (2 H, m),
4.14–4.02 (5 H, m), 3.90 (1 H, br t, J 9.1), 3.60 (1 H, t, J 9.3), 3.25 (1 H t,
J 9.0), 2.33–2.24 (4 H, m), 1.55–1.44 (4 H, m), 1.28–1.19 (48 H, m), 0.85
(6 H, br t, J 6.7); dP (101 MHz, [2H6]DMSO) 1.64, 1.00, 20.16; Found
(FAB MS): (M + H + H)+, 972.4794. C41H83O19P3 requires (M + H + H)+,
972.4741.
Received in in Glasgow, UK, 6th May 1997; 7/03045B
1636
Chem. Commun., 1997