Synthesis and biological evaluation of phosphatidylinositol phosphate affinity probes

Article abstract of DOI:10.1039/b913399b

The synthesis of the complete family of phosphatidylinositol phosphate analogues (PIPs) from five key core intermediates A-E is described. These core compounds were obtained from myo-inositol orthoformate 1 via regioselective DIBAL-H and trimethylaluminium-mediated cleavages and a resolution-protection process using camphor acetals 10. Coupling of cores A-E with phosphoramidites 34 and 38, derived from the requisite protected lipid side chains, afforded the fully-protected PIPs. Removal of the remaining protecting groups was achieved via hydrogenolysis using palladium black or palladium hydroxide on carbon in the presence of sodium bicarbonate to afford the complete family of dipalmitoyl- and amino-PIP analogues 42, 45, 50, 51, 58, 59, 67, 68, 76, 77, 82, 83, 92, 93, 99 and 100. Investigations using affinity probes incorporating these compounds have identified novel proteins involved in the PI3K intracellular signalling network and have allowed a comprehensive proteomic analysis of phosphoinositide interacting proteins. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b913399b

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Organic &
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Chemistry
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Volume 8 | Number 1 | 7 January 2010 | Pages 1–284
ISSN 1477-0520
FULL PAPER
PERSPECTIVE
Andrew B. Holmes et al.
Margaret A. Brimble et al.
Synthesis of natural products
containing spiroketals via
Synthesis and biological evaluation
of phosphatidylinositol phosphate
affinity probes
intramolecular hydrogen abstraction
1477-0520(2010)8:1;1-J

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of phosphatidylinositol phosphate
affinity probes†
Stuart J. Conway,a James Gardiner,*b Simon J. A. Grove,a Melloney K. Johns,a Ze-Yi Lim,a Gavin F. Painter,a
Diane E. J. E. Robinson,*b Christine Schieber,b Jan W. Thuring,a Leon S.-M. Wong,b Meng-Xin Yin,b
Antony W. Burgess,c Bruno Catimel,c Phillip T. Hawkins,d Nicholas T. Ktistakis,d Leonard R. Stephensd and
Andrew B. Holmes*b
Received 6th July 2009, Accepted 29th September 2009
First published as an Advance Article on the web 9th November 2009
DOI: 10.1039/b913399b
The synthesis of the complete family of phosphatidylinositol phosphate analogues (PIPs) from five key
core intermediates A–E is described. These core compounds were obtained from myo-inositol
orthoformate 1 via regioselective DIBAL-H and trimethylaluminium-mediated cleavages and a
resolution–protection process using camphor acetals 10. Coupling of cores A–E with phosphoramidites
34 and 38, derived from the requisite protected lipid side chains, afforded the fully-protected PIPs.
Removal of the remaining protecting groups was achieved via hydrogenolysis using palladium black or
palladium hydroxide on carbon in the presence of sodium bicarbonate to afford the complete family of
dipalmitoyl- and amino-PIP analogues 42, 45, 50, 51, 58, 59, 67, 68, 76, 77, 82, 83, 92, 93, 99 and 100.
Investigations using affinity probes incorporating these compounds have identified novel proteins
involved in the PI3K intracellular signalling network and have allowed a comprehensive proteomic
analysis of phosphoinositide interacting proteins.
Introduction
Phosphatidylinositol phosphates (PtdInsPs or PIPs) are an im-
portant class of membrane phospholipids.1,2 These lipids are
involved in intracellular signalling mechanisms that are known
to play a central role in fundamental cellular functions such as
vesicle trafficking, apoptosis, cell proliferation and metabolism.3
Furthermore, lack of regulation of these pathways is known to
result in a range of disease conditions, such as cancer, diabetes,
Alzheimer’s disease and autoimmune disorders.4
PIPs are phosphorylated derivatives of myo-inositol. There are
eight known naturally occurring classes of PIPs differing only
in the degree of phosphorylation of the hydroxyl groups at the
3-, 4- and 5-positions of the inositol rings (Fig. 1). The natural
materials carry a range of unsaturated fatty acid derivatives at the
sn-1 and sn-2 positions. The nature and degree of saturation of
the lipid chain is organism dependent. In this work we focus on
saturated analogues and largely on palmitoyl derivatives, which,
in our experience, have shown no marked influence on the binding
properties of proteins to the synthetic PIPs. To understand the
roles of each of these PIPs in their signalling pathways and their
effect on downstream processes, it is necessary to identify proteins
Fig. 1 General structure of phosphatidylinositol phosphates.
that exhibit binding specificity. However, the inability to isolate
practically useful amounts of these PIP compounds from cells has
led to a need for efficient syntheses.5-25 Furthermore, analogues of
these compounds that can be used as affinity probes to investigate
and isolate particular binding proteins, have become attractive
targets1,5,7,12,13,24,26–28 Our approach has been the incorporation of
an w-amino group on the sn-1 position of a saturated lipid side
chain (abbreviated as NH2-PIP) to facilitate immobilisation of the
R
PIP by covalent linkage through an amide bond onto Affi-Gelꢀ
10 beads.12,28
Herein, we describe our contribution over the last two decades
to the synthesis of the complete family of PIP analogues, which
we have used as probes for biological systems.28-35 We have
previously reported the synthesis of PI(3,4,5)P3 and PI(4,5)P2
affinity beads.12,16
aDepartment of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, CB2 1EW, UK
bSchool of Chemistry, Bio21 Institute, University of Melbourne, Building
102, 30 Flemington Road, Parkville, Victoria 3010, Australia
cLudwig Institute for Cancer Research, Melbourne Tumour Biology Branch,
Royal Melbourne Hospital, Parkville, Victoria 3052, Australia
dBabraham Institute, Department of Signalling, Inositide Laboratory
Cambridge, CB2 4AT, UK
Synthetic approach
† Electronic supplementary information (ESI) available: Experimental
procedures for 27–29, 39–45, 49, 51–69, 71, 75, 77, 81, 83–85, 90, 92,
102, 104–110. See DOI: 10.1039/b913399b
Our synthetic approach is simple and divergent and involves
the late-stage coupling of phosphoramidites, derived from the
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Scheme 1 Core building blocks for the synthesis of all the PIPs.
requisite protected lipid side chains, to the suitably protected
phosphorylated inositol cores followed by a global deprotection.
All eight PIPs and their analogues can be synthesised from
five inositol core compounds (A–E, Scheme 1).36 Each of these
intermediates can in turn be synthesised from the readily available
myo-inositol orthoformate 137 by judicial choice of protection,
resolution and deprotection strategies. It should be noted that
cores D and E can also be obtained from core C by suitable
protecting group manipulation; furthermore, in principle all the
PIPs can be obtained from core C, though this is not the most
efficient route. This report focuses primarily on the most efficient
routes to the target compounds.
of the inositol ring of 8 as the p-methoxybenzyl ether, followed by
deprotection of either the 3,4 and/or 5-positions afforded cores
A, C and E.
The synthesis of cores B and D begins with the complete
benzylation of myo-inositol orthoformate 1 to form benzyl ether
11. The differentially protected inositol acetals 12 and 17 can be
obtained via selective Lewis acid-mediated reductive or alkylative
cleavage of trisbenzyl orthoformate 11.39,42 As seen in the syntheses
of cores A, C and E, the use of DIBAL-H selectively afforded the
1,3-acetal, 12. In contrast, the use of trimethyl aluminium resulted
in the 3,5-acetal, 17. Protection of the free hydroxyl groups in 12
and 17 followed by removal of the acetal groups gave diols 14 and
19 respectively. Subsequent mono-protection of the 1-position of
14 gave racemic p-methoxybenzyl ether 15. Racemic alcohols 15
and 19 were then resolved as their camphanate esters. Subsequent
hydrolysis of esters 16 and 20 afforded enantiomerically pure cores
B and D respectively.
The stereoselectivity of these Lewis acid-mediated reductive and
alkylative cleavages of orthoformate 11 have been investigated and
the mechanisms proposed (Scheme 3).42 In the DIBAL-H reduc-
tion of 2 to 3, and 11 to 12, it was determined that at least 2 molar
equivalents of DIBAL-H are required for the reaction to proceed
to completion. It is proposed that the first equivalent of DIBAL-H
acts as a Lewis acid, coordinating to the C-5 oxygen, presumably
the most sterically accessible oxygen. Subsequent cleavage of the
orthoformate affords the oxocarbenium ion 22, the unfavourable
1,3-steric interactions in which can be accommodated by a ring
flip to the boat conformation 23. Reduction of this oxocarbenium
ion by a second equivalent of DIBAL-H from the less hindered
face produces the 1,3-acetal 12 exclusively.
The stereoselectivity of the hydride delivery to the orthoformate
was established through two deuterium-labelling experiments
(Scheme 4). Firstly, reduction of orthoformate 11 using 2.5 mo-
lar equivalents of diisobutylaluminium deuteride43 (DIBAL-D)
resulted in acetals 28 and 29 in a 92 : 8 ratio. The reciprocal
reaction in which deuterated orthoformate 2744 was treated
with diisobutylaluminium hydride (DIBAL-H), afforded the two
products with an inverted selectivity of 10 : 90 (28 : 29). The
structures were confirmed by nOe experiments. This switch in
selectivity between the two diastereomers 28 and 29, indicates
that the reductive cleavage is stereoselective in the delivery of the
Synthesis of cores A–E
The syntheses of core molecules A–E are shown in Scheme 2
and have previously been reported.12,16,38,39 To gain access to cores
A, C and E, selective mono PMB-protection of 1 followed by
benzylation of the remaining hydroxyl groups provides ortho-
formate 2. Selective cleavage of the orthoformate by treatment
with DIBAL-H affords 1,3-acetal 3. Benzylation or allylation and
subsequent removal of the acetal and PMB protecting groups
give the racemic triols 6 and 7. Resolution of the racemic triols 6
and 7 with simultaneous protection of the corresponding vicinal
diols according to an elegant procedure previously reported
by Bruzik40,41 afforded an efficient approach to the unnatural
(1L-series) and natural (1D-series) of PIPs. Thus, treatment of
triol 7 with the camphor acetal (1R)-10 afforded a mixture of four
diastereomers of 3,4-acetal 8, one of which could be separated
by column chromatography from the mixture of the other three;
deprotection of the acetal allows preparation of the unnatural
enantiomer of the triol 7 corresponding to the 1L-series (for the use
of this enantiomer see Scheme 12). The remaining mixture is there-
fore enriched in the desired enantiomer, which when protected with
dimethylacetal (1S)-10 provides diastereomer 8 with the correct
absolute configuration corresponding to the natural 1D-series. It
should be noted that direct resolution/protection of 6 or 7 with
dimethylacetal (1S)-10 also provides the required diastereomer of
8 in comparable yields. This route also allows separation of the
two enantiomers of the inositol ring and provides access to the
unnatural enantiomers of the PIPs.21 Protection of the 1-position
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Scheme 2 Synthesis of the core building blocks A–E. Reagents and conditions: i. NaH, PMBCl, DMF, 0 ◦C → rt; ii. NaH, BnBr, DMF, 0 ◦C → rt;
iii. DIBAL-H (2.5 eq.), CH2Cl2/hexanes, 0 ◦C → rt; iv. NaH, allyl bromide, DMF, 0 ◦C → rt; v. HCl, MeOH, reflux; vi. (1R)-10, TsOH·H2O (cat.),
CH2Cl2, reflux, separate, AcCl, CH2Cl2–MeOH (2/1 v/v), then (1S)-10, TsOH·H2O (cat.), CH2Cl2, reflux; vii. AcCl, CH2Cl2–MeOH (2/1 v/v), viii. From
9b: (Ph3P)3RhCl, DABCO, EtOH–toluene/H2O (7/3/1 v/v/v), reflux, then AcCl, CH2Cl2–MeOH (2/1 v/v); ix. (1S)-(-)-camphanic chloride, Et3N or
pyridine, CH2Cl2, rt; x. LiOH, THF–H2O (10/1 v/v), rt; xi. Me3Al (2.5 eq.), CH2Cl2/hexanes, 0 ◦C → rt; xii. HCl, CH2Cl2–MeOH (2/1 v/v), rt.
Scheme 3 Proposed mechanisms of the Lewis acid-mediated regioselective cleavage of orthoformate 1142
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Scheme 5 Synthesis of dipalmitoyl phosphoramidite 34. Reagents and
conditions: i. NaH, PMBCl, DMF, 0 ◦C → rt, quant.; ii. p-TsOH, MeOH,
reflux, 82%; iii. C15H31COCl, pyr., DMAP, CH2Cl2, 0 ◦C → rt, 88%; iv.
DDQ, CH2Cl2–H2O (16/1, v/v), rt, 85%; v. (BnO)P(NiPr2)2, 1H-tetrazole,
CH2Cl2, rt, 92%.
Scheme 4 Deuterium labelling experiments for the cleavage of the
orthoformates 11 and 27. Reagents and conditions: i. From 11: DIBAL-D
(2.5 eq.), CH2Cl2, 0 ◦C → rt, then Ac2O, Et3N, DMAP, CH2Cl2, rt, 83%,
28 : 29: 92 : 8; ii. From 27: DIBAL-H (2.5 eq.), CH2Cl2, 0 ◦C → rt, then
Ac2O, Et3N, DMAP, CH2Cl2, rt, 93%, 28 : 29: 10 : 90.
hydride (or deuteride) occurring preferentially on the endo face,
syn to the broken C–O bond. This retention of stereochemistry
discounts a mechanism pathway in which the orthoformate in 21
(Scheme 3) is cleaved via inter- or intramolecular delivery of the
hydride. Whilst these experiments do not allow the distinction
between intramolecular delivery of the hydride (or deuteride) in
22 and intermolecular delivery in 23, the latter is a more feasible
pathway when the unfavourable 1,3-steric interactions of 22 are
considered.
In contrast, the trimethylaluminium-mediated cleavage of the
orthoformate 11 exclusively affords the acetal 17, which results
from the cleavage of one of two identical alternative C–O bonds
in the orthoformate 24. This outcome indicates that trimethylalu-
minium is presumably sufficiently small to coordinate to one or
both of the equivalent oxygens at the 1- or 3-positions (perhaps
through intramolecular delivery by the equatorial benzyloxy
group), allowing an alternative cleavage pathway resulting in the
oxocarbenium ion 25. Delivery of a methyl group to the exo face
of the oxocarbenium 26 affords the 3,5-acetal 17.
Scheme 6 Synthesis of amino phosphoramidite 38. Reagents and condi-
tions: i. CbzNHC11H22CO2H, DCC, DMAP, CH2Cl2, 0 ◦C → rt, 87%;
ii. C15H31COCl, pyr., DMAP, CH2Cl2, 0 ◦C → rt, quant.; iii. DDQ,
CH2Cl2–H2O (16/1, v/v), rt, 80%; iv. (BnO)P(NiPr2)2, 1H-tetrazole,
CH2Cl2, rt, 92%.
diisopropylamino) phosphine gave phosphoramidite 38, providing
access to the NH2-PIP analogues.
With all the necessary building blocks in hand, the whole family
of the PIP analogues could be synthesised.
Synthesis of PI,7,8,10 PI(3)P,7,9,10,14–1619,23 PI(4)P10,19 and
PI(5)P5,10,14,18 analogues
Synthesis of phosphoramidites
The simplest member of this family of compounds is phos-
phatidylinositol (PI), which contains the phospholipid side chain
at the 1-position and none of the 3-, 4- or 5-positions are
phosphorylated (Fig. 1).
To install the lipid side chains, the phosphoramidites 3445 and
3812 were synthesised from (+)-1,2-O-isopropylidene-glycerol 30
(Scheme 5 and Scheme 6). The dipalmitoyl derivative 32 was
obtained by esterification of the diol 31, which resulted from PMB
protection of the free alcohol in 30 followed by methanolysis of
the acetal (Scheme 5). Subsequent removal of the PMB group
followed by a 1H-tetrazole mediated coupling of the alcohol 33
with (benzyloxy)bis(N,N-diisopropylamino)phosphine46 afforded
the phosphoramidite 34, which is used for the synthesis of the
dipalmitoyl PIP analogues.
In order to prepare the PIP derivatives that can be attached
to beads for use in protein affinity experiments, side chains
containing an w-amino group at the sn-1-position were also
synthesised (Scheme 6). A DCC-mediated coupling of diol 31
allowed selective esterification of the primary alcohol to afford the
alcohol 35 in good yield. Esterification of the free alcohol with
palmitoyl chloride and removal of the PMB group provided the
primary alcohol 37 which when coupled with (benzyloxy)bis(N,N-
The inositol moiety of PI (40) was obtained from core A
by benzylation of the 3- and 4-hydroxyl groups and removal
of the PMB protecting group from the 1-position (Scheme 7).
Coupling of the alcohol 40 with the amino-phosphoramidite
38 in the presence of an excess of 1H-tetrazole, followed by
mCPBA oxidation of the intermediate phosphite (not isolated)
provides the fully protected phosphate 41. A global deprotection
via hydrogenolysis in the presence of palladium black and sodium
hydrogen carbonate16,24 gave NH2-PI sodium salt 42,47 which was
R
coupled to NHS-activated Affi-Gelꢀ 10 beads in the presence of
sodium hydrogen carbonate to give affinity probe 43.48
To demonstrate the versatility of our synthetic strategy the enan-
tiomer of core B was treated with dipalmitoyl phosphoramidite 34
and 1H-tetrazole followed by oxidation with mCPBA to give the
fully protected dipalmitoyl-PI 44 (Scheme 8). Global deprotection
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Scheme 7 Synthesis of immobilised NH2-PI 43. Reagents and conditions:
i. NaH (3 eq.), BnBr, (3 eq.), TBAI, DMF, 49%; ii. CAN, MeCN–H2O
(4/1, v/v), 85%; iii. phosphoramidite 38, 1H-tetrazole, CH2Cl2, rt, then
mCPBA, -78 ◦C → rt, 73%; iv. H2 (4.1 bar), Pd black, NaHCO3,
tBuOH/H2O (6/1), 76%; v. Affi-GelꢀR 10, NaHCO3, CHCl3–MeOH/H2O
(4/5/1, v/v/v), 0 ◦C → rt, 9% loading.
Scheme
9
Synthesis of dipalmitoyl-PI(3)P 50 and immobilised
NH2-PI(3)P 52. Reagents and conditions: i. (BnO)2P(NiPr2), 1H-tetrazole,
CH2Cl2, rt, then mCPBA, -78 ◦C → rt, 87%; ii. CAN, MeCN–H2O
(4/1, v/v), 83%; iii. phosphoramidite 34, 1H-tetrazole, CH2Cl2, rt, then
mCPBA, -78 ◦C → rt, 48: 90%; iv. phosphoramidite 38, 1H-tetrazole,
CH2Cl2, rt, then mCPBA, -78 ◦C → rt, 49: 84%; v. H2 (4.1 or 3.8 bar),
Pd black, BuOH, 50: 89% from 48; 51: 62% from 49; vi. Affi-GelꢀR 10,
t
NaHCO3, CHCl3–MeOH/H2O (4/5/1, v/v/v), 0 ◦C → rt, 2% loading.
R
52 was obtained by coupling of 51 with NHS-activated Affi-Gelꢀ
10 beads.
The analogues of PI(4)P were obtained from core A (Scheme 10).
This synthesis required the selective protection of the hydroxyl
group at the 3-position of the inositol ring, which was achieved
using conditions developed by Gigg et al.50 In situ generation of a
stannane acetal in the presence of tetrabutylammonium bromide
and benzyl bromide gave rise to the 3-benzylated derivative 53
and the corresponding 4-benzylated regioisomer in a 4 : 1 ratio (as
judged by 1H NMR analysis).
Subsequent phosphorylation of the remaining hydroxyl group
at the 4-position of 53, followed by removal of the PMB group,
coupling to the requisite phosphoramidite, oxidation, and global
deprotection smoothly provided both the dipalmitoyl analogue 58
and the amino analogue 59 of PI(4)P. The immobilised PI(4)P
affinity probe 60 was synthesised in the standard manner.
The synthesis of PI(5)P analogues 67 and 68 from core C first
required protection of the 3- and 4-hydroxyl groups (Scheme 11).
The allyl group at the 5-position was then removed to allow instal-
lation of the 5-phosphate. This deprotection was achieved through
isomerisation of the allyl ether 61 with Wilkinson’s catalyst
followed by acid catalysed methanolysis of the resulting enol ether.
The standard phosphorylation, PMB removal, phosphoramidite
coupling with in situ oxidation and global deprotection methods
were then employed to obtain both analogues of PI(5)P. Amino-
Scheme 8 Synthesis of dipalmitoyl-PI analogue 45. Reagents and con-
ditions: i. phosphoramidite 34, 1H-tetrazole, CH2Cl2, rt, then m-CPBA,
-78 C → rt, 55% (ii) Pd black, NaHCO3, BuOH/H2O (6 : 1), H2 (3.1
bar), rt, 48 h, >99%.
◦
t
via hydrogenolysis provides the dipalmitoyl-PI sodium salt 45 in a
good yield.
The next group of phosphatidylinositols to consider is that
where, in addition to the phospholipid side chain at the 1-position,
one of either the 3-, 4- or 5-positions is phosphorylated.
For example, PI(3)P is obtained by first phosphorylating core B
with bis(benzyloxy)(N,N-diisopropylamino)phosphine49 followed
by in situ oxidation with mCPBA, to install the phosphate group
at the 3-position (Scheme 9). Removal of the PMB group in 46 and
subsequent 1H-tetrazole-mediated coupling with the appropriate
phosphoramidite afforded the fully protected dipalmitoyl-PI(3)P
4816 and NH2-PI(3)P 49. Standard global deprotection conditions
afforded both analogues of PI(3)P (50 and 51). The affinity probe
R
PI(5)P 68 was then immobilised onto Affi-Gelꢀ 10 beads in the
same manner as above.
Intermediate 61 in the synthesis of the PI(5)P analogues, can
also be accessed from the unnatural L-enantiomer of triol 7
through facile protecting group manipulation (Scheme 12). This
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Scheme 11 Synthesis of dipalmitoyl-PI(5)P 67 and immobilised
NH2-PI(5)P 69. Reagents and conditions: i. NaH, BnBr, DMF, rt, 94%;
ii. RhCl(PPh3)3, DIPEA, EtOH–toluene/H2O (7 : 3 : 1 v : v : v) then AcCl,
MeOH–CH2Cl2, 83%; iii. (BnO)2P(NiPr2), 1H-tetrazole, CH2Cl2, rt, then
mCPBA, -78 ◦C → rt, 91%; iv. CAN, MeCN–H2O (4/1, v/v), 79%; v.
phosphoramidite 34, 1H-tetrazole, CH2Cl2, rt, then m-CPBA, -78 ◦C
→ rt, 65: 62%; vi. phosphoramidite 38, 1H-tetrazole, CH2Cl2, rt, then
mCPBA, -78 ◦C → rt, 66: 64%; vii. H2 (3.5 or 35 bar), Pd black, NaHCO3,
tBuOH/H2O (6/1), 67: 86% from 65; 68: 83% from 66; viii. Affi-GelꢀR 10,
NaHCO3, CHCl3–MeOH/H2O (4/5/1, v/v/v), 0 ◦C → rt, 8% loading.
Scheme 10 Synthesis of dipalmitoyl-PI(4)P 58 and immobilised
NH2-PI(4)P 60. Reagents and conditions: i. Bu2SnO, BnBr, Bu4NBr, MeCN,
i
˚
3 A MS, reflux, 76%; ii. (BnO)2P(N Pr2), 1H-tetrazole, CH2Cl2, rt, then
mCPBA, -78 ◦C → rt, 93% iii. CAN, MeCN–H2O (4/1, v/v), 84%; iv.
phosphoramidite 34, 1H-tetrazole, CH2Cl2, rt, then m-CPBA, -78 ◦C
→ rt, 56: 78%; v. phosphoramidite 38, 1H-tetrazole, CH2Cl2, rt, then
mCPBA, -78 ◦C → rt, 57: 77%; vi. H2 (15 or 4.1 bar), Pd black, NaHCO3,
tBuOH/H2O (6/1), 58: 59% from 56; 59: 75% from 57; vii. Affi-GelꢀR 10,
NaHCO3, CHCl3–MeOH/H2O (4/5/1, v/v/v), 0 ◦C → rt, 16% loading.
enantiomer was obtained from diastereomer 70, resulting from
the initial resolution step of racemic diol 7 with camphor (R)-
10 (Scheme 2), by acid catalysed methanolysis of the acetal 70.
Selective protection of the hydroxyl group at the 1-position as the
PMB ether via the stannane acetal, followed by benzylation of the
remaining hydroxyl groups afforded intermediate 61 in good yield
with an optical rotation in agreement with an authentic sample (see
Electronic Supporting Information for more details). Though this
is not a direct route to PI(5)P, it makes good use of an otherwise
unused by-product.
Synthesis of analogues of PI(3,4)P2,9,10,16,20,21,23,25 PI(3,5)P27,9,10,13–18
and PI(4,5)P210–12,22,25
Scheme 12 Alternative synthesis of intermediate 61. Reagents and
conditions: i. AcCl, MeOH–CH2Cl2, (2/3 v/v), rt, 17 h, 97%; ii. Bu2SnO,
PMBCl, NaBr, Bu4NBr, MeCN–toluene (2/1 v/v), 3 A MS, reflux, 17 h,
PI(X,Y)P2, the third subclass of phosphatidylinositols contains
those in which two of the 3-, 4- or 5-positions are phosphorylated.
There are three possible permutations for the bisphosphates:
PI(3,4)P2, PI(3,5)P2 and PI(4,5)P2. PI(3,4)P2 and PI(3,5)P2 are
easily obtained from cores A and D respectively (Scheme 13).
This route follows the standard methods for phosphorylation and
deprotection to afford the dipalmitoyl (7616,21 and 8216) and amino
(77 and 83) analogues of PI(3,4)P2 and PI(3,5)P2 respectively. It
should be noted that alternative hydrogenolysis conditions using
˚
72%; iii. NaH, BnBr, DMF, rt, 17 h, 66%.
palladium hydroxide on carbon in butanol were employed for the
global deprotection of both the dipalmitoyl analogues 75 and 81,
affording 76 and 82 in comparable yields. Affinity probes 84 and
85 were then obtained in the manner as described above.
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Scheme 13 Synthesis of dipalmitoyl-PI(3,4)P2, 76, dipalmitoyl-PI(3,5)P2 82 and immobilised NH2-PI(3,4)P2 84 and NH2-PI(3,5)P2 85. Reagents and
conditions: i. (BnO)2P(NiPr2), 1H-tetrazole, CH2Cl2, rt, then mCPBA, -78 ◦C → rt, 72: 91%; 78: 71%; ii. CAN, MeCN–H2O (4/1, v/v), 73: 90%; 79:
72%; iii. phosphoramidite 34, 1H-tetrazole, CH2Cl2, rt, then m-CPBA, -78 ◦C → rt, 74: 91%; 80: 93%; iv. phosphoramidite 38, 1H-tetrazole, CH2Cl2, rt,
then mCPBA, -78 ◦C → rt, 75: 83%; 81: 88%; v. H2 (3.5 or 4.1 bar), Pd(OH)2-C, tBuOH, 76: 97% from 74; 82: quant. from 80; vi. H2 (3.6 bar), Pd black,
NaHCO3, tBuOH/H2O (6/1), 77: 80% from 75; 83: 88% from 81; vii. Affi-GelꢀR 10, H2O, 4 ◦C, 84: 3% loading; 85: 3% loading.
˚
Scheme 14 Synthesis of dipalmitoyl-PI(4,5)P2 92 and immobilised NH2-PI(4,5)P2 94. Reagents and conditions: i. Bu2SnO, BnBr, Bu4NBr, CH3CN, 3 A
MS, reflux, 75% ii, Rh(PPh3)3Cl, EtNiPr2, EtOH–toluene/H2O (7/3/1), reflux, then AcCl, CH2Cl2–MeOH (2/1), 58%; iii. (BnO)2P(NiPr2), 1H-tetrazole,
CH2Cl2, rt, then mCPBA, -78 ◦C → rt, 75%; iv. CAN, MeCN–H2O (4/1, v/v), 80%; v. phosphoramidite 34, 1H-tetrazole, CH2Cl2, rt, then mCPBA,
-78 ◦C → rt, 90: 83%; vi. phosphoramidite 38, 1H-tetrazole, CH2Cl2, rt, then mCPBA, -78 ◦C → rt, 91: 82%; vii. H2 (25 or 15 bar), Pd black, NaHCO3,
tBuOH/H2O (6/1), 92: 78% from 90; 93: 59% from 91; viii. Affi-GelꢀR 10, NaHCO3, H2O, 0 ◦C, 3% loading.
The synthesis of the analogues of PI(4,5)P2, required some
manipulation of the protecting groups to provide the correct
substitution pattern (Scheme 14). Thus, the hydroxyl group in
the 3-position of core C was selectively benzylated using the
dibutyltin oxide strategy to afford alcohol 86. Removal of the allyl
group was achieved via isomerisation with Wilkinson’s catalyst
and methanolysis of the resulting enol ether, affording diol 87.
Subjection to the standard phosphorylation and deprotection
conditions gave dipalmitoyl-PI(4,5)P2 92 and NH2-PI(4,5)P2 93.12
Affinity probe 9412 was synthesised in the manner described
previously.
Synthesis of PI(3,4,5)P3,6,9,10,16,21–25,28 and related analogues
Lastly, analogues of PI(3,4,5)P3 were obtained from core E
by a simple global phosphorylation (Scheme 15). The standard
methods for the coupling with the side chain phosphoramidites,
oxidation and global deprotection were used to gain access to both
the analogues 9916,21 and 10028 of PI(3,4,5)P3.
To demonstrate the versatility of this approach to the synthesis
of PIP analogues, shorter chain analogues of PI(3,4,5)P3 108 and
109, were synthesised using the corresponding hexanoyl51 and
octanoyl24 phosphoramidites 102 and 103, respectively (Fig. 2,
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Scheme 16 Synthesis of short chain analogues of PI(3,4,5)P3 108–110.
Reagents and conditions: i. phosphoramidite 102, 1H-tetrazole, CH2Cl2,
rt, then mCPBA, -78 ◦C → rt, 105: 67%; ii. phosphoramidite 103,
1H-tetrazole, CH2Cl2, rt, then m-CPBA, -78 ◦C → rt, 106: 75%; iii.
phosphoramidite 104, 1H-tetrazole, CH2Cl2, rt, then mCPBA, -78 ◦C →
rt, 107: 79%; iv. H2 (3.5, 3.1 or 25 bar), Pd black, NaHCO3, tBuOH/H2O
(6/1), 108: 96% from 105; 109: 80% from 106; 110: 78% from 107.
tion by directly activating upstream kinases. These investigations
confirmed the role of Akt/PKB in PI3K-signalling.
Work using PI(3)P affinity probes in 2001, led to the discovery
of the interactions of phosphatidylinositol phosphates with the
PX domain and its role in the formation of reactive oxygen species
(ROS) by neutrophils.32 It was determined that PI(3)P specifically
stimulates ROS formation by binding to the PX domain of
p40phox which in turn can interact with p67phox. Furthermore,
p40phox was shown to target PI(3)P-containing membranes. In
other work, two novel proteins FENS1 and DFCP1, containing
one and two FYVE domains, respectively, were shown to bind
PI(3)P specifically.33 These proteins are involved in intracellular
trafficking.53
In 2002, investigations with immobilised PI(3)P, PI(3,4)P2
and PI(3,5)P2 discovered five novel proteins and seven proteins
previously uncharacterised as phosphoinositide-binding.31 Inter-
estingly, three of the proteins that were not known to bind PIPs,
ATTP, MEG2 and Cdc42GAP, have different functions, but all
proved to have an SEC14-like domain in common. This domain
had not previously been known to bind to PIPs. One of the
novel PI(3,4,5)P3 and PI(3,4)P2-binding proteins, ARAP3, is a
PI3K-dependent, GTPase activating protein and was shown to
be a genuine effector of PI3K signalling responsible for PI3K-
dependent changes in the cell cytoskeleton and shape.
Scheme 15 Synthesis of dipalmitoyl-PI(3,4,5)P3 99 and immobilised
NH2-PI(3,4,5)P3 101. Reagents and conditions: i. (BnO)2P(NiPr2),
1H-tetrazole, CH2Cl2, rt, then mCPBA, -78 ◦C → rt, 78%; ii. CAN,
MeCN–H2O (4/1, v/v), 87%; iii. phosphoramidite 34, 1H-tetrazole,
CH2Cl2, rt, then m-CPBA, -78 ◦C → rt, 97: 78%; iv. phosphoramidite
38, 1H-tetrazole, CH2Cl2, rt, then mCPBA, -78 ◦C → rt, 98: 63%; v. H2
(3.5 bar), Pd(OH)2-C, tBuOH, 99: 85% from 97; vi. H2 (4.1 or 3.5 bar), Pd
black, NaHCO3, tBuOH/H2O (6/1), 100: 92% from 98; vii. Affi-GelꢀR 10,
NaHCO3, H2O, 0 ◦C, 3% loading.
Scheme 16). An w-amino analogue 110 with a hexanoyl chain
at the secondary position of the glycerol side chain was also
synthesised using the phosphoramidite 104.52 These shorter chain
analogues are known to be more water-soluble than the longer
chain PIPs.19,27
Investigations into the role of PI(3,5)P2 in vesicle recycling from
vacuole/lysosomal compartments, using immobilised PI(3,5)P2
identified Svp1p as a specific PI(3,5)P2-binding protein.30 This
protein participates in the recycling of membrane proteins from
the vacuole to the late endosome and at the time of discovery,
represented a new family of phosphoinositide-binding proteins. It
was shown that the binding of PI(3,5)P2 by Svp1p is necessary for
normal function in vacuole membrane trafficking.
Fig. 2 Short chain phosphoramidites 102–104.
Biological evaluation
During the course of our investigations we have used both the
dipalmitoyl and amino analogues of the PIPs in the investigation
of the PI3K pathway.30-35 Our original experiments used cytosolic
and membrane extracts from sheep brain or porcine neutrophils
and leukocytes. Initial investigations using the dipalmitoyl ana-
logue of PI(3,4,5)P3 revealed that Akt-1/PKB interacts directly
with PI(3,4,5)P3 via the PH domain.35 PI(3,4,5)P3-binding to
Akt/PKB was shown to be necessary for upstream phosphoryla-
More recently we have used analogues of PI(3,5)P2, PI(4,5)P2
and PI(3,4,5)P3 to perform a comprehensive proteomic analysis of
the phosphoinositide interactome in cell extracts from LIM1215
colorectal carcinoma cells.29 The procedure for these experiments is
outlined in Fig. 3. Phosphoinositide interacting protein complexes
were pulled down using analogues of PI(3,5)P2, PI(4,5)P2 and
PI(3,4,5)P3, either incorporated into liposomes or immobilised
R
onto Affi-Gelꢀ 10 beads in affinity-based assays. Proteins were
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Fig. 4 Venn diagram analysis of the purified phosphoinositide interacting
proteins showing the number of common and unique proteins pulled down
by PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3.
identified in our study were able to interact directly with their
phosphoinositide targets, suggesting that the remaining proteins
were presumably purified as part of interactome complexes.
Interestingly, the majority of purified proteins had molecular
function and processes corresponding to previously well char-
acterized cellular functions of phosphoinositide (e.g. molecular
transport, protein trafficking, vesicle mediated transport, actin
cytoskeletal regulation and GTPases regulated function, cell-to-
cell signalling and interaction, cellular development, movement,
assembly organization, growth and proliferation). A large number
of purified proteins were also reported to have direct and indirect
interactions with the PI3K/Akt signalling pathway.
Furthermore, proteins with different functions were found
to bind to the liposomes and beads. Broadly speaking, those
that require a membrane environment, such as GTPases and
cell adhesion molecules, bound to the liposomes, whereas those
involved in functions such as transport and trafficking were
identified using beads. Phosphatases and kinases were identified
in both pull-down experiments. This is depicted in Fig. 5 for
PI(4,5)P2. Experiments are continuing in an effort to further our
understanding of the PIP interactomes.
Fig. 3 Schematic of procedure for pull down experiments with liposomes
incorporating PIPs and immobilised PIPs.29
then identified using nano-RP-HPLC ESI MS/MS analysis. The
affinity probes were characterised using Biosensor technology.
Thus, the phosphoinositide-containing liposomes were charac-
terised using recombinant GST-tagged PH domains of General
Receptor 1, Phospholipase C delta 1 and Dynamin, whilst the
loading of the NH2-PIP on the beads was assessed by the
analysis of the supernatants from the immobilisation reactions
and comparison with calibration curves generated by injecting
various concentrations of each PIP over immobilised neomycin.29
The NH2-PIP analogues can also be immobilised onto a sensor
chip allowing the characterisation of their interaction with their
protein-binding partners.
From the cytosolic extracts of LIM1215 colon cancer cells we
identified 529 proteins/protein complexes that appeared to inter-
act specifically with their phosphoinositide targets: 69 proteins
interact specifically with PI(3,5)P2, 146 with PI(4,5)P2, 141 with
PI(3,4,5)P3, 32 with both PI(3,5)P2 and PI(4,5)P2, 36 with both
PI(3,5)P2 and PI(3,4,5)P3, 41 with both PI(4,5)P2 and PI(3,4,5)P3,
and 64 with all 3 phosphoinositides (Fig. 4).
Fig. 5 Venn diagram analysis of the purified PI(4,5)P2 interacting
proteins showing the number of common and unique proteins pulled down
by immobilised-NH2-PI(4,5)P2 and PI(4,5)P2-liposomes.
Our affinity method was validated by the purification of a
large number of proteins that possess known phosphoinositide
or phospholipid binding domains (for example: PH, PX, FERM,
PTB/PID, Phosphatidylinositol 3- and 4-kinase, RANBP1, C1,
C2, MARCKS, PDZ, PHD, Septin, Annexin, Clathrin adaptor,
Calponin, Phosphatidylinositol transfer protein, HEAT, Inositol
monophosphatase family, Cral-Trio, SPC2, START, SPFH). Fur-
thermore, small GTPases of Ras, Rab, Arf and Rho families that
are known to interact with phosphoinositide phosphates, were
also purified. Thus at least 30% of the total number of proteins
Conclusions
The complete family of phosphatidylinositol phosphate analogues
has been synthesised by a divergent strategy. Over the course of
these investigations, affinity probes incorporating these analogues
have been used to identify a range of important proteins in the
PI3K signalling pathway, which were novel at the time of discovery.
A comprehensive proteomic analysis of phosphoinositide inter-
acting proteins in cell extracts from LIM1215 colorectal carcinoma
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cells has also been carried out. Our studies provide an initial
detailed assessment of the phosphoinositide interactome which
can be used in the study of the cellular responses mediated
by phosphoinositides, to identify signalling pathways that are
affected by a change of phosphoinositide cellular concentration
(e.g. 3-phosphoinositide signalling cascade) and as a tool in drug
discovery and biomarker development.
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We thank the BBSRC and EPSRC for financial support and
for provision of the Swansea Mass Spectrometry Service. This
work was supported in part by the Australian Research Council,
Discovery Project, Grant DP0770668 and an NHMRC Program
Grant, 487922. General support was provided by CSIRO and
VESKI (Victorian Endowment for Science, Knowledge and Inno-
vation). We also thank Daiso Co., Ltd, Japan for the generous gift
of (+)-1,2-O-isopropylidene-glycerol. SJC thanks Hughes Hall,
Cambridge for a Research Fellowship. SJAG thanks GSK for
a CASE studentship. Z-YL thanks Cambridge Commonwealth
Trust, Universities UK (ORS award), New Hall, Cambridge
and Tan Kar Kee Foundation, Singapore for generous financial
support.
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51 Synthesised in a similar manner to dipalmitoyl phosphoramidite 34,
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52 Phosphoramidite 104 was synthesised from 35 following the procedure
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76 | Org. Biomol. Chem., 2010, 8, 66–76
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