Synthesis of highly water-soluble adamantyl phosphoinositide derivatives

Article abstract of DOI:10.1071/CH14543

Phosphatidylinositol phosphates are key regulators of cell signalling pathways and membrane trafficking in eukaryotic cells, and there is a need for new chemical probes to further understand how they interact with lipid-binding proteins. Here, the synthes

Full text of DOI:10.1071/CH14543

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem.
Full Paper
http://dx.doi.org/10.1071/CH14543
Synthesis of Highly Water-Soluble Adamantyl
Phosphoinositide Derivatives*
A
A
B
Mark Gregory, Meng-Xin Yin, Malcolm J. McConville,
C
C
D
Eleanor Williams, Alex N. Bullock, Stuart J. Conway,
Antony W. Burgess, Bruno Catimel, and Andrew B. Holmes
E
E
A F
,
A
School of Chemistry, Bio21 Institute, University of Melbourne, 30 Flemington Road,
Parkville, Vic. 3010, Australia.
B
Department of Biochemistry and Molecular Biology, Bio21 Institute, The University of
Melbourne, 30 Flemington Road, Parkville, Vic. 3010, Australia.
C
Structural Genomics Consortium, University of Oxford, Old Road Campus Research
Building, Roosevelt Drive, Oxford OX3 7DQ, UK.
D
Department of Chemistry, Chemistry Research Laboratory, University of Oxford,
Mansfield Road, Oxford OX1 3TA, UK.
E
Walter and Eliza Hall Institute of Medical Research, Parkville, Vic. 3052, Australia.
Corresponding author. Email: aholmes@unimelb.edu.au
F
Phosphatidylinositol phosphates are key regulators of cell signalling pathways and membrane trafficking in eukaryotic
cells, and there is a need for new chemical probes to further understand how they interact with lipid-binding proteins. Here,
the synthesis of phosphatidylinositol phosphate analogues containing adamantyl carboxylic ester groups, in place of the
natural lipid side chains, is described. These derivatives are considerably more soluble in water than analogues containing
other lipid side chains and do not form large aggregates such as liposomes or micelles. These adamantyl analogues bind to
known phosphoinositide-binding proteins with similar affinities to native ligands and will facilitate future studies on the
substrate specificities of these proteins involving cocrystallisation studies with proteins.
Manuscript received: 2 September 2014.
Manuscript accepted: 23 September 2014.
Published online: 3 December 2014.
Introduction
(as shown in Fig. 1). However, in plants, a range of carboxylic
acids from palmitic acid (16 : 0) and stearic acid to linoleic acid
(18 : 2) can be found at the sn-1 position with linoleic acid at the
sn-2 position.[8,9]
Phosphatidylinositol phosphates (PtdInsPs or PIPs) play sig-
nificant roles in multiple biological processes within the
cell.[1–3] They function as precursors for secondary messengers
in cell signalling pathways and act as membrane receptors for a
variety of proteins involved in intracellular membrane traf-
ficking and organelle biogenesis. These processes underlie
normal cellular and tissue development, while defects can lead
to disease.[4] The development of new probes for investigating
phosphoinositide function is therefore of high importance from
a biological and a pharmaceutical view point.[5–7]
There are eight known naturally occurring PIPs, varying only
in the phosphorylation status of the 3-, 4-, and 5-positions of
the myo-inositol head group of the phospholipid (summarised in
Fig. 1). The polar head group of PIPs is linked to a diacylglycerol
moiety via the myo-inositol C-1 hydroxyl through a phospho-
diester linkage. The fatty acids found in naturally occurring
PIPs have varying lengths and levels of unsaturation, and the
precise constitution is species-dependent. In animals, the major
molecular species contains stearic acid (18 : 0) at the sn-1
position and arachidonic acid (20 : 4) at the sn-2 position
We have previously described the synthesis of all eight
derivatives of the PIP family of compounds and analogues
carrying an v-N-terminal substituent on the sn-1 chain for each
compound.[10] This modification was used to immobilised these
lipids onto affinity beads for proteomic studies and onto a
BiacoreTM chip for detailed evaluation of the affinity of differ-
ent PIP-binding proteins for their cognate ligands.[11] The
synthesis of these derivatives was made possible through the
development of a reductive selective cleavage of myo-inositol
orthoformate to allow regioselective protection of the six
hydroxyl groups around the inositol ring.[12] Enantiomerically
pure compounds were obtained through diastereomeric resolu-
tion, using camphanic derivatives, in order to achieve separation
of the diastereomers.[8]
The synthetic derivatives of these molecules were used to
form liposomes for characterisation purposes.[13,14] However,
it is not always desirable to form liposomes or higher-order
*Dedicated to the memory of the late Sir John (Kappa) Cornforth, who inspired everyone he worked with.
Journal compilation Ó CSIRO 2014
www.publish.csiro.au/journals/ajc

B
M. Gregory et al.
OCOR2
OCOR1
O
OH
i ꢁ ii
iii
O
O
O
O
OH
HO
P
PMBO
HO
OH
6
1
2
1
2
OH
OR5
R3O
5
4
3
OR4
Ad
Ad
iPr2N
OBn
O
O
O
O
O
P
O
where R1 ꢀ C17H35 and R2 ꢀ C19H31
PI: R3 ꢀ R4 ꢀ R5 ꢀ H PI(3,4)P2: R3 ꢀ R4 ꢀ PO(OH)2, R5 ꢀ H
v
RO
Ad
O
Ad
PI(3)P: R3 ꢀ PO(OH)2, R4 ꢀ R5 ꢀ H PI(3,5)P2: R3 ꢀ R5 ꢀ PO(OH)2, R4 ꢀ H
PI(4)P: R4 ꢀ PO(OH)2, R3 ꢀ R5 ꢀ H PI(4,5)P2: R4 ꢀ R5 ꢀ PO(OH)2, R3 ꢀ H
PI(5)P: R5 ꢀ PO(OH)2, R3 ꢀ R4 ꢀ H PI(3,4,5)P3: R3 ꢀ R4 ꢀ R5 ꢀ PO(OH)2
O
O
5
3 R ꢀ PMB
4 R ꢀ H
iv
Fig. 1. General structure of the family of naturally occurring phosphati-
Ad ꢀ
dylinositol phosphates.
aggregates when studying such compounds. Furthermore, the
long-acyl-chain derivatives are not particularly soluble in water
owing to their hydrophobicity, which can cause problems when
studying the interactions of such compounds with polar macro-
molecules. Derivatives with shorter alkyl chains such as buta-
noyl ester side chains have been developed and applied to
protein cocrystallisation studies.[15] However, these compounds
have also been reported to be unstable. Cocrystals of known PIP-
binding proteins with the equivalent lipid-free inositol phos-
phate, such as myo-inositol 1,4,5-trisphosphate (IP3), have also
been prepared but these analogues completely lack the diacyl-
glycerol lipid moiety that is likely to contribute to the affinity of
these compounds with certain proteins.
Here, the adamantane-carboxylic ester groups have been
used as alternatives to the long fatty acid chains. Adamantane-
carboxylic acid has been used previously to fabricate semisyn-
thetic analogues of glycolipids.[16] The aqueous solubility of the
resulting compounds has been reported to be significantly
increased, which could be attributed to the formation of low-order
aggregates.[16,17] In addition, the rigid globular hydrocarbon
frameworkof adamantane groups may facilitate the crystallisation
processes. Furthermore, they may also mimic the actual behaviour
and conformation of the long acyl chains of naturally occurring
phosphoinositides in vivo when exposed to the buffer-like cyto-
solic environments.
Scheme 1. Synthesis of diadamantyl phosphoramidite 5. Reagents and
conditions: (i) NaH, paramethoxybenzyl chloride (PMBCl), DMF, 08C -
room temperature (rt), quantitative; (ii) p-TsOH (Ts ¼ toluenesulfonyl),
MeOH, reflux, 82 %; (iii) AdCOCl (Ad ¼ adamantyl), pyridine, DMAP,
CH2Cl2, 08C - rt - reflux, 86 %; (iv) CAN, MeCN/H2O, 2 h, 79 %; (v)
(BnO)P(NiPr)2, 1H-tetrazole, CH2Cl2, rt, 90 %.
P(NiPr)2,[8] in the presence of 1H-tetrazole, provided the di-
adamantyl glycerol-side chain phosphoramidite 5 in 90 % yield.
The protected inositol head group intermediates for both
PI(4,5)P2 6 and PI(3)P 7 were synthesised in 13 and 10 steps
respectively from myo-inositol.[10] Coupling the side chain
phosphoramidite 5 with the C-1 hydroxyl derivatives 6 and 7
gave the fully protected intermediates 8 and 9 in moderate yields
after purification by flash chromatography (Scheme 2). The
final step employed global hydrogenolytic cleavage under
pressure in the presence of a palladium catalyst.[8] In the case
of the PI(4,5)P2 derivative, conditions using Pd black (20 equiv.)
in a 6 : 1 tert-butanol/water mixture containing sodium bicar-
bonate gave the adamantane-PI(4,5)P2 derivative 10 as the
sodium salt in 62 % yield. For the PI(3)P derivate, Pd(OH)2
was used in a 3 : 2 THF/methanol mixture to give the adaman-
tine-PI(3)P derivative 11 in quantitative yield. Indicative 31P
NMR signals for compound 11 at ꢀ0.35 and ꢀ0.90 ppm, shifted
1
from further downfield, and no aromatic resonances in the H
Results and Discussion
NMR spectrum confirmed that the benzyl protecting groups had
been removed. A high-resolution mass spectroscopy (HRMS)
peak of m/z 737.2336, corresponding to the (M ꢀ H)ꢀ ion
(theoretical value 737.2336), confirmed the identity of the final
product 11.
As predicted, these analogues were considerably more solu-
ble in water than the naturally occurring compounds and no
liposomes or micelle formation was observed even at high
concentration. However, it was suggested that, owing to their
bulk, the adamantyl groups could potentially affect the binding
of the head group to the protein binding pocket. It was therefore
important to test the binding nature of these compounds before
further study.
The adamantane-containing side chain phosphoramidite 5 was
synthesised successfully from (þ)-1,2-O-isopropylidene glycerol
1 (Scheme 1), which was also used as a precursor in the previous
synthesis of the N-terminal, and unsaturated, side chain analogues
of the full family of PIPs.[10] Diol 2 was obtained by para-
methoxybenzyl (PMB) protection of 1 followed by subsequent
methanolysisoftheacetalgroup.[18] Exhaustiveesterificationof2
with adamantyl chloride gave the desired PMB-protected lipid 3
in 86% yield.
Traditionally, esterification of compound 2 with other acid
chlorides forms the ester at room temperature but in this case,
owing to the steric demand of the adamantane moieties, heating
under reflux was required to induce the reaction to reach
completion. Ceric ammonium nitrate (CAN) oxidation
successfully removed the PMB group to afford the 1,2-di-O-
adamantanecarbonyl-sn-glycerol 4 in 79 % yield. Phosphityla-
tion of the resulting alcohol with the bisphosphoramidite (BnO)
Isothermal titration calorimetry (ITC) studies were carried
out to compare the binding kinetics of the free head group
(inositol 1,3-diphosphate) and the PI(3)P analogue 11. The
affinity of compound 11 for the known PI(3)P-binding
FVYE domain-containing protein HGS (hepatocyte growth

Synthesis of Water-Soluble Adamantyl PIP Derivatives
C
myo-inositol
ref. [8]
O
O
O
O
O
O
BnO
O
O
O
O
OH
i
ii
OBn
OR3
OR2
BnO
HO
P
P
R1O
OBn
OBn
OH
OH
O
O
O
O
R1O
R1O
OR3
OR3
6 R1 ꢀ H, R2 ꢀ R3 ꢀ P(O)(OBn)2
OR2
OR2
7 R1 ꢀ P(O)(OBn)2, R2 ꢀ R3 ꢀ H
8 R1 ꢀ H, R2 ꢀ R3 ꢀ P(O)(OBn)2
10 R1 ꢀ H, R2 ꢀ R3 ꢀ P(O)(ONa)2
11 R1 ꢀ P(O)(ONa)2, R2 ꢀ R3 ꢀ H
9 R1 ꢀ P(O)(OBn)2, R2 ꢀ R3 ꢀ H
Scheme 2. Synthesis of PI(4,5)P2 and PI(3)P adamantyl derivatives 10 and 11 from readily available myo-inositol. Reagents and
conditions: (i) (a) phosphoramidite 5, 1H-tetrazole, CH2Cl2, room temperature (rt), (b) mCPBA, ꢀ788C to rt, 6 ¼ 47 %, 7 ¼ 44 %; (ii) Pd
black, H2 (150 psi [1 MPa]), tBuOH/H2O and NaHCO3 or Pd(OH)2, H2 (150 psi), MeOH/THF (3 : 2), 10 ¼ 62 %, 11 ¼ quantitative.
Time [min]
Time [min]
ꢂ10
0.2
0
10 20 30 40 50 60 70 80
ꢂ10
0
10 20 30 40 50 60 70 80
0
0
ꢂ0.2
ꢂ0.4
ꢂ0.6
ꢂ0.8
ꢂ1.0
ꢂ1.2
ꢂ1.4
ꢂ5
ꢂ1.6
0
ꢂ2
0
ꢂ2
ꢂ4
ꢂ6
ꢂ4
ꢂ8
ꢂ6
ꢂ12
ꢂ10
ꢂ14
ꢂ16
ꢂ18
ꢂ20
ꢂ22
ꢂ24
ꢂ8
ꢂ10
ꢂ12
ꢂ14
0
0.5
1.0
1.5
ꢂ0.5
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Molar ratio
Molar ratio
Fig. 2. Isothermal titration calorimetry (ITC) of the free head group (IP2, left panel) and adamantyl lipid 11 (right panel) binding to
human HGS (hepatocyte growth factor-regulated tyrosine kinase substrate); both have comparable binding affinities, with kD values
of 1.8 and 6.1 mM respectively.
factor-regulated tyrosine kinase substrate) shows only a small
reduction in binding affinity when compared with the free head
group (Fig. 2); dissociation constant (kD) values of 6.1 and
1.8 mM respectively were observed. Furthermore, this protein
has a well-defined binding surface in comparison with other
known PIP-binding domain-containing proteins, indicating that
the adamantyl groups are indeed useful replacements of the
naturally occurring long-chain fatty acids.[19]
Studies are now focussed on obtaining cocrystallised structures
in order to fully characterise the binding of these phosphoino-
sitides with their protein counterparts and to determine the
importance of the diacylglycerol unit in protein recognition.
Experimental
1H NMR spectra were recorded on Varian Unity 400 (400 MHz)
or Varian Unity 500 (500 MHz) instruments, using deutero-
chloroform (or other indicated solvents) as reference or internal
deuterium lock. The chemical shift data for each signal are
given as d in units of parts per million (ppm) relative to tetra-
methylsilane (TMS) where d TMS ¼ 0.00 ppm. The multiplicity
of each signal is indicated by: s, singlet; br s, broad singlet; d,
doublet; t, triplet; q, quartet; dd, doublet of doublets; ddd,
doublet of doublet of doublets; dt, doublet of triplets; and m,
multiplet. The number of protons (n) for a given resonance is
Conclusion
In conclusion, highly water-soluble derivatives of the naturally
occurring phosphoinositides have been generated by replacing
the acyl fatty chains with adamantyl groups. These analogues
containing bulky adamantyl groups have been demonstrated to
retain the binding affinity of the phosphorylated myo-inositol
head groups for the FYVE domain-containing protein HGS.

D
M. Gregory et al.
indicated by nH. Coupling constants (J) are quoted in Hz and are
recorded to the nearest 1 Hz. 13C NMR spectra were recorded on
Varian Unity 400 (100 MHz) or Varian Unity 500 (125 MHz)
instruments using the central resonance of the triplet of CDCl3 at
d 77.0 ppm as an internal reference. The chemical shift data for
each signal are given as d. 31P NMR spectra were recorded on
Varian Unity 500 (202 MHz) instrument. The chemical shift
data for each signal are given in units as d ppm, using the
chemical shift of 85 % H3PO4 at d 0.0 ppm as the internal
reference.
Infrared (IR) spectra were recorded on a Perkin Elmer
Spectrum One Fourier-transform (FT)-IR spectrometer in the
region 4000–650 cmꢀ1. The samples were analysed as thin films
from dichloromethane or as compressed solid samples.
Mass spectra were obtained on an LCMS system containing
an Agilent 1100 HPLC and an Agilent 6220 electrospray
ionisation time of flight (esiTOF) mass spectrometer fitted with
a standard Agilent electrospray ion (ESI) source, in the Research
Transfer Facility (RTF) at Bio 21 Institute, or a hybrid linear ion-
trap and FT ion cyclotron resonance (FT-ICR) mass spectrome-
ter (Finnigan LTQ-FT San Jose, CA), which is equipped with
ESI, by the Mass Spectrometry Service at the University of
Melbourne Chemistry Department.
Where appropriate and if not stated otherwise, all reactions
were performed in flame-dried apparatus under an atmosphere
of dry nitrogen.
3-O-Methoxybenzyl-1,2-di-O-adamantanecarbonyl-sn-
glycerol (3)
To a stirred solution of diol 1 (500 mg, 2.13 mmol, 1 equiv.) in
dry dichloromethane (10 mL) under nitrogen was added
4-dimethylaminopyridine (33 mg, 0.27 mmol, catalytic). The
resulting solution was cooled to 08C and dry pyridine (508 mg,
0.524 mL, 6.40 mmol, 3 equiv.) was added dropwise. After
stirring for 30 min, adamantancarbonyl chloride (1.12 g,
5.88 mmol, 3 equiv.) was added dropwise. The reaction mixture
was then warmed up to room temperature and heated at 658C
overnight. The reaction was quenched, once no starting material
remained by TLC, by addition of water (30 mL) and the aqueous
phase was extracted with dichloromethane (4 ꢁ 30 mL). The
combined organic layers were washed with 2 M aqueous
hydrochloric acid (30 mL) and the acid phase was back-extracted
with dichloromethane (30 mL). The combined organic layers
were washed with brine (35 mL), dried over MgSO4, filtered, and
the solvent was removed under vacuum. Flash chromatography
(10–20% ethyl acetate/petroleum spirit v/v) afforded the diester
3 (983mg, 1.83mmol, 86%) as a colourless solid.
Melting points were determined with an Electrothermal
Engineering IA9100 or a Bu¨chi 510 melting point apparatus
and are uncorrected.
Analytical thin-layer chromatography (TLC) was completed
on precoated 0.2-mm thick Merck Kieselgel 60 F254 silica gel
plates and visualised by absorption of UV light and ethanolic
phosphomolybdic acid (PMA) or aqueous potassium perman-
ganate solution.
20
Rf 0.25 (10 % ethyl acetate/petroleum spirit). ½aꢂD þ7.38
(c 0.5, CHCl3). Mp 58–618C. nmax (neat)/cmꢀ1 2905, 2852,
1728, 1612, 1513, 1454, 1325, 1243, 1222, 1183, 1103, 1073,
1038, 819. dH (500 MHz, CDCl3) 7.24 (d, J 9, 2H), 6.86 (d, J 9,
2H), 5.23–5.17 (m, 1H), 4.49–4.44 (m, 2H), 4.33 (dd, J 12, 4,
1H), 4.14 (dd, J 12, 6, 1H), 3.80 (s, 3H), 3.55 (dd, J 5, 2, 2H),
2.03–1.96 (m, 6H), 1.86 (dd, J 19, 3, 12H), 1.74–1.66 (m, 12H).
dc (125 MHz, CDCl3) 177.3, 176.9, 159.5, 130.1, 129.5, 114.0,
73.1, 70.0, 68.1, 62.7, 55.4, 40.9, 39.0, 39.0, 36.7, 36.7, 28.1,
28.1. HRMS (ESIþ) m/z 559.3030; calc. for C33H44O6Na
(M þ Na)þ 559.3030.
Flash chromatography was carried out, unless stated other-
wise, on silica gel (Merck Kieselgel 60, 230–400 mesh) under a
pressure of nitrogen.
Hydrogenations at high pressure were carried out in a Bu¨chi
GlasUster ‘miniclave drive’ stainless steel vessel, 100 mL, with
a maximum operation pressure of 60 bar (6 MPa). Teflon inserts
were used and reactions were stirred using magnetic stirrer bars.
Protein expression and purification involved human HGS
protein (also known as Hrs, Uniprot O14964; residues 1–225),
which was prepared from bacterial expression and purified by
nickel-affinity and size-exclusion chromatography.
Isothermal titration calorimetry was performed at 158C using
a Microcal VP-ITC microcalorimeter. Proteins were buffered in
50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) pH 7.5, 100 mM NaCl. IP2 at 0.2 mM concentration
was titrated into 0.02 mM HGS protein. Adamantyl lipid at
1.4 mM concentration was titrated into 0.2 mM HGS. Data were
analysed using a single binding site model implemented in the
Origin software package provided with the instrument.
Solvents such as n-hexane and N,N-dimethylformamide
were purchased from Aldrich and dried over freshly activated
1,2-di-O-Adamantanecarbonyl-sn-glycerol (4)
To a stirred solution of the PMB-ether 2 (55 mg, 0.10 mmol,
1 equiv.) in acetonitrile (4 mL) and water (1 mL) was added
CAN (337 mg, 0.61 mmol, 6 equiv.). After stirring for 2 h, the
reaction mixture was diluted with water and ethyl acetate.
The separated aqueous phase was extracted with ethyl acetate.
The combined organic extract was washed with sat. NaHCO3
and brine, dried over MgSO4, and filtered. The solvent was
removed under vacuum to give the crude product. Flash chro-
matography eluting with 10–20 % ethyl acetate/petroleum spirit
afforded the alcohol 4 (33 mg, 0.079 mmol, 79 %) as a colourless
film.
20
Rf 0.26 (20 % ethyl acetate/petroleum spirit). ½aꢂD ꢀ1.82
(c 2.0, CHCl3). nmax (neat)/cmꢀ1 2905, 2849, 1730, 1453,
1344, 1326, 1269, 1225, 1183, 1104 1072. dH (500MHz, CHCl3)
5.07–5.03 (m, 1H), 4.30 (dd, J 12, 5, 1H), 4.20 (dd, J 12, 6, 1H),
3.71 (d, J 5, 2H), 2.04–1.98 (m, 6H), 1.89 (dd, J 9, 3, 12H), 1.76–
1.66 (m, 12H). dc (125 MHz, CDCl3) 71.9, 61.8, 61.7, 40.8, 40.8,
38.8, 38.8, 36.4, 27.8. HRMS (ESIþ) m/z 439.2455; calc. for
C25H36O5Na (M þ Na)þ 439.2455.
˚
4-A molecular sieves for 1 h. Anhydrous THF, diethyl ether,
and dichloromethane were dried by passage through a packed
column of activated neutral alumina under an argon atmosphere,
and toluene by passage through a column with additional R3–11
copper-based catalyst (Pure Process Technology). Moisture
levels of solvent from the drying columns were regularly
checked by Karl Fischer titration. Petroleum spirits refers to
the fraction of boiling point range 40–608C. Procedures using
moisture- or air-sensitive reagents were undertaken in a nitrogen-
filled dual manifold employing standard Schlenk line techniques.
Brine refers to a saturated aqueous solution of sodium chloride.
(1)-Benzyloxy(N,N-di-isopropylamino)(1,2-di-O-
adamantanecarbonyl-sn-glycer-3-yl)phosphine (5)
A solution of benzyloxy-bis(N,N-di-isopropylamino)phosphine
(548 mg, 1.62 mmol, 3 equiv.) and 1H-tetrazole (3.8 mL,
0.45 M, 1.73 mmol, 3.2 equiv.) in dry dichloromethane (5.0 mL)

Synthesis of Water-Soluble Adamantyl PIP Derivatives
E
under nitrogen was stirred for 30 min. To the solution was added
the diacyl glycerol 3 (225 mg, 0.54 mmol, 1 equiv.) in dry
dichloromethane (25 mL). After stirring at room temperature
overnight, the reaction mixture was diluted with dichloro-
methane (30 mL) and quenched by addition of sat. NaHCO3
solution (50 mL). The organic phase was separated and the
aqueous phase was extracted with dichloromethane
(3 ꢁ 30 mL). The combined organic phases were washed with
brine (50 mL), dried over MgSO4, filtered, and the solvent was
removed under vacuum. Flash chromatography (5 : 15 : 80 tri-
ethylamine/ethyl acetate/petroleum spirit) afforded the phos-
phoramidite 5 (320 mg, 90 %) as a colourless oil.
(46 mg, 0.069 mmol, 2.2 equiv.) in dry dichloromethane (1 mL)
under nitrogen. After stirring at room temperature for 30 min,
a solution of 7 (25 mg, 0.031 mmol, 1 equiv.) in dry dichlor-
omethane (2 mL) was added to the mixture dropwise. After
stirring at room temperature overnight, mCPBA (100 mg, 70–
75 %, 0.41 mmol) was added in a single portion and the mixture
was stirred for a further 2 h. The reaction mixture was diluted
with dichloromethane and washed with sat. aq. NaHSO3. The
aqueous phase was extracted with dichloromethane and the
combined dichloromethane extract was washed with sat.
NaHCO3, brine, and dried (MgSO4). After filtration the solvent
was removed under vacuum to give the crude product. Flash
chromatography (25–75 % ethyl acetate/petroleum spirit)
afforded the protected lipid 9 (19 mg, 0.014 mmol, 44 %) as a
colourless oil.
Rf 0.24 (50 % ethyl acetate/petroleum spirits). [a]D ꢀ1.0
(c 0.6, CHCl3). nmax (neat)/cmꢀ1 2908, 2854, 1730, 1455, 1269,
1218, 1071, 1017, 738, 696. dH (500 MHz, CDCl3) 7.65–7.17
(m, 35H), 5.14–4.70 (m, 13H), 4.54–4.48 (m, 1H), 4.37–3.99
(m, 7H), 3.94–3.89 (m, 1H), 3.77 (dd, J 9, 6, 1H), 3.57–3.45 (m,
2H), 2.04–1.85 (m, 6H), 1.83–1.77 (m, 12H), 1.75–1.58 (m,
12H). dC (125 MHz, CDCl3) 174.2, 173.8, 135.8, 135.5, 133.1,
126.0, 125.9, 125.8, 125.7, 125.3, 125.25, 125.2, 125.1, 124.9,
124.7, 124.6, 77.3, 75.3, 74.6, 74.3, 74.0, 73.5, 73.1, 72.8, 66.9,
58.9, 38.1, 36.2, 36.1, 33.9, 25.3, 25.2, 20.4. dP (202 MHz,
CDCl3) ꢀ1.6, ꢀ1.67, ꢀ1.71, ꢀ1.75. HRMS (ESIþ) m/z
1391.5586; calc. for C80H90O16P2Na (M þ Na)þ 1391.5596.
18
Rf 0.9 (30 % ethyl acetate/petroleum spirit). ½aꢂD þ5.7 (c 1.0,
CHCl3). nmax (neat)/cmꢀ1 2906, 2852, 1732, 1454, 1221, 1184,
1103, 1073, 1024, 976. dH (500 MHz, CHCl3) 7.42–7.30 (m,
5H), 5.18–5.12 (m, 1H), 4.76–4.59 (m, 2H), 4.35–4.30 (m, 1H),
4.16–4.10 (m, 1H), 3.82–3.60 (m, 4H), 2.00 (s, 6H), 1.88–1.86
(m, 12H), 1.73–1.66 (m, 12H), 1.22–1.15 (m, 12H). dC
(125 MHz, CDCl3) 177.3, 176.9, 128.4, 127.4, 127.1, 70.8,
70.7, 65.6, 65.5, 65.4, 62.6, 62.5, 62.1, 61.9, 46.4, 43.3, 43.2,
40.9, 39.0, 36.7, 28.1, 24.9, 24.8, 24.7, 11.8. dP (202 MHz,
CDCl3) 149.6, 149.5. HRMS (ESIþ) m/z 676.3736; calc. for
C38H56N1O6PNa 676.3738.
1D-2,3,6-Tri-O-benzyl-myo-inositol-(10,20-di-O-
adamantanecarbonyl-glycer-30-yl benzyl phosphate)
4,5-Bis(dibenzyl)phosphate (8)
1H-Tetrazole solution (0.29 mL, 0.45 M, 0.13 mmol, 3 equiv.) in
acetonitrile was added to a solution of phosphoramidite 5 (63 mg,
0.095 mmol, 2.2 equiv.) in dry dichloromethane (1 mL) under
nitrogen. After stirring at room temperature for 30 min, a solution
of inositol head group 6 (42 mg, 0.043 mmol, 1 equiv.) in dry
dichloromethane (1.2 mL) was added to the mixture dropwise.
After stirring at room temperature overnight, meta-chloroperoxy-
benzoic acid (mCPBA) (140 mg, 70–75 %, 0.57 mmol, 6 equiv.)
was added in a single portion and the mixture was stirred for a
further 2 h. The reaction mixture was diluted with dichlor-
omethane and washed with sat. aq. NaHSO3. The aqueous
phase was extracted with dichloromethane and the combined
dichloromethane extract was washed with sat. NaHCO3, brine,
and dried (MgSO4). After filtration, the solvent was removed
under vacuum to give the crude product. Flash chromatography
(40–80 % ethyl acetate/petroleum spirit) afforded the protected
lipid 6 (30 mg, 0.020 mmol, 47 %) as a colourless oil.
1D-2,3,6-Trihydroxy-myo-inositol-(10,20-di-O-
adamantanecarbonyl-glycer-30-yl phosphate)
4,5-Bisphosphate (10)
To a solution of the protected lipid 8 (30 mg, 0.0195 mmol,
1 equiv.) in tert-butanol (4.2 mL) and water (0.7 mL) was added
NaHCO3 (8.2 mg, 0.0975 mmol, 5 equiv.) followed by Pd
black (41 mg, 20 equiv.). The reaction vessel (Bu¨chi GlasUster
hydrogenator) was degassed with N2 three times and then filled
with hydrogen (150 psi [1 MPa]) and stirred for 65 h at room
temperature. The mixture was centrifuged and the supernatant
was removed. The residue was washed with ethyl acetate and
centrifuged again to remove the starting material or by-products
left. The solid residue was stirred with water (5 mL) and cen-
trifuged and the aqueous supernatant was collected. This pro-
cedure was repeated three times (1300 g, 10 min). The combined
aqueous phases were passed through a plug of Celite and freeze-
dried to afford product 7 (11 mg, 62 %) as a fluffy colourless
amorphous solid.
[a]D22 ꢀ7.0 (c 0.3, D2O). nmax (neat)/cmꢀ1 3389, 2906, 1717,
1657, 1235, 1085, 979. dH (500 MHz, CDCl3) 5.35–5.27 (m, 1H,
CH2CHCH2), 4.49 (dd, J 12, 3, 1H), 4.30–4.22 (m, 3H), 4.15–
3.90 (m, 5H), 3.71 (dd, J 10, 3, 1H), 2.05–2.00 (m, 6H), 1.93–
1.86 (m, 12H), 1.89–1.68 (m, 12H). dC (125 MHz, CDCl3)
180.2, 179.9, 77.8, 77.7, 75.9, 75.8, 71.3, 71.2, 70.9, 70.7,
70.6, 63.7, 62.6, 40.9, 40.8, 38.3, 35.8, 35.7, 27.5, 27.4. dP
(202 MHz, CDCl3) 3.3, 2.0, ꢀ0.8. HRMS (ESI–) m/z 817.2026;
calc. for C31H48O19P3 (M ꢀ H)ꢀ 817.2018; m/z 839.1841; calc.
for C31H48 NaO19 P3 (M þ Na – H – H)ꢀ 839.1828.
Rf 0.30 (50 % ethyl acetate/petroleum spirit). [a]2D2 ꢀ3.6 (c 1.0,
CHCl3). nmax (neat)/cmꢀ1 2909, 2857, 1736, 1455, 1270, 1217,
1070, 1035, 1021, 737, 698. dH (500 MHz, CDCl3) 7.40–6.90
(m, 40H), 5.10–3.45 (m, 27H), 2.03–1.92 (m, 6H), 1.85–1.78
(m, 12H), 1.75–1.58 (m, 12H). dC (125 MHz, CDCl3) 176.7,
176.3, 138.2, 138.1, 137.4, 137.3, 136.1, 128.6, 128.5, 128.4,
128.3, 128.2, 128.1, 128.0, 127.8, 127.7, 127.6, 127.5, 127.2,
127.1, 127.0, 79.1, 79.0, 78.1, 78.0, 77.9, 77.7, 75.3, 75.2, 74.5,
74.4, 72.4, 72.3, 69.7, 69.6, 69.5, 69.4, 69.3, 69.1, 69.0, 68.9,
65.8, 65.5, 61.3, 60.3, 40.7, 38.7, 38.6, 36.4, 36.3, 27.8, 27.8,
27.7. dP (202 MHz, CDCl3) ꢀ0.6, ꢀ0.7, ꢀ0.9. HRMS (ESIþ)
m/z 1539.5914; calc. for C87H98O19P3 (M þ H)þ 1539.5915; m/z
1561.5742; calc. for C87H97O19P3Na (M þ Na)þ 1561.5735.
1D-2,4,5,6-Tetrahydroxy-myo-inositol-(10,20-di-O-
adamantanecarbonyl-glycer-30-yl phosphate)
3-Phosphate (11)
1D-2,4,5,6-Tetra-O-benzyl-myo-inositol-(10,20-di-O-
adamantanecarbonyl-glycer-30-yl benzyl phosphate)
3-(Dibenzyl)phosphate (9)
To a suspension of the protected derivative 9 (8 mg, 6 mmol,
1 equiv.) in methanol and THF (3 : 2) was added palladium(II)
hydroxide on carbon (20 wt-%, 8 mg), and the mixture was
1H-Tetrazole solution (0.21 mL, 0.45 M, 0.93 mmol, 3 equiv.)
in acetonitrile was added to a solution of phosphoramidite 5

F
M. Gregory et al.
placed in a high-pressure hydrogenation vessel. The vessel was
purged with nitrogen (10ꢁ, 3 bar [0.3 MPa]) and hydrogen
(10ꢁ, 10 bar [1 MPa]) before stirring rapidly (900 rpm) at room
temperature for 48 h. The reaction vessel was then purged with
nitrogen (3ꢁ, 3 bar [0.3 MPa]) before filtration of the crude
reaction mixture through Celite, rinsing with methanol (50 mL)
and milliQ water (50 mL). All solvent was removed (under
vacuum and by lyophilisation) and the crude product was sus-
pended in diethyl ether (10 mL) and milliQ water (15 mL). The
organic layer was extracted with milliQ water (3 ꢁ 10 mL) and
the combined aqueous layers were lyophilised to give a pure
colourless solid that was then stirred with sodium hydrogen
carbonate (3 equiv.) in milliQ water overnight. The solution was
then lyophilised to give the pure product 11 (4.0 mg, quantita-
tive) as a fluffy colourless solid.
Mp 146–1488C (dec.) (lyophilised from water). [a]D ꢀ34.5
(c 0.42, D2O). nmax (neat)/cmꢀ1 3363, 2906, 2851, 1727, 1452,
1224, 1035. dH (500 MHz, D2O) 5.45–5.35 (m, 1H), 4.64–4.52
(m, 2H), 4.37–4.27 (m, 1H), 4.26–4.01 (m, 4H), 3.96–3.82 (m,
2H), 3.54–3.43 (m, 1H), 2.18–2.05 (m, 6H), 2.05–1.90 (m, 12H),
1.90–1.74 (m, 12H). dC (125 MHz, D2O) 75.8, 75.6, 73.4, 71.2,
70.6, 70.5, 70.0, 63.9, 62.6, 61.4, 40.8, 38.3, 35.8, 27.5. dP
(202 MHz, CDCl3) ꢀ0.35. ꢀ0.90. HRMS (ESI–) m/z 737.2336;
calc. for C31H48O16P2 (M ꢀ H)ꢀ 737.2345.
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Acknowledgements
We thank the Australian Research Council for supporting this work through
Discovery Project (DP1094497). We would also like to thank The Univer-
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Abraham Trust (Oxford) for financial support. MJM is a National Health
and Medical Research Council (NHMRC) Principal Research Fellow. The
Structural Genomics Consortium (SGC) is a registeredcharity (no. 1097737)
that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim,
the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline,
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Ministry of Economic Development and Innovation, Pfizer, Takeda, and the
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