Synthesis and biological evaluation of a novel cardiolipin affinity matrix

Article abstract of DOI:10.1039/b909306k

Cardiolipin (1) is a dimeric phospholipid found in the mitochondrial membranes of both plants and animals. In order to understand better its role, we report the preparation of an immobilised analogue (2) using phosphoramidite chemistry; the probe has been used successfully to bind a recombinant protein containing a cardiolipin-binding domain.

Full text of DOI:10.1039/b909306k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of a novel cardiolipin affinity matrix†
Melloney K. Johns,a Meng-Xin Yin,b Stuart J. Conway,a,c Diane E. J. E. Robinson,b Leon S.-M. Wong,b
Rebecca Bamert,d,e Richard E. H. Wettenhalle and Andrew B. Holmes*b
Received 12th May 2009, Accepted 9th June 2009
First published as an Advance Article on the web 14th July 2009
DOI: 10.1039/b909306k
Cardiolipin (1) is a dimeric phospholipid found in the mitochondrial membranes of both plants and
animals. In order to understand better its role, we report the preparation of an immobilised analogue
(2) using phosphoramidite chemistry; the probe has been used successfully to bind a recombinant
protein containing a cardiolipin-binding domain.
Cardiolipin (1) (Fig. 1) represents a family of dimeric phos-
pholipids that comprise three glycerol molecules connected
via the primary hydroxyl groups through two phosphodiester
linkages.1 The primary and secondary hydroxyl groups of the
two terminal glycerol moieties are acylated, giving rise to its
alternative name, diphosphatidylglycerol. This phospholipid is
particularly abundant in mammalian heart tissue, although it is
found in the mitochondria of all animal and plant tissues, and
throughout the prokaryotic kingdom. In general, cardiolipins
from mammalian tissues are rich in unsaturated fatty acid side
chains whilst those from bacteria contain mostly saturated chains
and those from plants have varying proportions of both.2 The
majority of cardiolipin (1) is found in membranes that generate
an electrochemical potential for substrate transport and ATP
synthesis.3 It comprises around 20% of the phospholipids present
in mitochondrial membranes4 and is located mainly on the inner
membrane, where it interacts with a variety of mitochondrial
proteins. Cardiolipin (1) is believed to be involved in the activation
of a number of enzymes, in particular those involved in the
production of energy by oxidative phosphorylation.5,6 It also plays
a role in mitochondrial lipid and protein import, programmed
cell death and the regulation of gene expression.3,7 Aberrant
cellular levels of cardiolipin have been linked to disease states
including autoimmune disorders such as arthritis, antiphospho-
lipid syndrome (APS) and systematic lupus erythematosus (SLE),
ischemic heart disease, thyroid dysfunction, Barth’s syndrome and
ageing.3,8
We have previously shown9–11 that immobilised phospho-
lipid analogues (including the phosphatidylinositol phosphates
[PIPns]9–11 and phosphatidic acid [PA]10) can be used as affinity
matrices to identify the intracellular protein targets of the lipid.
Applying this technique to cardiolipin (1) may assist in elucidating
its intracellular role, its involvement in disease and the intracellular
proteins with which it interacts. Several probes have been reported
for the purification of anticardiolipin antibodies, in which the
phospholipid is attached to a solid support via non-covalent
hydrophobic interactions.12 However, previous studies have shown
that covalent attachment of the phospholipids to the solid phase
has a number of advantages.13 These include the improved removal
of the protein from the matrix and the ability to reuse the matrix
a number of times. It has also been demonstrated that covalent
immobilisation of the lipid allows control over, and estimations of,
the number of sites on the solid phase that are occupied, termed
the percentage loading.10 Additionally, covalently immobilising
the phospholipid through a functional group at the end of one of
the fatty acid chains orients the molecule away from the surface
of the bead, in a manner that is designed to mimic its natural
orientation in cellular membranes.
Phosphoramidite chemistry has been used by our group
in the synthesis of numerous inositol polyphosphates14,15 and
phospholipids.9,10,15,16 Herein, we report the application of this
methodology to the synthesis of the cardiolipin analogue 3 and
the subsequent immobilisation of this compound onto an agarose-
based solid support, to form a covalently bound cardiolipin affinity
matrix 2.
Fig. 1 The general structure of cardiolipin 1. R = fatty acid.
Retrosynthetic analysis of the immobilised phospholipid gives
two phosphoramidite fragments 4 and 5, and a differentially
protected glycerol fragment 6 (Scheme 1). PMB protection of
(+)-1,2-O-isopropylidene-glycerol 7 followed by acid catalysed
methanolysis of the acetal afforded, in good yield, diol 8, a key
intermediate for all three fragments (Scheme 2). Esterification
of diol 8 with palmitoyl chloride followed by removal of the
PMB group with DDQ furnished alcohol 10 in good yield.
This compound was then transformed into phosphoramidite
fragment, 4, via a 1H-tetrazole mediated coupling with (ben-
zyloxy)bis(N,N-diisopropylamino)phosphine.17 The synthesis of
aDepartment of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, CB2 1EW, UK
bSchool of Chemistry, Bio21 Institute, Building 102, 30 Flemington Road,
University of Melbourne, Parkville, VIC 3010, Australia
cDepartment of Chemistry, Chemistry Research Laboratory, University of
Oxford, Mansfield Road, Oxford, OX1 3TA, UK
dDepartment of Biochemistry and Molecular Biology, Faculty of Medicine,
Nursing and Health Sciences, Monash University, Victoria 3800, Australia
eBio21 Institute, Building 102, 30 Flemington Road, University of Melbourne,
Parkville, VIC 3010, Australia
† Electronic supplementary information (ESI) available: NMR spectra of
3, 4, 15–19. See DOI: 10.1039/b909306k
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Scheme 1 Retrosynthetic analysis of the affinity matrix.
Scheme 2 Synthesis of fragment 4. Reagents and conditions: i. NaH,
PMBCl, DMF, 0 ◦C → rt, quant.; ii. p-TsOH·H2O, MeOH, reflux,
82%; iii. C15H31COCl, pyr., DMAP, CH2Cl2, 0 ◦C → rt, 94%; iv. DDQ,
CH2Cl2/H2O (16/1, v/v), rt, 85%; v. (BnO)P(NiPr2)2, 1H-tetrazole,
CH2Cl2, rt, 97%.
Scheme
3 Synthesis of fragment 5. Reagents and conditions: 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%.
fragment 5 (Scheme 3) was conducted in a similar manner and was
identical to that reported for the synthesis of immobilised phos-
phatidylinositol 4,5-bisphosphate (PI(4,5)P2) and immobilised
PA.10 Thus, selective DCC-mediated coupling of the primary al-
cohol of 8 with 12-N-(benzyloxycarbonyl)aminododecanoic acid
and subsequent esterification with palmitoyl chloride afforded
diester 12, which was deprotected and coupled with (benzy-
loxy)bis(N,N -diisopropylamino)phosphine to afford fragment 5.
The synthesis of the central fragment 6 commenced with
protection of the primary alcohol of the diol 8 (Scheme 4) as
the corresponding trityl ether 14. This reaction proceeded in high
yield and with complete chemoselectivity, as adjudged by 1H NMR
spectroscopy. A one-pot procedure involving the introduction
of the benzyl ether group at the secondary position and subse-
quent removal of the trityl protecting group by treatment with
p-toluenesulfonic acid monohydrate afforded the desired alcohol
6 in 97% yield.
With all three fragments in hand, assembly of the cardiolipin
analogue was undertaken (Scheme 5). Coupling of bis-protected
glycerol moiety 6 with the phosphoramidite 5 in the presence
of 1H-tetrazole followed by in situ oxidation with mCPBA
proceeded smoothly to afford the protected phosphatidylglycerol
15 in 74% yield. The PMB protecting group was cleaved by
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affinity matrix was incubated with purified recombinant PRK2
HR1 domain and the binding was analysed (Fig. 2b) and com-
pared with a control experiment with blank beads (Fig. 2a). As can
be seen from Fig. 2b, the majority of PRK2 HR1 domain bound to
the immobilised cardiolipin with high affinity with relatively little
detected in the unbound fraction. The control experiment revealed
that a small amount of the HR1 domain bound to the blank
beads, reflecting non-specific (probably hydrophobic) interactions,
however the clear band seen in both the washing steps indicates
that the majority of the protein was only weakly bound to the beads
and easily removed by phosphate buffer solution (W1 and W2,
Fig. 2a). These experiments confirm the validity of our approach
and that the HR1 domain of PRK2 is involved in the binding of
cardiolipin.
Scheme 4 Synthesis of the central fragment 6. Reagents and conditions:
i. Ph3CCl, pyr., DMAP, CH2Cl2, rt, 84%; ii. NaH, BnBr, TBAI, DMF, rt
then p-TsOH·H2O, MeOH, rt, 97%.
DDQ oxidation to furnish the alcohol 16 and the fully pro-
tected cardiolipin derivative 17 was obtained in 75% yield upon
coupling of phosphoramidite 4 with alcohol 16 in a similar
manner.18,19
All that remained was to remove the four protecting groups
in 17. It is necessary for the benzyl phosphate esters to be
removed before the benzyl ether, to prevent a 1,2-migration of
the phosphatidyl ester to the secondary hydroxyl. Thus, disodium
salt 18 was obtained in 61% yield by treatment of 17 with sodium
iodide. Hydrogenolysis of the benzyl ether and N-Cbz groups
afforded hydroxyamine 3 in 65% yield. It was subsequently found
that ammonium salt 19 could be prepared in an improved 91%
yield via a one-pot hydrogenation strategy (Scheme 5).20
The cardiolipin affinity matrix 2 was prepared by coupling
R
amine 3 with N-hydroxysuccimidyl (NHS)-activated Affi-Gelꢀ
Fig. 2 SDS-PAGE analysis of the binding of recombinant PRK2 HR1
domain with (a) blank beads and (b) immobilised cardiolipin. Total: total
amount of protein; unbound: residual protein in supernatant; W1: protein
in first wash; W2: protein in second wash; bound: protein bound to beads.
10 resin (BioRad) in a mixture of chloroform, methanol and
water in the presence of an excess of sodium hydrogen carbonate.
The matrix was washed thoroughly with the reaction solvent
and then with water, and the washings were lyophilised and
1
analysed by H NMR. In this manner, the percentage loading
Conclusions
of phospholipid on the solid phase was found to be 1.7%, based
on the recovery of unreacted cardiolipin analogue 3, which was
quantified through use of an internal standard (benzyloxy-myo-
inositol orthorformate).
The efficacy of cardiolipin beads as affinity probes was tested
using the purified recombinant HR1 domain of PRK2, a protein
kinase containing a negative regulatory domain known to be acti-
vated seven-fold by cardiolipin in in vitro assays.21 The expressed
and purified HR1 domain of PRK2 has been shown to interact
with cardiolipin in surface plasmon resonance experiments with an
11 mM affinity.22 To further confirm these results, the cardiolipin
An w-amine functionalised analogue of cardiolipin has been syn-
R
thesised and immobilised onto Affi-Gelꢀ 10 beads. The use of these
beads as a tool for the isolation of cardiolipin-binding proteins
has been demonstrated, which may help improve understanding
of the physiological roles of cardiolipin in both healthy and disease
states. Furthermore, the HR1 domain of a cardiolipin-activated
protein kinase has been proven to bind to cardiolipin. To our
knowledge, this is the first example of a cardiolipin affinity probe
being used to investigate the HR1 domain of protein kinase C
related kinases. Such an affinity matrix may also be used for
Scheme 5 Final coupling of the fragments and immobilisation of the cardiolipin analogue (3). Reagents and conditions: i. 5, 1H-tetrazole, CH2Cl2, rt,
then mCPBA, -78 ◦C → rt, 74%; ii. DDQ, CH2Cl2/H2O (16/1, v/v), rt, 82%; iii. 4, 1H-tetrazole, CH2Cl2, rt, then mCPBA, -78 ◦C → rt, 75%; iv. NaI,
acetone, 90 ◦C, 3 h, microwave, 61%; v. From 18: Pd black, H2 (150 psi), THF, rt, 3: 65%; vi. From 17: 10% Pd/C, THF, H2 (150 psi), rt, then NH4OH
19: 91%; vii. From 3: Affi-GelꢀR 10, chloroform/methanol/water (4/5/1, v/v/v), 4 ◦C, 1.7% loading.
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diagnostic purposes and for the identification of potential ther-
apeutic agents.
in petrol) afforded the diester (+)-9 (4.58 g, 6.67 mmol, 94%) as a
white solid with data in agreement with the literature.25
(-)-1,2-Di-O-hexadecanoyl-sn-glycerol 10
Experimental
To a stirred solution of PMB ether (+)-9 (1.50 g, 2.18 mmol,
1 equiv) in dry dichloromethane (87 mL) and deionised water
(5.5 mL) was added 2,3-dichloro-5,6-dicyanobenzoquinone
(DDQ) (1.01 g, 4.46 mmol, 2.05 equiv) and the reaction mixture
was stirred overnight at room temperature. The reaction was di-
luted with dichloromethane (50 mL) and washed with a saturated
aqueous solution of sodium hydrogen carbonate (100 mL) and
brine (2 ¥ 75 mL). The combined aqueous layers were extracted
with dichloromethane (30 mL). The combined organic phases were
dried (MgSO4), filtered and the solvent was removed in vacuo.
Flash chromatography (40% diethyl ether in petrol) afforded the
alcohol (-)-10 (1.06 g, 1.83 mmol, 85%) as a white solid with data
in agreement with the literature.25
General experimental techniques
Unless otherwise specified, all 1H, 13C and 31P NMR spectra
were recorded on a Bruker AV400 spectrometer at 400 MHz,
100 MHz and 162 MHz respectively, or a Bruker DRX500
spectrometer at 500 MHz, 125 MHz or 202 MHz respectively,
using deuterochloroform (or other indicated solvents) as reference
(d = 7.26 ppm and 77.0 ppm for 1H and 13C NMR respectively).
Chemical shifts (d) are measured in ppm. The chemical shift data
for 31P signals are reported in ppm relative to an external standard
of 85% H3PO4 (d = 0.00 ppm). Coupling constants (J) are quoted
in Hz and are recorded to the nearest 0.1 Hz. Infrared spectra were
recorded on Perkin-Elmer 1600 series FTIR and Perkin-Elmer
Spectrum One ATR-FTIR spectrometers in the region 4000–
600 cm-1. The sample was prepared as a solution in the indicated
solvent. Mass spectra were recorded at the EPSRC National Mass
Spectroscopy Centre, University of Wales, Swansea, the Mass
Spectrometry Service at the University of Cambridge Chemical
Laboratory and the University of Melbourne Mass Spectrometry
Service. Melting points were determined using a Reichert-Koeffler
hot stage or Bu¨chi melting point apparatus and are uncorrected.
Optical specific rotations were measured using a JASCO DIP-
1000 digital polarimeter or a Perkin-Elmer 241 polarimeter, in
a microcell of 1 dm path length for 1 mL of solution, units are
10-1 deg cm2 g-1 and concentration is expressed in g/100 cm3.
Microwave reactions were carried out in sealed reaction vessels
using a Biotage Initiator 2.0 (400 W). Solvents and reagents
were purified and dried where necessary by standard techniques.
Where appropriate and if not stated otherwise, all reactions were
performed in flame-dried apparatus under an inert atmosphere of
nitrogen or argon. Petrol refers to the fraction of petroleum ether
of boiling point range 40–60 ◦C.
(+)-Benzyloxy(N,N-diisopropylamino)(1,2-di-O-hexadecanoyl-sn-
glycer-3-yl)phosphine 4
A mixture of the alcohol (-)-10 (0.50 g, 0.88 mmol, 1 equiv), (ben-
zyloxy)bis(N,N -diisopropylamino)phosphine (0.75 g, 2.20 mmol,
2.5 equiv) and 1H-tetrazole (0.19 g, 2.64 mmol, 3.0 equiv) was
placed under high vacuum for 5 minutes and then under an atmo-
sphere of argon. After addition of dry dichloromethane (40 mL),
the reaction mixture was stirred at room temperature overnight.
The reaction mixture was diluted with dichloromethane (30 mL)
and quenched by addition of saturated aqueous sodium hydrogen
carbonate 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 (MgSO4), filtered and the solvent was removed
in vacuo. Flash chromatography (5/15/80 triethylamine/ethyl
acetate/hexane) afforded the phosphoramidite (+)-4 (0.69 g,
0.85 mmol, 97%) as a waxy solid: Rf 0.69 (20% ethyl acetate in hex-
24
ane); [a]D = +6.2 (c 1.21 in CHCl3); dH (400 MHz; CDCl3) 7.36–
The following compounds were synthesised following literature
procedures: (+)-1,2-O-isopropylidene-3-O-(4¢-methoxybenzyl)-
sn-glycerol,10 12-N-(benzyloxycarbonyl)aminododecanoic acid,23
5,10 8,10 11,10 12,10 13,10 14.24
7.24 (5 H, m, C6H5), 5.19–5.15 (1 H, m, CH2CHCH2), 4.75–4.65
(2 H, m, OCH2C6H5), 4.36–4.30 (1 H, m, CH2CHCHH), 4.17–
4.10 (1 H, m, CH2CHCHH), 3.78–3.45 (4 H, m, CH2CHCH2, 2 ¥
CH(CH3)2), 2.28 (4 H, t, J 7.4, 2 ¥ OCOCH2), 1.61–1.55 (4 H,
m, 2 ¥ OCOCH2CH2), 1.29–1.16 (48 H, br s, 2 ¥ C15H31), 1.02
(12 H, d, J 7.1, CH(CH3)2), 0.87 (6 H, t, J 6.8, 2 ¥ CH2CH3); dP
(162 MHz; CDCl3) 151.65, 151.50.
(+)-3-O-(4-Methoxybenzyl)-1,2-di-O-hexadecanoyl-sn-glycerol 9
To a stirred solution of the diol (-)-8 (1.50 g, 7.10 mmol, 1 equiv)
in dry dichloromethane (25 mL) was added dry pyridine (1.40 mL,
1.40 g, 17.75 mmol, 2.5 equiv) and 4-dimethylaminopyridine
(0.043 g, 0.3 mmol, 0.05 equiv). The solution was cooled to
(+)-2-O-Benzyl-3-O-(4¢-methoxybenzyl)-sn-glycerol 6
To a stirred solution of the alcohol (-)-14 (0.050 g, 0.11 mmol,
1 equiv) in dry THF (2 mL) under argon was added sodium hydride
(0.006 g, 60% suspension in mineral oil, 0.15 mmol, 1.4 equiv),
followed after 15 minutes by tetrabutylammonium iodide (0.004 g,
0.01 mmol, 0.1 equiv) and benzyl bromide (0.016 mL, 0.023 g,
0.13 mmol, 1.2 equiv). After stirring at room temperature for
2 h, the reaction was quenched with methanol (2 mL) and
diluted with diethyl ether (10 mL). The solution was washed
with saturated aqueous ammonium chloride solution (10 mL)
and the aqueous layer was separated and extracted with diethyl
ether (2 ¥ 10 mL). The combined organic layers were washed
with water (20 mL) and the solvent was removed in vacuo. The
0
◦C and palmitoyl chloride (4.70 mL, 4.29 g, 15.62 mmol,
2.2 equiv) was added dropwise. After stirring for 10 minutes at
this temperature, the solution was allowed to warm to room
temperature and stirred overnight. The reaction was quenched
by addition of water (25 mL). The aqueous phase was extracted
with dichloromethane (3 ¥ 30 mL) and the combined organic
layers were washed with 2 M aqueous hydrochloric acid (20 mL)
and water (25 mL), and these washings were back-extracted
with dichloromethane (30 mL). The organic phase was washed
with brine (30 mL), dried (MgSO4), filtered and the solvent was
removed in vacuo. Flash chromatography (50% dichloromethane
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residue was taken up in methanol (10 mL) and p-toluenesulfonic
acid monohydrate (0.002 g, 0.01 mmol, 0.1 equiv) was added.
After stirring at room temperature for 1 h, the reaction was
quenched with triethylamine (1 mL) and the solvent was removed
in vacuo. Flash chromatography (30% ethyl acetate in petrol) gave
the desired alcohol (+)-6 (0.032 g, 0.106 mmol, 97%) as a colourless
oil with data in agreement with the published literature values.26
2.0 equiv). After stirring at room temperature overnight, the reac-
tion mixture was diluted with dichloromethane (40 mL) and sat-
urated aqueous sodium hydrogen carbonate solution (5 mL) was
added. The aqueous phase was extracted with dichloromethane
(3 ¥ 20 mL), and the combined organic phases were washed with
water (20 mL) and brine (2 ¥ 20 mL), dried (MgSO4), filtered and
the solvent was removed in vacuo. The resulting yellow oil was
purified by flash chromatography (45% ethyl acetate in hexane)
to afford the alcohol (-)-16 (0.082 g, 0.08 mmol, 82%) as a white
solid: mp 34–36 ◦C (from diethyl ether/dichloromethane); Rf 0.12
(+)-1-O-(1¢-O-[12¢¢-N-(Benzyloxycarbonylamino)dodecanoyl]-2¢-
O-hexadecanoyl-sn-glycer-3¢-yl-O-benzyloxyphosphoryl)-2-O-
benzyl-3-O-(4¢-methoxybenzyl)-sn-glycerol 15
25
(50% ethyl acetate in hexane); [a]D = -4.2 (c 4.0 in CHCl3); nmax
-1
(CDCl3)/cm 3451, 2926, 2855, 1735, 1515, 1456, 1255, 1147,
To a stirred solution of the alcohol (+)-6 (0.043 g, 0.14 mmol,
1 equiv) and 1H-tetrazole (0.030 g, 0.42 mmol, 3 equiv) in dry
dichloromethane (4 mL) under argon was added a solution of
the phosphoramidite (+)-5 (0.390 g, 0.43 mmol, 3.1 equiv) in
dry dichloromethane (6 mL). After stirring at ro◦om temperature
for 2 h, the reaction mixture was cooled to -78 C and mCPBA
(0.072 g, 0.42 mmol, 3.0 equiv) was added. The resulting mixture
was stirred at -78 ◦C for 10 minutes and then for a further 1 h once
it had attained room temperature. The reaction was quenched by
addition of 10% (w/v) aqueous sodium hydrogen sulfite solution
(20 mL), and after stirring for 10 minutes, the aqueous phase was
separated and extracted with dichloromethane (3 ¥ 20 mL). The
combined organic extracts were washed with aqueous saturated
sodium hydrogen carbonate solution (20 mL) and brine (2 ¥
30 mL), dried (MgSO4), filtered and the solvent was removed in
vacuo. Flash chromatography (30% ethyl acetate in hexane) of the
resulting oil afforded the lipid (+)-15 (0.115 g, 0.10 mmol, 74%) as
1109, 1035, 1024; dH (250 MHz; CDCl3) 7.37–7.27 (15 H, m, 3 ¥
C6H5), 5.18 (1 H, qn, J 5.0, CH2CH(OCOC15H31)CH2), 5.09–
5.05 (4 H, m, HNC(O)OCH2C6H5, POCH2C6H5), 4.78 (1 H, br s,
NH), 4.65 (1 H, dd, J 11.7 and 6.1, CHOCHAHBC6H5), 4.57 (1
H, dd, J 11.7 and 3.7, CHOCHAHBC6H5), 4.30–4.22 (1 H, m,
CH2CHCHHOCO), 4.20–4.01 (5 H, m, CH2CHCHHOCO, 2 ¥
CH2CHCH2OP), 3.75–3.68 (1 H, m, CH2CH(OBn)CH2), 3.67–
3.56 (2 H, m, HOCH2CHCH2), 3.17 (2 H, dt, J 6.4 and 6.4,
CH2NH), 2.44 (1 H, br s, HOCH2CHCH2), 2.30–2.25 (4 H, m,
2 ¥ OCOCH2), 1.60–1.55 (4 H, m, 2 ¥ OCOCH2CH2), 1.50–1.45
(2 H, m, CH2CH2NH), 1.33–1.21 (38 H, m, C15H31, C11H22NH),
0.88 (3 H, t, J 6.8, CH2CH3); dC (100 MHz; CDCl3) 173.2, 172.8,
156.4, 137.8, 136.7, 135.5 (2 ¥ d), 128.7, 128.5, 128.1, 128.0, 127.9,
127.8, 77.6 (d), 77.5 (d), 72.1, 69.7 (d), 69.3 (d), 66.5, 66.1 (d), 65.5
(m), 61.6, 61.0 (d), 41.1, 34.1, 34.0, 31.9, 29.9, 29.7, 29.6, 29.5,
29.4, 29.3, 29.2, 29.1, 26.7, 24.8, 22.7, 14.1; dP (162 MHz; CDCl3)
0.17; m/z (EI+) 1013.6230 (M + NH4)+, C56H90N2O12P requires
1013.6231.
25
a yellow oil: Rf 0.45 (50% ethyl acetate in hexane); [a]D = +2.6
(c 5.8 in CHCl3); nmax (CDCl3)/cm-1 3452, 2927, 2855, 1732, 1612,
1586, 1514, 1465, 1249, 1173, 1150, 1102, 1035, 1022; dH (400 MHz;
CDCl3) 7.35–7.19 (17 H, m, C6H5, C6H4OCH3), 6.87–6.83 (2 H,
m, C6H4OCH3), 5.16 (1 H, qn, J 5.0, CH2CHCH2), 5.08 (2 H,
br s, HNC(O)OCH2C6H5), 5.03 (1 H, d, J 8.2, POCHAHBC6H5),
5.02 (1 H, dd, J 8.2 and 1.4, POCHAHBC6H5), 4.77 (1 H, br s,
NH), 4.63 (1 H, dd, J 11.8 and 4.1, CHOCHAHBC6H5), 4.60 (1
H, dd, J 11.8 and 3.8, CHOCHAHBC6H5), 4.44–4.43 (2 H, m,
CH2C6H4OCH3), 4.27–4.16 (2 H, m, CH2CHCH2OCO), 4.15–
4.00 (4 H, m, CH2CHCH2OP, POCH2CHCH2), 3.78–3.77 (3 H,
m, C6H4OCH3), 3.76–3.72 (1 H, m, CH2CH(OBn)CH2), 3.53–
3.51 (2 H, m, OCH2CHCH2), 3.17 (2 H, dt, J 6.7 and 6.7,
CH2NH), 2.29–2.23 (4 H, m, 2 ¥ OCOCH2), 1.58–1.54 (4 H, m,
2 ¥ OCOCH2CH2), 1.49–1.44 (2 H, m, CH2CH2NH), 1.33–1.20
(38 H, m, C15H31, C11H22NH), 0.87 (3 H, t, J 6.9, CH2CH3); dC
(100 MHz; CDCl3) 172.2 (d), 171.8 (d), 158.3, 155.4, 137.1, 135.7
(d), 134.7 (d), 129.0, 128.3, 127.6, 127.5, 127.4, 127.1, 126.9, 126.8,
126.7, 112.8, 75.5 (d), 72.1, 71.2, 68.5 (d), 68.4 (d), 68.4 (d), 68.3
(d), 67.6, 66.3 (d), 66.2 (d), 65.5, 64.5–64.4 (m), 60.6, 54.2, 40.1,
33.1, 33.0, 30.9, 29.0, 28.7, 28.6, 28.5, 28.4, 28.3, 28.2, 28.1, 23.8,
21.7, 13.1; dP (162 MHz; CDCl3) -0.37; m/z (ES+) 1133.6796 (M
+ NH4)+, C64H98N2O13P requires 1133.6807.
(+)-1-O-(1¢-O-[12¢¢-N-(Benzyloxycarbonylamino)dodecanoyl]-2¢-
O-hexadecanoyl-sn-glycer-3¢-yl-O-benzyloxyphosphoryl)-2-O-
benzyl-3-O-(1¢,2¢-di-O-hexadecanoyl-sn-glycer-3¢-yl-O-
benzyloxyphosphoryl)-sn-glycerol 17
To a stirred solution of the phosphoramidite (+)-4 (0.08 g,
0.10 mmol, 2.5 equiv) and 1H-tetrazole (0.08 g, 0.12 mmol,
3.0 equiv) in dry dichloromethane (2 mL) under argon was added
a solution of alcohol (-)-16 (0.04 g, 0.04 mmol, 1 equiv) in dry
dichloromethane (3 mL) and the resulting mixture was stirred
overnight at room temperature. The reaction mixture was then
cooled to -78 ◦C and mCPBA (0.02 g, 0.12 mmol, 3.0 equiv)
was added. After stirring at this temperature for 30 minutes, the
reaction mixture was allowed to attain room temperature and
stirred for 1 h. The reaction was diluted with dichloromethane
(10 mL) and quenched by addition of 10% (w/v) aqueous
sodium hydrogen sulfite solution (10 mL). After stirring at room
temperature for 10 minutes, the aqueous phase was separated
and extracted with dichloromethane (3 ¥ 20 mL). The combined
organic extracts were washed with saturated aqueous sodium
hydrogen carbonate solution (10 mL) and brine (2 ¥ 20 mL),
dried (MgSO4), filtered and the solvent was removed in vacuo. The
resulting residue was purified by flash chromatography (30–50%
ethyl acetate in hexane) to afford the product (+)-17 (0.050 g,
0.03 mmol, 75%) as a colourless oil: Rf 0.22 (50% ethyl acetate
(-)-1-O-(1¢-O-[12¢¢-N-(Benzyloxycarbonylamino)dodecanoyl]-2¢-
O-hexadecanoyl-sn-glycer-3¢-yl-O-benzyloxyphosphoryl)-2-O-
benzyl-3-lyso-sn-glycerol 16
in hexane); [a]D = +2.3 (c 2.3 in CHCl3); nmax (CDCl3)/cm-1
25
To a stirred solution of the PMB ether (+)-15 (0.114 g, 0.10 mmol,
1 equiv) in dichloromethane (5 mL) and deionised water (0.25 mL)
under air was added portionwise DDQ (0.046 g, 0.20 mmol,
3451, 2927, 2855, 1737, 1602, 1515, 1456, 1268, 1159, 1112, 1038,
1024; dH (250 MHz; CDCl3) 7.35–7.29 (20 H, m, 4 ¥ C6H5),
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Org. Biomol. Chem., 2009, 7, 3691–3697 | 3695
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5.19–5.15 (2 H, m, 2 ¥ CH2CH(OCOC15H31)CH2), 5.09 (2 H, s,
HNC(O)OCH2C6H5), 5.05–5.02 (4 H, m, 2 ¥ POCH2C6H5),
4.75 (1 H, br s, NH), 4.60–4.59 (2 H, m, CHOCH2C6H5),
4.27–4.22 (2 H, m, CH2CHCH2OCO), 4.16–4.01 (10 H, m,
CH2CHCH2OCO, 4 ¥ CH2CHCH2OP), 3.75–3.71 (1 H, m,
CH2CH(OBn)CH2), 3.18 (2 H, dt, J 6.3 and 6.3, CH2NH), 2.27 (8
H, t, J 7.4, 4 ¥ OCOCH2), 1.58–1.56 (8 H, m, 4 ¥ OCOCH2CH2),
1.50–1.46 (2 H, m, CH2CH2NH), 1.33–1.21 (86 H, br s, 3 ¥ C15H31,
C11H22NH), 0.88 (9 H, t, J 6.9, 3 ¥ CH2CH3); dC (100 MHz; CDCl3)
173.2, 172.8, 156.4, 137.4 (d), 136.7, 135.5 (2 ¥ d), 128.7, 128.5,
128.4, 128.1, 128.0, 127.8, 77.2 (m), 72.3, 69.6 (d), 69.2 (2 ¥ d) 68.4,
66.6, 65.8, 65.5 (2 ¥ m), 61.6, 41.1, 34.1, 34.0, 31.9, 30.0, 29.7, 29.5,
29.4, 29.3, 29.1, 26.7, 24.8, 22.7, 14.1; dP (162 MHz; CDCl3) -0.28,
-0.33; m/z (ES+) 1739.0874 (M + Na)+, C98H159NNaO19P2 requires
1739.0841].
0.3 equiv). The reaction vessel was then pressurised and vented
with hydrogen (10 times) then finally pressurised to 150 psi and
the reaction stirred overnight at room temperature. The catalyst
R
was removed by filtration through Celiteꢀ and the filtrate treated
with 28% aqueous ammonia solution (0.1 mL). This mixture was
allowed to stir for 1 h before the solvent was removed in vacuo.
The residue was taken up in chloroform and the product pre-
cipitated with acetone. The resulting suspension was centrifuged
(10 minutes, 3000 rpm) and the supernatant was removed. The
remaining solid was taken up in chloroform, precipitated with
acetone and the supernatant removed by centrifuging (10 minutes,
3000 rpm). Drying the solid under vacuum afforded the desired
diammonium cardiolipin salt (+)-19 (0.037 g, 0.011 mmol, 91%)
25
as a white amorphous solid: [a]D = +4.4 (c 1.0 in CHCl3); nmax
(neat)/cm-1 3368, 2919, 2851, 1738, 1641, 1467, 1215, 1171, 1088,
1064, 835, 758, 721, 664; dH (500 MHz; CDCl3) 5.20–5.18 (2 H, m,
2 ¥ CH2CH(OCOC15H31)CH2), 4.46–4.43 (1H, m, one CH2CH),
4.39–4.36 (1 H, m, one CH2CH), 4.16–4.07 (2 H, m, CH2CH),
3.95–3.80 (9 H, m, 4 ¥ CH2CH, CH2CH(OH)CH2), 2.87 (2 H,
br s, CH2NH2), 2.34–2.27 (8 H, m, 4 ¥ OCOCH2), 1.85 (8 H, br s,
2 ¥ NH4), 1.70–1.53 (10 H, m, 4 ¥ OCOCH2CH2, CH2CH2NH2),
1.40–1.25 (89 H, m, OH, NH2, 3 ¥ C15H31, C11H22NH2), 0.88
(9 H, t, J 7.0, 3 ¥ CH2CH3); dC (125 MHz; CDCl3) 173.4, 173.3,
173.0, 172.9(5), 70.3, 69.6, 66.9, 63.6, 63.5, 62.6, 62.2, 39.5, 34.2(7),
34.2(5), 34.1, 33.9, 31.9, 29.7(2), 29.6(7), 29.6(0), 29.5(8), 29.4(1),
29.3(6), 29.2(2), 29.1(9), 24.9(4), 24.8(9), 22.7, 14.1; dP (202 MHz,
CDCl3) 1.30, 0.98; MS (ESI+) m/z 1312.9280 (M - 2NH3 + H)+,
C69H136NO17P2 requires, 1312.9278; 1334.9097 (M - 2NH3 + Na)+,
C69H135NO17P2Na requires 1334.9098; MS (ESI-) m/z 1310.9126
(M - 2NH3 - H)-, C69H134NO17P2 requires 1310.9133.
(+)-1-O-(1¢-O-[12¢¢-N-(Benzyloxycarbonylamino)dodecanoyl]-2¢-
O-hexadecanoyl-sn-glycer-3¢-yl-O-phosphoryl)-2-O-benzyl-3-O-
(1¢,2¢-di-O-hexadecanoyl-sn-glycer-3¢-yl-O-phosphoryl)-
sn-glycerol disodium salt 18
To a stirred solution of sodium iodide (0.011 g, 0.073 mmol,
6.0 equiv) in dry acetone (0.2 mL) was added a solution of lipid
(+)-17 (0.021 g, 0.012 mmol, 1 equiv) in dry acetone (0.1 mL)
under nitrogen and the resulting mixture was heated at 90 ◦C
for 3 h in a microwave. The reaction mixture was allowed to
cool to room temperature, diluted with acetone (10 mL) and
centrifuged (3000 rpm, 15 minutes). The yellow supernatant
was removed and the remaining white solid was washed with
acetone (2 ¥ 10 mL) by centrifugation. The resulting white
solid was taken up in chloroform (2 mL) and precipitated with
acetone (10 mL), the supernatant was removed by centrifugation
(3000 rpm, 15 minutes). The solid was dried under vacuum,
affording the disodium salt (+)-18 (0.011 g, 0.007 mmol, 61%) as a
white amorphous solid: Rf 0.44 (65/25/4 CHCl3/MeOH/H2O);
(-)-1-O-(1¢-O-[12¢¢-Aminododecanoyl]-2¢-O-hexadecanoyl-sn-
glycer-3¢-yl-O-phosphoryl)-3-O-(1¢,2¢-di-O-hexadecanoyl-
sn-glycer-3¢-yl-O-phosphoryl)-sn-glycerol disodium salt 3
[a]D = +4.5 (c 0.74 in CHCl3); nmax (CDCl3)/cm-1 2927, 2854,
25
To a stirred suspension of protected cardiolipin analogue (+)-18
(0.020 g, 0.013 mmol, 1.0 equiv) in dry tetrahydrofuran (5 mL)
was added palladium black (0.014 g, 0.13 mmol, 10.0 equiv). The
reaction vessel was then pressurised and vented with hydrogen
(10 times) then finally pressurised to 150 psi and stirred for
2 days at room temperature. After degassing, the reaction mixture
was centrifuged (3 minutes, 3000 rpm) and the supernatant was
removed. The palladium pellet was washed with THF (15 mL) and
centrifuged again. After removing the supernatant, this washing
process was repeated with more THF (10 mL). The combined
1732, 1603, 1516, 1456, 1259, 1223, 1169, 1106, 1078, 1015; dH
(400 MHz; CDCl3) 7.35–7.27 (10 H, m, 2 ¥ C6H5), 5.21 (2 H, br s,
2 ¥ CH2CH(OCOC15H31)CH2), 5.08 (2 H, s, HNC(O)OCH2C6H5),
4.78 (1 H, br s, NH), 4.59 (2 H, s, CHOCH2C6H5), 4.38–4.35
(2 H, m, glycerol CH2), 4.16–4.12 (2 H, m, glycerol CH2), 4.01
(4 H, br s, 2 ¥ glycerol CH2), 3.90 (4 H, br s, 2 ¥ glycerol
CH2), 3.66 (1 H, m, CH2CH(OBn)CH2), 3.18 (2 H, dt, J 6.4
and 6.4, CH2NH2), 2.27–2.21 (8 H, m, 4 ¥ OCOCH2), 1.57–1.48
(10 H, m, 4 ¥ OCOCH2CH2, CH2CH2NH), 1.25 (86 H, br s,
3 ¥ C15H31, C11H22NH2), 0.87 (9 H, t, J 6.9, 3 ¥ CH2CH3); dC
(125 MHz; CDCl3) 173.5, 156.3, 138.0, 136.7, 128.5, 128.3, 128.1,
128.0, 127.6, [A signal was expected at around 77 ppm but it was
obscured by the chloroform peaks], 71.2, 70.6, 66.5, 63.5, 62.8,
41.1, 34.3, 34.1, 31.9, 30.0, 29.8, 29.7, 29.4, 29.2, 26.8, 24.9, 24.8,
22.7, 14.1; dP (162 MHz; CDCl3) 1.56; m/z (ES+) 1580.9754 (M +
H)+, C84H146NNa2O19P2 requires 1580.9740.
R
supernatants were passed through a plug of Celiteꢀ and concen-
trated in vacuo. The residue was then taken up in chloroform
and precipitated with acetone. The resulting suspension was
centrifuged (10 minutes, 3000 rpm) and the supernatant was
removed. The remaining solid was redissolved in chloroform,
precipitated with acetone and the supernatant was removed by
centrifuging (10 minutes, 3000 rpm). The solid was dried under
vacuum to give the desired cardiolipin analogue (-)-3 (0.011 g,
(+)-1-O-(1¢-O-[12¢¢-Aminododecanoyl]-2¢-O-hexadecanoyl-sn-
glycer-3¢-yl-O-phosphoryl)-3-O-(1¢,2¢-di-O-hexadecanoyl-
sn-glycer-3¢-yl-O-phosphoryl)-sn-glycerol diammonium 19
25
0.008 mmol, 65%) as a white amorphous solid: [a]D = -2.0 (c
-1
0.2 in CHCl3); nmax (neat)/cm 3384, 2957, 2918, 2851, 1737,
1664, 1467, 1260, 1221, 1170, 1092, 1040, 801, 758, 721, 662; dH
(500 MHz; CDCl3) 5.22 (2 H, br s, 2 ¥ CH2CH(OCOC15H31)CH2),
4.48–4.35 (2H, m, CH2CH), 4.20–4.10 (2H, m, CH2CH), 4.10–
3.82 (9 H, m, NH, 4 ¥ CH2CH, CH2CH(OH)CH2), 3.09 (3 H, br s,
To a stirred suspension of the protected cardiolipin analogue (+)-
17 (0.050 g, 0.030 mmol, 1.0 equiv) in dry tetrahydrofuran (0.5 mL)
was added 10% Pd on activated charcoal (0.008 g, 0.008 mmol,
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CH2NH2, OH), 2.89–2.82 (2 H, m, CH2NH), 2.35–2.26 (8 H, m,
4 ¥ OCOCH2), 1.59 (10 H, br s, 4 ¥ OCOCH2CH2, CH2CH2NH2),
1.26 (86 H, br s, 3 ¥ C15H31, C11H22NH), 0.88 (9 H, t, J 7.0, 3 ¥
CH2CH3); dC (125 MHz; CDCl3) 173.5, 173.3, 70.5, 69.6, 66.7,
63.5, 62.8, 34.3, 34.1, 31.9, 30.9, 29.8, 29.7, 29.4, 29.3, 24.9, 22.7,
14.1; dP (202 MHz; CDCl3) 1.87; m/z (ES+) 1356.8917 (M + H)+,
C69H134NNa2O17P2 requires 1356.8922.
was provided by CSIRO and VESKI (Victorian Endowment for
Science, Knowledge and Innovation). We also thank Daiso Co.,
Ltd, Japan for the generous gift of (+)-1,2-O-isopropylidene-
glycerol.
References
1 M. Schlame, S. Brody and K. Y. Hostetler, Eur. J. Biochem., 1993, 212,
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1-O-(1¢-O-[12¢¢-N-Affi-Gel-10-aminododecanoyl]-2¢-O-
hexadecanoyl-sn-glycer-3¢-yl-O-phosphoryl)-3-O-(1¢,2¢-di-O-
hexadecanoyl-sn-glycer-3¢-yl-O-phosphoryl)-sn-glycerol disodium
salt 2
R
Affi-Gelꢀ 10 resin (1 mL, 0.015 mmol) was washed with cold chlo-
roform/methanol/water (4/5/1). A solution of the cardiolipin
analogue (-)-3 (0.002 g, 0.0015 mmol) and NaHCO3 (0.006 g,
0.071 mmol) in chloroform/methanol/water (1 mL, 4/5/1) was
shaken at room temperature for 10 minutes and then incubated
8 G. M. Hatch, Biochem. Cell Biol., 2004, 82, 99.
◦
R
with Affi-Gelꢀ 10 beads at 4 C in chloroform/methanol/water
(3 mL, 4/5/1) for 2 days. The beads were washed successively with
cold chloroform/methanol/water (4/5/1), cold methanol, and
cold water by centrifugation (2000 rpm, 5 minutes). The beads
were blocked by incubation with 0.1 mL of 1 M ethanolamine
(pH = 8.0) in chloroform/methanol/water (2 mL, 4/5/1) for
2 h at room temperature. The beads were then washed with
cold chloroform/methanol/water (4/5/1), cold methanol, and
cold water by centrifugation (2000 rpm, 5 minutes). The beads
were stored (at 4 ◦C) and used as a 50% slurry in water. The
loading of cardiolipin on the beads was 1.7% as determined by
1H NMR analysis of the residual cardiolipin in the supernatant
after lyophilisation, using benzyloxy myo-inositol orthorformate
(0.0074 mmol) as an internal standard.
9 G. F. Painter, J. W. Thuring, Z. Y. Lim, A. B. Holmes, P. T. Hawkins
and L. R. Stephens, J. Chem. Soc., Chem. Commun., 2001, 645.
10 Z.-Y. Lim, J. W. Thuring, A. B. Holmes, M. Manifava and N. T.
Ktistakis, J. Chem. Soc., Perkin Trans. 1, 2002, 1067.
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Catimel, D. E. J. E. Robinson, L. S.-M. Wong, E. C. Nice, A. B. Holmes
and A. W. Burgess, J. Proteome Res., 2009, 8, 3712; B. Catimel, C.
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Nice, A. W. Burgess and A. B. Holmes, J. Proteome Res., 2008, 7,
5295–5313.
12 T. Exner, N. Sahman and B. Trudinger, Biochem. Biophys. Res.
Commun., 1988, 155, 1001; V. Pengo and A. Biasiolo, Thromb. Res.,
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J. Coadwell, K. Davidson, A. Eguinoa, C. D. Ellson, P. Lipp, M.
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Z. Y. Lim, A. B. Holmes, S. K. Dove, R. H. Michell, A. Grewal, A.
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Hawkins, Mol. Cell, 2002, 9, 95.
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Binding experiment
The control blank beads were first blocked by incubation
with 0.10 mL of 1 M ethanolamine (pH = 8.0) in chloro-
form/methanol/water (2 mL, 4/5/1) for 2 h at room temperature.
The E. coli expressed and purified recombinant HR1 domain from
rat PRK2 at a concentration of 0.2 g/mL was then incubated
R
with either the blank Affi-Gelꢀ 10 beads or cardiolipin-conjugated
15 S. J. Conway and G. J. Miller, Nat. Prod. Rep., 2007, 24, 687.
16 G. F. Painter, S. J. A. Grove, I. H. Gilbert, A. B. Holmes, P. R. Raithby,
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Trans. 1, 1999, 923.
matrix at 37 ◦C fo◦r 10 minutes. The mixture was then centrifuged
at 5000 rpm at 4 C for 5 minutes and the supernatant fraction
(containing unbound protein) carefully removed. The sedimented
matrix was resuspended in ice-cold phosphate buffer solution
(PBS) and centrifuged again, prior to removal of the supernatant.
This was repeated and both PBS washes retained for SDS-PAGE
analysis. The final protein bound matrix was then resuspended in
PBS. Equal loadings of the pre-incubation HR1 protein, unbound
fraction and resuspended bound matrix, as well as a 1/10 loading
of the washes were analysed on SDS-PAGE and stained with
Coomassie Blue.
17 Synthesised from phosphorus trichloride by the method of W.
Bannwarth and A. Trzeciak, Helv. Chim. Acta, 1987, 70, 175.
18 All new compounds were characterised by 1H, 13C, 31P NMR spec-
troscopy, IR, mp and mass spectrometry.
19 As expected, two signals were observed in the 31P NMR spectrum
(dP -0.28 and -0.33 ppm), corresponding to the diastereomers arising
from the two stereogenic phosphate ester groups. Only one signal is
observed in the 31P NMR of sodium salts 18 and 3.
20 U. M. Krishna, M. U. Ahmad, S. M. Ali and I. Ahmad, Lipids, 2004,
39, 595; M. U. Ahmad, U. M. Krishna, S. M. Ali, S. Choudhury and I.
Ahmad, Lipids, 2007, 42, 291.
21 W. Yu, J. Liu, N. A. Morrice and R. E. H. Wettenhall, J. Biol. Chem.,
1997, 272, 10030.
Acknowledgements
22 R. Bamert, and R. E. H. Wettenhall, unpublished results.
23 D’ Ale´o, J.-L. Pozzo, K. Heuze´, F. Vo¨gtle and F. Fages, Tetrahedron,
2007, 63, 7482.
We thank the BBSRC and EPSRC for financial support and
for provision of the Swansea Mass Spectrometry Service. SJC
thanks Hughes Hall (Cambridge) for a Research Fellowship.
This work was supported in part by the Australian Research
Council, Discovery Project, Grant DP0770668. General support
24 D. Qin, H.-S. Byun and R. Bittman, J. Am. Chem. Soc., 1999, 121,
662.
25 C. Vilchtze and R. Bittman, J. Lipid, Res., 1994, 35, 734.
26 K. Fukase, T. Matsumoto, N. Ito, T. Yoshimura, S. Kotani and S.
Musumoto, Bull. Chem. Soc. Jpn., 1992, 65, 2643.
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 3691–3697 | 3697
©