Synthesis and biological evaluation of a PtdIns(3,4,5)P3 affinity matrix

Article abstract of DOI:10.1039/b101320n

New PtdIns(3,4,5)P₃ binding proteins have been identified utilising PtdIns(3,4,5)P₃ modified affinity matrix 1 which was synthesised from myo-inositol derivative 2, phosphoramidite 9 and an agarose based solid support.

Full text of DOI:10.1039/b101320n

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Synthesis and biological evaluation of a PtdIns(3,4,5)P3 affinity matrix
Gavin F. Painter,a,b Jan. W. Thuring,a,b Ze-Yi Lim,a Andrew B. Holmes,*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. E-mail: abh1@cam.ac.uk; Fax: +44 1223 336362; Tel: +44 1223 336404
b Melville Laboratory, Department of Chemistry, University of Cambridge, Pembroke Street, Cambridge, UK
CB2 3RA. E-mail: abhl@cam.ac.uk; Fax: +44 1223 334866; Tel: +44 1223 334370
c
Babraham Institute, Department of Signalling, Inositide Laboratory, Cambridge, UK CB2 4AT
Received (in Cambridge, UK) 9th February 2001, Accepted 27th February 2001
First published as an Advance Article on the web 20th March 2001
New PtdIns(3,4,5)P3 binding proteins have been identified
utilising PtdIns(3,4,5)P3 modified affinity matrix 1 which
was synthesised from myo-inositol derivative 2, phosphor-
amidite 9 and an agarose based solid support.
to afford the perbenzylated compound 10 (Scheme 3). Re-
ductive debenzylation was readily effected using H2 (50 psi) in
the presence of Pd-black and NaHCO3 in tBuOH–H2O (6+1) as
the solvent, to afford the amine 11 in good yield.† This was then
coupled with the N-hydroxysuccinimide (NHS) activated ester
resin, Affi-Gel 10,‡ to afford the PtdIns(3,4,5)P3 modified
matrix 1. Excess resin (ca. 5 equiv.) was required to ensure the
complete consumption of the amine which was determined by a
negative Kaiser test.§
The role of myo-inositol phospholipids in cell signalling
systems is well established.1–4 One such signal transduction
mechanism involves the in vivo production of PtdIns(3,4,5)P3
via phosphorylation of PtdIns(4,5)P2 mediated by PI3K.5
Although PtdIns(3,4,5)P3 binding proteins are known,6 many of
the cellular processes downstream of PI3K activation do not yet
have a defined lipid binding protein mapped above them. For
this reason we embarked on the preparation and evaluation of an
affinity matrix based on PtdIns(3,4,5)P3.
The phospholipid was attached to an agarose matrix by an
amide linkage formed between a carboxylic acid-terminated
side chain on the agarose and a 3-(w-aminoacyl)glycerol
derivative on the phospholipid (Scheme 1). The phospholipid
11 was prepared by coupling the alcohols 27 and 3 (from the
commercially available (S)-(+)-1,2-O-isopropylideneglycerol
4) through a phosphodiester linkage.
Pilot experiments showed that PKB (25 mM) [a known6,10
PtdIns(3,4,5)P3 binding protein] would bind to the matrix 1 and
could be completely displaced by 10 mM
D
, -PtdIns(3,4,5)P3¶
D
but not at all by 10 mM
L,L
-PtdIns(3,4,5)P3, thus establishing the
potential specificity of the matrix. When applied to a pig
neutrophil cytosol a number of proteins have been identified
that bind to the resin 1 in a PtdIns(3,4,5)P3 sensitive manner.
We initially protected the primary alcohol 4 as the tert-
butyldiphenylsilyl ether, but encountered difficulties in its
removal at a later stage of the synthesis. A more efficient
process involved 4-methoxybenzylation of the primary alcohol
4 (Scheme 2),8 followed by acetonide removal to give the PMB-
ether 5 (PMB = p-methoxybenzyl). Selective esterification of
the primary alcohol in 5 with the Cbz-protected (Cbz =
benzyloxycarbonyl) w-amino acid 6, followed by palmitoyla-
tion of the secondary alcohol 7 gave the diester 8. Oxidative
removal (CAN) of the PMB protecting group and phosphityla-
tion of 3 with BnOP(NiPr2)29 gave the phosphoramidite 9.†
The lipid side chain, in the form of the phosphoramidite 9,
was then coupled with the enantiomerically pure alcohol (2)-2
Scheme 2 Reagents and conditions: i, NaH, p-MeOC6H4CH2Cl (PMBCl),
DMF; ii, PTSA, MeOH; iii, HOOCC11H22NHCBz 6, DCC, DMAP,
CH2Cl2; iv, palmitoyl chloride, DMAP, pyridine, CH2Cl2; v, CAN, MeCN–
H2O (4+1); vi, BnOP(NiPr2)2, 1H-tetrazole, CH2Cl2.
Scheme 3 Reagents and conditions: i, 1H-tetrazole, 9, CH2Cl2, then
MCPBA; ii, Pd-black, H2 (50 psi), tBuOH–H2O (6+1), NaHCO3; iii, Affi-
Gel 10, NaHCO3, H2O.
Scheme 1
DOI: 10.1039/b101320n
Chem. Commun., 2001, 645–646
This journal is © The Royal Society of Chemistry 2001
645

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H, m); dP (101.25 MHz, D2O), 5.81, 4.79, 3.55, 0.80; m/z (2ve FAB) 1142
[(M 2 Na)2, 25%], 1119 (50), 1098 (100), 1076 (90).
Several novel proteins were identified and the full biological
results will be disclosed in a separate publication. One of these
proteins was subsequently shown to be identical to the recently
characterised protein, DAPP1, possessing a Src homology
(SH2) domain and a pleckstrin homology (PH) domain. This
novel protein had been independently identified from a database
search by comparison of the PH domain sequences with known
PtdIns(3,4,5)P3 binding proteins.11 The fact that the ‘functional
screen assay’ identified several proteins including DAPP1,
which is involved in endosomal trafficking or sorting,12 is
noteworthy and exemplifies the strength of the approach. Very
recently biotinylated PtdIns(3,4,5)P3 has been used as an
affinity ligand for the purification of recombinant
PtdIns(3,4,5)P3 binding proteins.13
‡ Affi-Gel 10 was purchased from BioRad.
§ The matrix 1 was constructed by reacting 60 mmol of N-hydroxy-
succinimide activated resin (4 mL) with 12.2 mmol of the amine 11 in the
presence of 122 mmol NaHCO3 at 0 °C overnight.
¶ D,D-PtdIns(3,4,5)P3 refers to the dipalmitoyl analogue of PtdIns(3,4,5)P3
containing the 1( )-myo-inositol ring stereochemistry and sn-2-diacylgly-
D
cerol side chain; L,L-PtdIns(3,4,5)P3 refers to the enantiomer.
1 C. L. Carpenter and L. C. Cantley, Curr. Opin. Cell Biol., 1996, 8,
153.
2 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.
In summary we have demonstrated a synthesis of a
PtdIns(3,4,5)P3-modified matrix and demonstrated its use as a
tool for the identification of proteins binding to
PtdIns(3,4,5)P3.14 The flexible nature of the methodology and
the biological success of resin 1 warrants further investigation
into the preparation and biological evaluation of other D-3
phosphorylated myo-inositol phospholipid modified matri-
ces.15
We thank the BBSRC, the Cambridge Commonwealth Trust,
the CVCP (ORS to Z.-Y. L), Astra-Zeneca and Tan Kar Kee
Foundation, Singapore (Z.-Y. L.) for financial support, and the
EPSRC for provision of the Swansea Mass Spectrometry
Service. We thank Dr Corinne Kaye for helpful advice on the
use of agarose supports.
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Notes and references
† All new compounds exhibited spectroscopic and analytical data in accord
with the assigned structure. Selected data (J values in Hz) for 9: [a]2D2 + 7.0
(c 1.9 in CHCl3); dH (250 MHz, CDCl3), 7.38–7.28 (10 H, m, Ph), 5.20–5.10
(1 H, m), 5.10 (2 H, br s, OCH2Ph), 4.80–4.60 (3 H, m), 4.36 (1 H, m), 4.12
(1 H, m), 3.85–3.55 (4H, m), 3.18 [2 H, dt, (apparent quartet), J 6.7, 6.7,
CH2CH2NH], 2.29 (4 H, two overlapping t, J 7.3), 1.64–1.40 (6 H, m),
1.30–1.20 (38 H, m), 1.18 (6 H, d, J 6.8, 2 3 Me), 1.17 (6 H, d, J 6.8, 2 3
Me), 0.87 (3 H, t, J 6.9, Me); dP (101.25 MHz, CDCl3), 149.2, 149.1; m/z
(FIB) [Found: (M + Na)+ 921.6022. C52H87N2O8PNa requires 921.6098].
For 11: [a]2D2 +3.0 (c 0.1 in H2O); nmax (KBr/cm21) 3403, 2920, 2850, 1742,
1238, 1094; dH (250 MHz, D2O), 5.25 (1 H, br s), 4.45–3.80 (10 H, m),
2.95–2.85 (2 H, m), 2.40–2.25 (4 H, m), 1.65–1.05 (44 H, m), 0.85–0.70 (3
15 Z.-Y. Lim, J. W. Thuring, A. B. Holmes, M. Manifava and N. T.
Ktistakis, manuscript submitted for publication.
646
Chem. Commun., 2001, 645–646