Synthesis of short and long chain cardiolipins

Article abstract of DOI:10.1016/j.tet.2006.04.071

A phosphoramidite approach using 2-cyanoethyl N,N-diisopropylchlorophosphoramidite was utilized for the first time to synthesize short chain cardiolipins. The approach was extended to synthesize long chain and their ether analogue. Optically active 1,2-di

Full text of DOI:10.1016/j.tet.2006.04.071

Tetrahedron 62 (2006) 6990–6997
Synthesis of short and long chain cardiolipins
Shoukath M. Ali, Moghis U. Ahmad, Peter Koslosky, Krishnudu Kasireddy,
*
U. Murali Krishna and Imran Ahmad
NeoPharm Inc., 1850 Lakeside Drive, Waukegan, IL 60085, USA
Received 21 February 2006; revised 20 April 2006; accepted 21 April 2006
Available online 26 May 2006
Abstract—A phosphoramidite approach using 2-cyanoethyl N,N-diisopropylchlorophosphoramidite was utilized for the first time to synthe-
size short chain cardiolipins. The approach was extended to synthesize long chain and their ether analogue. Optically active 1,2-di-O-acyl-
sn-glycerol or 1,2-di-O-myristyl-sn-glycerol was coupled with phosphoramidite reagent and 2-benzyloxy-1,3-propanediol in presence of
1H-tetrazole, followed by in situ oxidation, to give the corresponding protected cardiolipin analogues. The above intermediates were
converted into cardiolipin analogues in two steps by deprotection of cyanoethyl and benzyl groups.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
therapeutic agents ranging from difficult-to-formulate, water-
insoluble drugs to delivery of molecules to intracellular
targets. The encapsulation of chemotherapeutic agents3,4 in
a macromolecular carrier, such as liposomes, significantly
reduces the volume of distribution in normal tissues and
thereby increases the concentration of drug in the tumor.
This results in a decrease in toxicities and an increase in ther-
apeutic efficacy. The potential effects of the length and nature
of cardiolipin fatty acid chains (i.e., saturated or unsaturated)
on liposome aggregation have not been elucidated. However,
cardiolipin having short chain fatty acids are unknown till
now. Recognizing the need for the development of short
chain cardiolipins to improve drug delivery, we undertook
a program to design and synthesize a new class of short chain
cardiolipins (C8:0–C12:0), long chain cardiolipin (C14:0), and
their ether analogue (C14:0).
A need for synthetic phospholipids has developed, in part,
their use in liposomes, which have become useful carriers
of active therapeutic agents, enzymes, antibiotics, antigens,
hormones, and anticancer drugs.1 Cardiolipin (also known as
diphosphatidyl glycerol; Fig. 1) constitutes a class of
complex anionic phospholipids, typically purified from
cell membranes of tissues associated with high metabolic
activity, including the mitochondria of heart and skeletal
muscles.2 However, known chromatographic purification
techniques cannot resolve cardiolipin into discrete mole-
cular species. As a result, the use of this component in
drug formulations has limited potential because the resulting
formulations are not homogeneous. In animal tissues cardi-
olipin contains up to 90% linoleic acid (C18:2). Yeast cardi-
olipin differs in having more oleic (C18:1) and palmitoleic
(C16:1) fatty acids, while the bacterial lipid contains saturated
and monoenoic fatty acids with 14–18 carbons. Liposomes
containing cardiolipin encapsulate a broad spectrum of
The known methodologies for synthesizing cardiolipin are
mainly divided into two groups: (a) coupling the primary
hydroxyl groups of a 2-protected glycerol with 1,2-O-di-
acyl-sn-glycerol using a phosphorylating agent and (b) con-
densation at both primary hydroxyl groups of a 2-protected
glycerol with phosphatidic acid in the presence of 2,4,6-
triisopropylbenzenesulfonylchloride.5 Cardiolipin has also
been generated via a reaction between the silver salt of di-
acylglycerophosphoric acid benzyl ester6 with 1,3-diiodo-
propanol benzyl ether or 1,3-diiodopropanol t-butyl ether.
Although the schemes were suitable for the preparation of
small quantities of cardiolipin, these were unattractive for
the routine preparation of larger quantities due to the many
steps involved, the requirement for careful purification of
intermediates, the use of highly photosensitive silver salt
derivatives, and unstable iodo intermediates.
O
O
R
R
O
O
R
R
O
O
O
P
OH
O
O
O
O
P
O
O
O
OH
OH
Cardiolipin Analogues
R = Alkyl Groups
Figure 1. General structure of cardiolipin.
* Corresponding author. Tel.: +1 847 887 0800; fax: +1 847 887 9281;
e-mail: imran@neophrm.com
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.04.071

S. M. Ali et al. / Tetrahedron 62 (2006) 6990–6997
6991
phospholipids is known,17 their usage in the synthesis of
cardiolipin analogues having varying fatty acid chain
lengths is not well established.
N
P
OR
N
N
P
R1
OR2
N
P
OR
OR
New synthetic methods are needed that can be used to pre-
pare large quantities of cardiolipin analogue species having
varying fatty acid chain length, particularly ‘short chain car-
diolipins’. Such methods would increase the availability of
a wider variety of cardiolipin species and would diversify
the lipids available for the development of new liposomal
formulations containing active agents, which will have
more defined compositions than those currently available.
The short chain cardiolipin may also be useful for cosmetics
and skin care products. As part of ongoing research, we
developed new synthetic methods to synthesize cardiolipin
and its analogues using the phosphoramidite approach18,19a
as well as O-chlorophenyl dichlorophosphate.19b Herein,
we report for the first time the use of novel phosphoramidite
reagent, 2-cyanoethyl N,N-diisopropylchlorophosphorami-
dite 10 to prepare short chain cardiolipins, long chain cardio-
lipin, and cardiolipin ether analogues.
1. R = CH3
5. R = CH3
8. R1 = Cl, R2 = CH3
9. R1 = Cl, R2 = CH2Ph
10. R1 = Cl, R2 = C2H4CN
2. R = CH2Ph
6. R = CH2Ph
7. R = t-C4H9
3. R = C2H4CN
4. R = C4H8NHCOCF3
Figure 2. Phosphoramidite reagents.
Several phosphoramidite reagents (Fig. 2) such as methyl-
N,N,N,N-tetraisopropyl phosphorodiamidite7 1, benzyl-
N,N,N,N-tetraisopropylphosphorodiamidite8 2, 2-cyanoethyl
N,N,N,N-tetraisopropyl phosphorodiamidite9 3, (N-trifluoro-
acetylamino)butyl-N,N,N,N-tetraisopropyl phosphoramidite10
4, dimethyl-N,N-diisopropyl phosphoramidite11 5, dibenzyl-
N,N-diisopropyl phosphoramidite12 6, di-t-butyl-N,N-di-
isopropyl phosphoramidite13 7, methyl-N,N-diisopropyl-
chlorophosphoramidite14 8, benzyl-N,N-diisopropylchloro-
phosphoramidite15 9, and 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite16 10 have been used extensively in
the synthesis of oligonucleotides,16 phosphatidylinositols15
[PtdIns(4,5)P2 and PtdIns(3,4,5)P3], and to a lesser extent,
in phospholipids’ synthesis. Although the use of phosphate
triesters and phosphoramidite esters in preparing
2. Synthesis
The synthesis of cardiolipin analogues 18a, 18b, 18c, and
18d are outlined in Scheme 1. The synthetic methodology
that we have developed involves the application of
O
O
Py, DMAP, 55°C, 48h
HO
R
R
O
O
N
R
R
O
O
10% Pd-C, H , 50 psi
2
n-C H COCl
7
15
O
10
P
HO
CN
EtOH:EtOAc:AcOH (9:1:0.1)
n-C H COCl
9
19
Cl
OBn
OH
OBn
O
Et N, DMAP, CH Cl , rt, 12h
3
2
2
DIPEA, CH Cl
O
2
2
11
13a R = n-C H
13b R = n-C H
7
15
12a R = n-C H
7
15
19
9
19
13c R = n-C
13d R = n-C
O
H
12b R = n-C H
11 23
9
H
13 27
O
O
R
R
R
O
O
OBn
R
O
O
HO
OH
R
O
N
O
R
O
O
P
OBn
O
P
Et N
3
15
P
O
O
O
O
O
O
O
CH CN
3
O
CN
Tetrazole, CH Cl
2
2
2
O
O
35% H O
2
14a R = n-C H
7
15
CN
16a R = n-C H
14b R = n-C H
7
15
9
19
CN
16b R = n-C H
14c R = n-C
14d R = n-C
H
9
19
11 23
16c R = n-C
16d R = n-C
H
H
13 27
11 23
H
13 27
O
O
O
O
O
R
R
O
O
R
R
O
R
R
O
O
R
R
O
O
10% Pd-C, H , 50 psi
2
O
O
OH
O
P O
O NH
O
OBn
O
P
O NH
THF
O
P O
O
O
P O
O
O
O
O
O
-
+
-
+
-
+
O NH
-
+
4
O NH
4
4
4
18a R = n-C H
7
15
17a R = n-C H
7
15
18b R = n-C H
9
19
17b R = n-C H
9
19
18c R = n-C
18d R = n-C
H
11 23
17c R = n-C
17d R = n-C
H
11 23
H
13 27
H
13 27
Scheme 1. Synthesis of cardiolipin-ester analogues.

6992
S. M. Ali et al. / Tetrahedron 62 (2006) 6990–6997
O
O
O
O
N
+
DIPEA
O
P
N
O
CH Cl
2
2
Cl
P
OH
O
CN
10
CN
20
19
OBn
HO
OH
O
O
O
15
O
Tetrazole
35% H O , CH Cl
O
P
O
OBn
O
P
2
2
2
2
O
O
O
O
21
O
CN
CN
Et N
3
CH OH
3
O
O
O
O
O
P
O NH
OBn
O
P
O NH
O
O
O
O
-
+
-
+
4
22
4
10% Pd-C, H , 50 psi
2
THF
O
O
O
O
O
OH
O
P
O
P O
O
O
-
+
-
+
O NH
O NH
4
4
23
Scheme 2. Synthesis of cardiolipin-ether analogues.
chlorophosphoramidite 10 as the phosphitylating agent
to build a cardiolipin core structure through a phosphotriester
approach. The method is attractive because phosphorus(III)
reagents are generally more reactive than phosphorus(V)
reagents. Commercially available (R)-(+)-3-O-benzyl-1,
2-propanediol 11 was treated with octanoyl chloride in pyr-
idine at 55 ꢀC to obtain 1,2-di-O-octanoyl-3-O-benzyl-sn-
glycerol 12a. The glycerol 11 on esterification with decanoyl
chloride and triethylamine in dichloromethane afforded 1,2-
di-O-decanoyl-3-O-benzyl-sn-glycerol 12b. Debenzylation
of 12a and 12b via hydrogenation over 10% Pd–C catalyst
in ethanol/ethyl acetate/acetic acid (9:1:0.1) gave the
corresponding glycerols 13a and 13b, respectively. 1,2-Di-
O-octanoyl-sn-glycerol 13a and 1,2-di-O-decanoyl-sn-glyc-
erol 13b were reacted with the bifunctional phosphitylating
reagent 2-cyanoethyl N,N-diisopropylchlorophosphorami-
dite 10 in dichloromethane in the presence of diisopropyle-
thylamine for 1 h to afford 1,2-diacyl-sn-glycerol-N,N-
diisopropylaminophosphoramidite intermediates 14a and
14b, respectively. Similarly commercially available 1,2-di-
O-lauroyl-sn-glycerol 13c and 1,2-di-O-myristoyl-sn-glyc-
erol 13d were reacted with the bifunctional phosphitylating
reagent 10, in dichloromethane in the presence of diisopro-
pylamine, for 1 h to afford 1,2-di-O-acyl-sn-glycerol-N,N-
diisopropylaminophosphoramidite intermediates 14c and
14d, respectively. The crude products were used as such
for the next reaction without any purification. The phosphor-
amidite intermediates 14a–d on coupling with 2-benzyloxy-
1,3-propanediol 15 in the presence of 1H-tetrazole gave
phosphite triester intermediates, which was oxidized in
situ with 35% H2O2 to afford protected cardiolipin ana-
logues 16a–d, respectively. Deprotection of the cyanoethyl
group in the presence of triethylamine by b-elimination
yields benzyl protected cardiolipin analogues 17a–d.
Finally, 17a–d subjected to hydrogenolysis with Pd–C, H2,
at 50 psi in tetrahydrofuran at room temperature for 6 h
furnished short chain cardiolipins 18a, 18b, 18c, and long
chain cardiolipin 18d, respectively.
The synthesis of cardiolipin ether analogue 23 is outlined in
Scheme 2. Commercially available (R)-(+)-3-O-benzyl-
1,2-propanediol was converted in two steps into 1,2-di-O-
myristyl-sn-glycerol20 19. The bifunctional phosphitylating
reagent 2-cyanoethyl N,N-diisopropylchlorophosphor-
amidite 10 mixed with 19 in dichloromethane in the presence
of diisopropylethylamine for 2 h afforded 1,2-bis-tetradecyl-
oxy-sn-glycerol-N,N-diisopropylaminophosphoramidite in-
termediate 20, which was used as such for the next reaction
without any purification. The phosphoramidite intermediate
20, on coupling with 2-benzyloxy-1,3-propanediol 15 in the
presence of 1H-tetrazole gave phosphite triester intermedi-
ate, which was oxidized in situ with hydrogen peroxide to
produce the desired protected cardiolipin ether analogue
21. Deprotection of the cyanoethyl group of 2-O-benzyl-
1,3-bis[(1,2-di-O-myristyl-sn-glycero-3)-phosphoryl] glyc-
erol dicyanoethyl ester 21 followed by hydrogenolysis gave
cardiolipin ether analogue 23.
3. Conclusions
We used commercially available, low cost, and efficient
phosphoramidite reagent, such as 2-cyanoethyl N,N-di-
isopropylchlorophosphoramidite, to produce cardiolipin
analogues in higher yield. The 2-cyanoethyl group pro-
vided three advantages.17c First, the high reactivity of the

S. M. Ali et al. / Tetrahedron 62 (2006) 6990–6997
6993
2-cyanoethyl N,N-diisopropylchlorophosphoramidite en-
sured that a bulky primary alcohol could form efficient new
O–P bonds. Second, it has been known that the 2-cyanoethyl
phosphite derivatives were more stable to chromatography
steps than the corresponding benzyl phosphites.15c Third,
not only could the 2-cyanoethyl group serve as the source
of the 3-amino-propyl linker, but also it could be removed
by b-elimination using a base to provide high yield of the
phosphodiesters. The deprotection can be accomplished by
mild basic conditions and catalytic hydrogenolysis. The
routes are short and proceed in good overall yield.
(SiO2) Rf¼0.34 (hexane/ethyl acetate, 9:1). 1H NMR
d (CDCl3, 500 MHz) 0.87 (t, J¼7.0 Hz, 6H), 1.22–1.34 (m,
16H), 1.52–1.66 (m, 4H), 2.22–2.34 (m, 4H), 3.58 (d,
J¼4.2 Hz, 2H), 4.18 (dd, J¼6.4 and 11.9 Hz, 1H), 4.34
(dd, J¼6.4 and 11.9 Hz, 1H), 4.51 (d, J¼12.2 Hz, 1H),
4.57 (d, J¼12.2 Hz, 1H), 5.21–5.28 (m, 1H), 7.28–7.36 (m,
5H, Ar–H). 13C NMR (CDCl3, 125 MHz) d 172.2, 172.1,
137.2, 137.2, 128.5, 128.1, 128.1, 128.1, 128.0, 75.7, 75.8,
70.4, 67.4, 34.3, 34.1, 31.7, 31.6, 29.1, 29.0, 28.9, 28.9,
24.9, 22.6, 22.6, 14.0. HRMS (ESI) Calcd for C26H42O5
(M+Na+) 457.2924; found 457.2947.
4.1.2. 1,2-Di-O-decanoyl-3-O-benzyl-sn-glycerol (12b).
To an oven dried, 250 mL three-neck round bottom flask
equipped with rubber septum, argon inlet, and ice bath
were added (R)-(+)-3-O-benzyl-1,2-propanediol 11 (7.38 g,
40.51 mmol), triethylamine(18.4 g, 182.39 mmol, 24.5 mL),
and anhydrous CH2Cl2 (110 mL) with mixing. To the cooled
solution was added dropwise decanoyl chloride (10.0 g,
91.15 mmol) over 15 min, followed by the addition of 4-
(dimethylamino) pyridine (0.495 g, 4.05 mmol) with mix-
ing. The reaction mixture was stirred at room temperature
for 12 h, diluted with CH2Cl2 (200 mL), washed successively
with water (3ꢁ100 mL), and (3ꢁ100 mL) brine, dried over
anhydrous Na2SO4, and concentrated down to deep red oil.
The oil was purified as such using flash chromatography
(SiO2) and eluted with step gradient from 3 to 10%, ethyl
acetate/hexane to give 12b (6.4 g, 32%) as an oil. TLC
(SiO2) Rf¼0.34 (hexane/ethyl acetate, 9:1). 1H NMR
(CDCl3, 500 MHz) d 0.87 (t, J¼6.8 Hz, 6H), 1.22–1.34 (m,
24H), 1.52–1.66 (m, 4H), 2.22–2.34 (m, 4H), 3.58 (d,
J¼4.2 Hz, 2H), 4.18 (dd, J¼6.4 and 11.9 Hz, 1H), 4.34
(dd, J¼6.4 and 11.9 Hz, 1H), 4.51 (d, J¼12.2 Hz, 1H),
4.57 (d, J¼12.2 Hz, 1H), 5.21–5.28 (m, 1H), 7.28–7.36 (m,
5H, Ar–H). 13C NMR (CDCl3, 125 MHz) d 172.9, 172.5,
137.2, 128.4, 128.3, 127.8, 127.7, 127.6, 75.9, 75.7, 71.1,
67.3, 33.1, 33.1, 35.5, 31.0, 30.0, 30.0, 29.7, 25.4, 23.1,
14.1. HRMS (ESI) Calcd for C30H50O5 (M+Na+)
513.3555; found 513.3571.
4. Experimental
4.1. General
1H NMR spectra were recorded on a Varian Inova NMR
spectrometer at 500 MHz. H chemical shifts are reported
1
in parts per million from internal tetramethylsilane. Mass
spectral analyses [electron spray ionization (ESI)] were
carried out on a Triple Quadruple LC/MS/MS mass spec-
trometer API 4000 (Applied Biosystems). Accurate mass
measurements (HRMS–ESI) were done at University of
Minnesota on a Bruker Daltonics BioTOF II spectrometer.
Melting points were determined at atmospheric pressure
and uncorrected. Infrared (IR) spectra were recorded on
a Nicolet Nexus 470 FTIR. Samples were prepared by
ATR method. Thin layer chromatography (TLC) was carried
out on Merck silica gel 60 F254 plates (250 mm) and devel-
oped with the appropriate solvents. The TLC spots were
visualized either by UV light or by heating the plates sprayed
with a solution of phosphomolybdic acid (5% ethanolic
solution). Flash column chromatography was carried out
on silica gel (230–400 mesh). All chemicals and anhydrous
solvents purchased from Aldrich Chemical Co. (Milwaukee,
WI), except 1,2-di-O-lauroyl-sn-glycerol and 1,2-di-O-myr-
istoyl-sn-glycerol (Genzyme Pharmaceutical, Cambridge,
MA). All of the extracts were dried over anhydrous
Na2SO4. Anhydrous dichloromethane, acetonitrile, ethanol,
and tetrahydrofuran were used as such without further
drying.
4.1.3. 1,2-Di-O-octanoyl-sn-glycerol (13a). To a solution
of 1,2-dioctanoyl-3-O-benzyl-sn-glycerol 12a (5.4 g, 12.42
mmol) in EtOH/EtOAc/AcOH (9:1:0.1, 24 mL) in a pressure
vessel containing a stir bar was added palladium catalyst
(0.7 g). The reaction mixture was stirred for 18 h at 40 psi
of hydrogen before filtering the reaction over Celite to re-
move the catalyst. The solvent was removed under reduced
pressure to afford an oil. The oil was purified as such using
flash chromatography (SiO2) and eluted with a step gradient
of ethyl acetate/hexane (1:1 to 4:1). The solvents were
removed and the resulting oil was kept under high vacuo
for 4 h to afford 13a (4.0 g, 94%) as pure oil. The compound
was used as such for further reactions. TLC (SiO2) Rf¼0.51
4.1.1. 1,2-Di-O-octanoyl-3-O-benzyl-sn-glycerol (12a). To
an oven dried, 100 mL three-neck round bottom flask equip-
ped with a stir bar, condenser, heating mantle, addition
funnel, and a temperature probe under an argon atmosphere
were added (R)-(+)-3-O-benzyl-1,2-propanediol 11 (4.5 g,
24.7 mmol) and anhydrous pyridine (45 mL). The solution
was mixed moderately while octanoyl chloride (10.0 g,
61.5 mmol) was added dropwise via an addition funnel
over 15 min maintaining the reaction temperature below
40 ꢀC. To this reaction mixture was added 4-(dimethyl-
amino) pyridine (0.320 g, 2.47 mmol) all at once. The reac-
tion mixture was stirred vigorously for 48 h while refluxing
at 55 ꢀC. Then pyridine was removed under reduced pressure
to afford a deep red oil. To this oil was added ethyl acetate
(300 mL) and extracted with water (3ꢁ100 mL), 0.5 N HCl
(3ꢁ100 mL), and brine solution (3ꢁ100 mL). The organic
layer was dried over Na2SO4, filtered, and concentrated to
afford 13.5 g of oil. The oil was purified as such using flash
chromatography (SiO2) and eluted (3–10% ethyl acetate/
hexane) to afford 12a (5.4 g, 50%) as a colorless oil. TLC
1
(ethyl acetate/hexane, 1:1). H NMR (CDCl3, 500 MHz)
d 0.87 (J¼7.0 Hz, 6H), 1.22–1.34 (m, 16H), 1.52–1.66 (m,
4H), 2.12 (t, J¼6.2 Hz, 1H, –OH), 2.32 (t, J¼7.6 Hz, 2H),
2.35 (t, J¼7.6 Hz, 2H), 3.73 (t, J¼6.0 Hz, 2H), 4.22 (dd,
J¼5.8 and 11.9 Hz, 1H), 4.33 (dd, J¼5.8 and 11.9 Hz,
1H), 5.08 (quintet, J¼5.1 Hz, 1H). 13C NMR (125 MHz,
CDCl3) d 173.6, 173.3, 77.9, 67.2, 63.3, 34.2, 34.1, 31.7,
31.7, 29.1, 29.0, 28.9, 28.9, 24.9, 24.8, 22.6, 22.6, 14.0.
HRMS (ESI) Calcd for C19H35O5 (M+Na+) 367.2455; found
367.2442.

6994
S. M. Ali et al. / Tetrahedron 62 (2006) 6990–6997
4.1.4. 1,2-Di-O-decanoyl-sn-glycerol (13b). To a solution
of 1,2-di-O-decanoyl-3-O-benzyl-sn-glycerol 12b (6.4 g,
13.04 mmol) in EtOH/EtOAc/AcOH (9:1:0.1, 55 mL) was
added palladium on carbon catalyst (1.92 g). The reaction
mixture was stirred vigorously for 18 h under 40 psi of
hydrogen. The catalyst was filtered over a Celite bed, and
the solvent was removed to afford oil. The oil was purified
as such using flash chromatography (SiO2) with a step gradi-
ent of ethyl acetate/hexane (1:1 to 4:1), concentrated, and
kept under high vacuo to yield 13b (4.9 g, 94%) as a oil.
86% yield as a colorless syrup following the procedure
used for the synthesis of 16a. TLC (SiO2) Rf¼0.38
(EtOAc/CH2Cl2, 1:3). 1H NMR (CDCl3, 500 MHz)
d 0.86–0.89 (t, J¼7.0 Hz, 12H), 1.26–1.28 (m, J¼10.5 Hz,
48H), 1.58–1.61 (m, J¼6.0 Hz, 8H), 2.28–2.34 (m,
J¼2.0 Hz, 8H), 2.70–2.72 (m, J¼6.0 Hz, 4H), 3.83–3.88
(m, 1H), 4.13–4.31 (m, 16H), 4.67–4.68 (s, 2H), 5.22–5.24
(m, 2H), 7.35–7.37 (m, 5H, Ar–H). 13C NMR (CDCl3,
125 MHz) d 173.2, 173.1, 172.8, 172.7, 137.2, 137.2,
128.5, 128.1, 128.0, 127.9, 116.3, 77.2, 77.0, 76.7, 75.4,
75.3, 72.3, 69.2, 69.1, 66.0, 66.0, 65.9, 65.8, 62.2, 62.2,
62.1, 61.5, 34.1, 33.9, 31.8, 29.4, 29.2, 29.2, 29.1, 29.0,
24.8, 22.6, 19.6, 19.5, 19.4, 14.0. HRMS (ESI) Calcd for
C62H106N2O17P2 (M+Na+) 1235.6879; found 1235.6893.
1
TLC (SiO2) Rf¼0.39 (hexane/ethyl acetate, 3:2). H NMR
(CDCl3, 500 MHz) d 0.87 (t, J¼7.0 Hz, 6H), 1.22–1.34
(m, 24H), 1.52–1.66 (m, 4H), 2.03 (t, J¼6.2 Hz, 1H,
–OH), 2.32 (t, J¼7.6 Hz, 2H), 2.35 (t, J¼7.6 Hz, 2H), 3.73
(t, J¼6.0 Hz, 2H), 4.22 (dd, J¼5.8 and 11.9 Hz, 1H), 4.33
(dd, J¼5.8 and 11.9 Hz, 1H), 5.08 (quintet, J¼5.1 Hz,
1H). 13C NMR (CDCl3, 125 MHz) d 172.4, 172.3, 78.1,
67.0, 63.5, 33.9, 33.6, 32.5, 32.4, 30.3, 30.3, 30.1, 30.1,
30.0, 30.0, 29.8, 29.7, 25.4, 25.4, 23.4, 23.1, 14.0. HRMS
(ESI) Calcd for C23H44O5 (M+Na+) 423.3081; found
423.3093.
4.1.7. 2-O-Benzyl-1,3-bis[(1,2-di-O-lauroyl-sn-glycero-
3)-phosphoryl] glycerol dicyanoethyl ester (16c).
Compound 16c was synthesized from 13c in an 87% yield
as a colorless syrup by following the procedure used for the
synthesis of 16a. TLC (SiO2) Rf¼0.48 (EtOAc/CH2Cl2,
1:3). 1H NMR (CDCl3, 500 MHz) d 0.88 (t, J¼7.0 Hz,
12H), 1.25–1.28 (m, 64H), 1.58–1.60 (m, 8H), 2.28–2.34
(m, 8H), 2.66–2.72 (m, 4H), 3.83–3.88 (m, 1H), 4.05–4.34
(m, 16H), 4.66–4.67 (s, 2H), 5.22–5.24 (m, 2H), 7.35–7.36
(m, 5H). 13C NMR (CDCl3, 125 MHz) d 173.2, 172.8,
137.2, 130.8, 128.8, 128.6, 128.5, 128.2, 128.1, 128.1,
128.0, 127.9, 127.7, 116.3, 77.2, 77.0, 76.7, 75.4, 75.3,
70.6, 70.5, 69.2, 68.1, 66.0, 65.9, 62.2, 62.2, 62.1, 61.6,
61.5, 38.7, 34.1, 33.9, 31.9, 30.3, 29.6, 29.5, 29.4, 29.3,
29.2, 29.0, 29.1, 28.9, 24.8, 23.7, 22.9, 22.6, 19.6, 19.5,
14.1, 14.0. HRMS (ESI) Calcd for C64H122N2O17P2
(M+Na+) 1241.7585; found 1241.7566.
4.1.5. 2-O-Benzyl-1,3-bis[(1,2-di-O-octanoyl-sn-glycero-
3)-phosphoryl] glycerol dicyanoethyl ester (16a). To
a solution of 1,2-di-O-octanoyl-sn-glycerol 13a (4.0 g,
11.6 mmol) in anhydrous dichloromethane (30 mL) were
added diisopropylethylamine (1.65 g, 12.76 mmol) and
dropwise 2-cyanoethyl N,N-diisopropylchlorophosphor-
amidite 10 (3.02 g, 12.76 mmol) under a steady stream of
dry argon. The reaction mixture was stirred moderately at
room temperature under an argon atmosphere for 1 h. To
this stirring solution were added 1H-tetrazole (0.97 g,
13.91 mmol, 30.9 mL of 0.45 M solution in acetonitrile)
and dropwise a solution of 2-benzyloxy-1,3-propanediol
15 (0.95 g, 5.23 mmol) in anhydrous dichloromethane
(30 mL). The reaction mixture was stirred at room tempera-
ture for 3 h and cooled to ꢂ40 ꢀC while hydrogen peroxide
(0.51 g, 15.11 mmol, 1.34 mL of 35 wt % H2O2) was added
dropwise. After stirring the reaction at ꢂ40 ꢀC for 15 min,
the reaction was allowed to come to room temperature over
2 h, diluted with CH2Cl2 (200 mL), and washed with 10%
sodium thiosulfate solution (50 mL). The organic layer was
extracted with water (2ꢁ50 mL), brine (2ꢁ50 mL), then
dried over anhydrous sodium sulfate, filtered, and concen-
trated under vacuum to yield 6.7 g as a colorless syrup. The
residue was purified on a (SiO2) column using a step gradient
from 50% ethyl acetate/hexane to 100% ethyl acetate, which
gave 16a (5.6 g, 85%). TLC (SiO2) Rf¼0.35 (EtOAc/
4.1.8. 2-O-Benzyl-1,3-bis[(1,2-di-O-myristoyl-sn-glycero-
3)-phosphoryl] glycerol dicyanoethyl ester (16d).
Compound 16d was synthesized from compound 13d in an
87% yield as a colorless oil following the procedure used
for the synthesis of 16a. TLC (SiO2) Rf¼0.26 (EtOAc/
1
CH2Cl2, 1:3). H NMR (CDCl3, 500 MHz) d 0.86–0.89 (t,
J¼7.0 Hz, 12H), 1.25 (m, 80H), 1.58–1.59 (m, 8H), 2.27–
2.33 (m, 8H), 2.64–2.75 (m, 4H), 3.90 (m, 1H), 4.13–4.17
(m, 16H), 4.67 (s, 2H), 5.19–5.28 (m, 2H), 7.28–7.38 (m,
5H, Ar–H). 13C NMR (CDCl3, 125 MHz) d 173.5, 173.2,
173.2, 138.0, 128.4, 127.7, 127.6, 77.2, 77.0, 76.7, 70.3,
70.3, 66.7, 63.6, 62.7, 34.3, 34.1, 31.9, 29.7, 29.7, 29.7,
29.7, 29.6, 29.6, 29.4, 29.3, 29.2, 29.1, 29.1, 24.9, 24.9,
22.7, 14.0. HRMS (ESI) Calcd for C78H138N2O17P2
(M+Na+) 1459.9363; found 1459.9394.
1
CH2Cl2, 1:3, v/v). H NMR (CDCl3, 500 MHz) d 0.88 (t,
J¼7.0 Hz, 12H, –CH3), 1.26–1.29 (m, 32H, –CH2), 1.58–
1.62 (m, 8H), 2.28–2.34 (m, 8H), 2.66–2.73 (m, 4H), 3.85–
3.88 (m, 1H), 4.07–4.32 (m, 16H), 4.67 (s, 2H), 5.22–5.25
(m, 2H), 7.35–7.36 (m, 5H, Ar–H). 13C NMR (CDCl3,
125 MHz) d 173.2, 173.1, 172.8, 137.2, 137.1, 128.5,
128.1, 128.1, 128.1, 128.0, 127.9, 127.9, 127.9, 116.3,
77.2, 77.0, 76.7, 75.4, 75.4, 75.3, 72.3, 69.2, 69.2, 66.0,
65.9, 65.9, 62.2, 62.1, 61.5, 34.1, 33.9, 31.6, 29.0, 28.9,
28.8, 24.7, 22.5, 19.5, 19.4, 14.0. HRMS (ESI) Calcd for
C54H90N2O17P2 (M+Na+) 1123.5607; found 1123.5610.
4.1.9. 1,3-Bis[(1,2-di-O-octanoyl-sn-glycero-3)-phos-
phoryl]-2-O-benzylglycerol diammonium salt (17a). A
solution of the protected cardiolipin analogue 16a (1.95 g,
1.77 mmol), Et3N (1.36 g, 13.48 mmol), and water (0.1 mL)
in acetonitrile (10 mL) was stirred overnight. The reaction
mixture was evaporated to dryness. The residue was con-
verted into ammonium salt by adding 2 mL NH4OH and
purified on SiO2 column (15% MeOH in CH2Cl2 containing
1% NH4OH) to give 17a (606 mg, 65%) as a colorless syrup
that slowly solidified. TLC (SiO2) Rfw0.46 (CHCl3/MeOH/
1
NH4OH, 6.5:2.5:0.5). H NMR d (CDCl3, 500 MHz) 0.88
4.1.6. 2-O-Benzyl-1,3-bis[(1,2-di-O-decanoyl-sn-glycero-
3)-phosphoryl] glycerol dicyanoethyl ester (16b).
Compound 16b was synthesized from compound 13b in an
(t, J¼7.0 Hz, 12H), 1.22–1.39 (m, 32H), 1.56–1.63 (m,
8H), 2.22–2.34 (m, 8H), 3.66–3.76 (m, 1H), 3.82–4.06
(m, 8H), 4.08–4.18 (m, 2H), 4.26–4.37 (m, 2H), 4.60 (s,

S. M. Ali et al. / Tetrahedron 62 (2006) 6990–6997
6995
2H), 5.14–5.26 (m, 2H), 7.22–7.36 (m, 5H), 7.49 (br, 8H).
13C NMR (CDCl3, 125 MHz) d 173.2, 173.1, 172.8, 172.7,
128.4, 127.7, 127.6, 77.2, 77.0, 76.7, 71.4, 70.4, 63.7, 62.7,
45.8, 34.3, 34.1, 31.7, 29.1, 29.0, 28.9, 24.9, 24.8, 22.6,
14.0. HRMS (ESI) Calcd for C48H90N2O17P2 (M+2NH+4)2ꢂ
496.2519; found 496.2519.
10% Pd–C (665 mg) for 10 h. The catalyst was filtered off
over Celite bed and concentrated. The residue was dissolved
in CHCl3, filtered through a 0.25 m filter, and precipitated
with acetone to give tetraoctanoyl (C8:0) cardiolipin 18a
(0.99 g, 85%) as a white semi solid. TLC (SiO2) Rfw0.43
1
(CHCl3/MeOH/NH4OH, 6.5:2.5:0.5). H NMR d (CDCl3,
500 MHz) 0.88 (t, J¼7.0 Hz, 12H), 1.22–1.34 (br s, 32H),
1.52–1.64 (m, 8H), 2.26–2.34 (m, 8H), 3.82–3.98 (m, 9H),
4.12–4.18 (m, 2H), 4.35–4.42 (m, 2H), 5.14–5.24 (m, 2H),
7.41 (br, 8H). 13C NMR (CDCl3, 125 MHz) d 173.6,
173.3, 77.2, 77.0, 76.7, 62.6, 34.2, 34.1, 31.7, 31.6, 29.1,
29.0, 28.9, 24.8, 22.6, 22.5, 14.0. FTIR (ATR) 3250, 3015,
4.1.10. 1,3-Bis[(1,2-di-O-decanoyl-sn-glycero-3)-phos-
phoryl]-2-O-benzylglycerol diammonium salt (17b).
Compound 17b was synthesized from 16b in an 88% yield
as a colorless syrup following the procedure used for the syn-
thesis of 17a. TLC (SiO2) Rf¼0.47 (CHCl3/MeOH/NH4OH,
65:25:5, v/v). 1H NMR (CDCl3, 500 MHz) d 0.86–0.89
(t, J¼7.0 Hz, 12H), 1.25–1.28 (m, J¼16 Hz, 48H), 1.56
(m, 8H), 2.24–2.30 (m, J¼3.0 Hz, 8H), 3.72 (m, 1H),
3.89–4.00 (m, J¼6.5 Hz, 8H), 4.10–4.14 (m, J¼4.0 Hz,
2H), 4.32–4.34 (m, 2H), 4.61 (s, 2H), 5.19 (m, 2H), 7.24–
7.33 (m, 5H, Ar–H), 7.55 (br, 8H). 13C NMR (CDCl3,
125 MHz) d 173.5, 173.2, 138.1, 128.3, 127.6, 77.2, 77.0,
76.7, 71.5, 70.4, 70.3, 63.5, 63.4, 62.7, 45.7, 34.2, 34.0,
31.8, 29.5, 29.4, 29.3, 29.1, 24.9, 24.8, 22.6, 14.0. HRMS
(ESI) Calcd for C56H106N2O17P2 (Mꢂ2NH+4)2ꢂ 552.3145;
found 552.3167.
2945, 2922, 2840, 1746, 1473, 1371, 1198, 1082, 1069 cmꢂ1
.
HRMS (ESI) Calcd for C41H84N2O17P2 (Mꢂ2NH+4)2ꢂ
451.2324; found 451.2266.
4.1.14. 1,3-Bis[(1,2-di-O-decanoyl-sn-glycero-3)-phos-
phoryl] glycerol diammonium salt (18b, tetradecanoyl
cardiolipin diammonium salt). Compound 18b was syn-
thesized from 17b in a 97% yield as a white solid by follow-
ing the procedure used for the synthesis of 18a. Mp
177–179 ꢀC. TLC (SiO2) Rf¼0.38 (CHCl3/MeOH/NH4OH,
65:25:5, v/v). 1H NMR (CDCl3, 500 MHz) d 0.88 (t,
J¼7.0 Hz, 12H), 1.26 (br s, 48H), 1.58–1.59 (m, J¼6.0 Hz,
8H), 2.27–2.33 (m, J¼7.0 Hz, 8H), 3.62 (br, 1H), 3.90–
3.91 (m, J¼4.5 Hz, 9H), 4.13–4.17 (m, J¼7.0 Hz, 2H),
4.35–4.37 (m, J¼10 Hz, 2H), 5.19–5.20 (m, J¼2.0 Hz,
2H), 7.44 (br, 8H, NH+4). 13C NMR (CDCl3, 125 MHz)
d 173.6, 173.3, 77.2, 77.0, 76.7, 62.6, 34.3, 34.1, 31.9,
31.8, 29.5, 29.3, 29.2, 29.1, 24.2, 24.8, 22.6, 14.0. FTIR
(ATR) 3228, 3022, 2946, 2914, 2850, 1744, 1466, 1387,
4.1.11. 1,3-Bis[(1,2-di-O-lauroyl-sn-glycero-3)-phos-
phoryl]-2-O-benzylglycerol diammonium salt (17c).
Compound 17c was synthesized from 16c in an 89% yield
as a colorless syrup by following the procedure used for
the synthesis of 17a. TLC (SiO2) Rf¼0.34 (CHCl3/MeOH/
1
NH4OH, 65:25:5). H NMR (CDCl3, 500 MHz) d 0.86–
0.89 (t, J¼7.0 Hz, 12H), 1.25–1.30 (m, J¼7.5 Hz, 64H),
1.56 (m, 8H), 2.24–2.29 (m, J¼4.0 Hz, 8H), 3.70–3.72 (m,
1H), 3.89–3.91 (m, 8H), 4.09–4.14 (m, 2H), 4.32–4.34 (m,
2H), 4.61 (s, 2H), 5.17–5.20 (m, 2H), 7.23–7.33 (m, 5H,
Ar–H), 7.25 (br s, 8H, NH+4). 13C NMR (CDCl3, 125 MHz)
d 173.5, 173.2, 138.0, 128.3, 127.7, 127.6, 77.2, 77.0,
76.7, 76.2, 76.1, 71.5, 70.4, 70.3, 63.6, 63.5, 62.7, 34.3,
35.0, 31.9, 29.7, 29.6, 29.5, 29.3, 29.3, 29.2, 29.1, 24.9,
24.8, 22.6, 14.0. HRMS (ESI) Calcd for C64H122N2O17P2
(Mꢂ2NH+4)2ꢂ 609.3821; found 609.3831.
1212, 1095, 1070 cmꢂ1
.
HRMS (ESI) Calcd for
C49H100N2O17P2 (Mꢂ2NH+4)2ꢂ 507.2962; found 507.2953.
4.1.15. Synthesis of 1,3-bis[(1,2-di-O-lauroyl-sn-glycero-
3)-phosphoryl] glycerol diammonium salt (18c, tetra-
lauroyl cardiolipin diammonium salt). Compound 18c
was synthesized from 17c in a 98% yield as a white solid
by following the procedure used for the synthesis of 18a.
Mp 156–158 ꢀC. TLC (SiO2) Rf¼0.31 (CHCl3/MeOH/
1
NH4OH, 65:25:5). H NMR (CDCl3, 500 MHz) d 0.86–
4.1.12. 1,3-Bis[(1,2-di-O-myristoyl-sn-glycero-3)-phos-
phoryl]-2-O-benzylglycerol diammonium salt (17d).
Compound 17d was synthesized from compound 16d in an
85% yield as a colorless oil following the procedure used
for the synthesis of 17a. TLC (SiO2) Rf¼0.54 (CHCl3/
MeOH/NH4OH, 65:25:5). 1H NMR (CDCl3, 500 MHz)
d 0.86–0.89 (t, J¼7.0 Hz, 12H), 1.25–1.31 (m, 80H), 1.56
(m, 8H), 2.23–2.30 (m, 8H), 3.71 (m, 1H), 3.90–3.98 (m,
8H), 4.10–4.14 (m, 2H), 4.31–4.34 (m, 2H), 4.60 (s, 2H),
5.18–5.20 (m, 2H), 7.24–7.32 (m, 5H, Ar–H), 7.51 (br, 8H,
NH+4). 13C NMR (CDCl3, 125 MHz) d 173.5, 173.2, 138.0,
128.4, 127.7, 127.6, 77.2, 77.0, 76.7, 71.5, 70.4, 70.3, 63.5,
62.7, 34.3, 34.0, 31.9, 29.7, 29.7, 29.6, 29.5, 29.4, 29.4,
29.3, 29.3, 29.2, 29.1, 24.0, 24.8, 22.7, 22.6, 14.0. HRMS
(ESI) Calcd for C72H138N2O17P2 (Mꢂ2NH+4)2ꢂ 665.4447;
found 665.4443.
0.89 (t, J¼7.0 Hz, 12H), 1.26 (m, 64H), 1.58–1.59 (m,
8H), 2.27–2.33 (m, 8H), 3.90–3.92 (m, 9H), 4.13–4.17 (m,
2H), 4.36–4.38 (m, 2H), 5.20–5.21 (m, 2H), 7.41 (br, 8H,
NH+4). 13C NMR (CDCl3, 125 MHz) d 173.6, 173.3, 167.7,
132.4, 130.8, 128.8, 70.3, 70.3, 69.5, 68.1, 66.6, 63.6,
63.5, 62.7, 38.7, 34.3, 34.1, 31.9, 30.3, 29.7, 29.6, 29.4,
29.3, 29.2, 28.9, 24.9, 24.8, 23.7, 22.9, 22.6, 14.0. FTIR
(ATR) 3207, 3035, 2956, 2918, 2850, 1737, 1467, 1378,
1206, 1092, 1067 cmꢂ1
.
HRMS (ESI) Calcd for
C57H116N2O17P2 (Mꢂ2NH+4)2ꢂ 563.3553; found 563.3540.
4.1.16. Synthesis of 1,3-bis[(1,2-di-O-myristoyl-sn-glyc-
ero-3)-phosphoryl] glycerol diammonium salt (18d, tet-
ramyristoyl cardiolipin diammonium salt). Compound
18d was synthesized from compound 17d in a 98% yield
as a white solid following the procedure used for the synthe-
sis of 18a. Mp 181–182 ꢀC. TLC (SiO2) Rf¼0.29 (CHCl3/
MeOH/NH4OH, 65:25:5). 1H NMR (CDCl3, 500 MHz)
d 0.86–0.89 (t, J¼7.0 Hz, 12H, –CH3), 1.25 (br, 80H,
–CH2–), 1.58–1.59 (m, 8H), 2.27–2.33 (m, 8H), 3.06 (br,
1H), 3.90 (m, 9H), 4.13–4.17 (m, 2H), 4.36–4.38 (m, 2H),
5.20–5.21 (m, 2H), 7.41 (br, 8H, NH+4). 13C NMR (CDCl3,
4.1.13. 1,3-Bis[(1,2-di-O-octanoyl-sn-glycero-3)-phos-
phoryl] glycerol diammonium salt (18a, tetraoctanoyl
cardiolipin diammonium salt). A solution of benzyl
protected cardiolipin analogue 17a (1.33 g, 1.29 mmol) in
tetrahydrofuran (15 mL) was hydrogenated at 50 psi over

6996
S. M. Ali et al. / Tetrahedron 62 (2006) 6990–6997
125 MHz) d 173.6, 173.3, 77.2, 77.0, 76.7, 70.34, 70.3, 66.7,
63.6, 62.7, 34.3, 34.1, 31.9, 29.7, 29.6, 29.4, 29.2, 29.1, 24.9,
24.8, 22.7, 14.0. FTIR (ATR) 3207, 3035, 2956, 2918, 1737,
1461, 1378, 1206, 1092, 1067 cmꢂ1. HRMS (ESI) Calcd for
C65H132N2O17P2 (Mꢂ2NH+4)2ꢂ 619.4199; found 619.4173.
3.98 (br, 4H), 4.60 (s, 2H), 7.28–7.31 (m, 5H, Ar–H), 7.56
(br s, 8H). 13C NMR (CDCl3, 125 MHz) d 128.3, 127.6,
77.2, 77.0, 76.7, 71.8, 71.4, 70.6, 31.9, 30.10, 29.8, 29.7,
29.7, 29.6, 29.4, 26.1, 22.7, 14.1. HRMS (ESI) Calcd for
C72H146N2O13P2 (Mꢂ2NH4+)2ꢂ 637.4901; found 637.4901.
4.1.17. Synthesis of 2-O-benzyl-1,3-bis[(1,2-di-O-myris-
tyl-sn-glycero-3)-phosphoryl] glycerol dicyanoethyl ester
(21). To a 250-mL three-neck round bottom flask equipped
with stir bar, rubber septum, and argon inlet was added
1,2-di-O-myristyl-sn-glycerol19 19 (9.2 g, 19.01 mmol) in
anhydrous dichloromethane (150 mL). To the mixture was
added diisopropylethylamine (5.46 g, 42.26 mmol) and
stirred for 10 min. To this mixture was added dropwise 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite 10 (5.0 g,
21.13 mmol), and the reaction mixture was stirred moder-
ately at room temperature. The reaction mixture was stirred
for 2 h before adding 1H-tetrazole (1.59 g, 22.77 mmol of
0.45 M solution in acetonitrile) and dropwise a solution of
2-benzyloxy-1,3-propanediol 15 (1.55 g, 8.54 mmol) in
anhydrous dichloromethane (20 mL). The reaction mixture
was stirred for 3 h, and then cooled to ꢂ20 ꢀC while
35 wt % H2O2 (0.42 g, 12.38 mmol, 1.09 mL) was added
dropwise. After stirring the reaction mixture at ꢂ20 ꢀC for
15 min, the reaction was allowed to come to room tempera-
ture over 2 h, diluted with CH2Cl2 (200 mL), and washed
with 10% sodium thiosulfate solution (50 mL). The organic
layer was extracted with water (2ꢁ50 mL), brine (2ꢁ
50 mL), dried over anhydrous Na2SO4, filtered, and concen-
trated under vacuum to afford a colorless syrup. The residue
was purified as such by flash chromatography on a (SiO2) col-
umn using a step gradient from 50 to 70% ethyl acetate/petro-
leum ether to yield 21 (7.1 g, 61%). TLC (SiO2) Rf¼0.27
4.1.19. 1,3-Bis[(1,2-di-O-myristyl-sn-glycerol-3)-phos-
phoryl] glycerol diammonium salt (23, tetradecyloxy
cardiolipin diammonium salt). To a solution of 2-O-ben-
zyl-1,3-bis-[(1,2-di-O-myristyl-sn-glycero-3)-phosphoryl]-
2-O-benzylglycerol diammonium salt 22 (1.2 g, 0.91 mmol)
in tetrahydrofuran (30 mL) in a pressure vessel, was added
palladium on carbon catalyst (600 mg). The reaction mix-
ture was stirred under 50 psi of hydrogen at room tempera-
ture for 6 h. The palladium catalyst was filtered over
Celite, washed with chloroform/methanol (1:1, 100 mL),
and concentrated under reduced pressure to afford white
solids. The product was purified on a flash chromatography
(SiO2) column using a step gradient of (CHCl3/MeOH/
NH4OH, 100:15:1, v/v) and (CHCl3/MeOH/NH4OH,
65:15:1, v/v) to yield 23 (1.1 g, 98%). Mp 174–176 ꢀC.
TLC (SiO2) Rf¼0.34 (CHCl3/MeOH/NH4OH, 65:25:5,
v/v). 1H NMR (CDCl3, 500 MHz) d 0.86–0.89 (t,
J¼7.0 Hz, 12H), 1.25–1.27 (br, 80H), 1.31 (br, 8H), 1.54–
1.55 (m, 8H), 3.42–3.43 (m, 4H), 3.53–3.54 (m, 4H),
3.57–3.58 (m, 4H), 3.85 (br, 4H), 7.43 (br s, 8H). 13C
NMR (CDCl3, 125 MHz) d 77.2, 77.0, 76.7, 71.8, 31.9,
30.0, 29.8, 29.8, 29.7, 29.6, 29.4, 26.2, 26.1, 22.7, 14.1.
HRMS (ESI) Calcd for C65H140N2O13P2 (Mꢂ2NH+4)2ꢂ
591.4613; found 591.4597.
Acknowledgements
1
(ethyl acetate/hexane, 3:1). H NMR (CDCl3, 500 MHz)
d 0.86–0.89 (t, J¼7.0 Hz, 12H), 1.25–1.31 (br, J¼7.5, 4.5,
and 9.5 Hz, 80H), 1.53–1.54 (m, 8H), 2.23–2.27 (m, 8H),
2.70–2.71 (m, 4H), 3.01 (m, 1H), 4.05–4.11 (m, 4H), 4.16–
4.23 (m, 4H), 4.24–4.30 (m, 4H), 4.61–4.63 (d, 2H), 4.67–
4.69 (m, 2H), 7.34–7.36 (m, 5H, Ar–H). 13C NMR
(125 MHz, CDCl3) d 128.5, 128.4, 128.0, 127.9, 127.8,
77.5, 77.2, 77.0, 76.7, 72.1, 71.8, 70.7, 70.6, 69.3, 69.3,
67.7, 61.9, 61.8, 60.8, 31.9, 30.0, 29.9, 29.6, 29.5, 29.4,
29.3, 26.0, 22.6, 19.5, 19.45, 14.0. HRMS (ESI) Calcd for
C78H146N2O13P2 (M+Na+) 1404.0197; found 1404.0234.
We thank the Bioanalytical group at NeoPharm for the Mass
spectral analyses.
References and notes
1. Tryell, A.; Heath, T. D.; Colley, C. M.; Ryman, B. E. Biochem.
Biophys. Acta 1976, 457, 259–302.
2. Grunner, S. M.; Jain, M. H. Biochem. Biophys. Acta 1985,
352–355.
4.1.18. 2-O-Benzyl-1,3-bis[(1,2-di-O-myristyl-sn-glyc-
erol-3)-phosphoryl] glycerol diammonium salt (22). To
a (250 mL) round bottom flask were added 2-O-benzyl-1,3-
bis[(1,2-di-O-myristyl-sn-glycero-3)-phosphoryl] glycerol
dicyanoethyl ester 21 (3.48 g, 2.51 mmol) and anhydrous
acetonitrile (50 mL). The solution was mixed vigorously
while triethylamine (2.18 g, 21.52 mmol, 3.0 mL) was
added. The reaction mixture was stirred vigorously for 24 h
at room temperature. The solvents were removed under
reduced pressure, and the crude oil was kept under high
vacuo for 4 h. The residue was purified by flash chromato-
graphy on a (SiO2) column using a step gradient of
(CHCl3/MeOH/NH4OH, 100:15:1, v/v) and (CHCl3/
MeOH/NH4OH, 65:15:1, v/v) to yield 22 (2.8 g, 88%).
TLC (SiO2) Rf¼0.43 (CHCl3/MeOH/NH4OH, 65:25:5, v/v).
1H NMR (CDCl3, 500 MHz) d 0.86–0.89 (t, J¼7.0 Hz, 12H),
1.25 (br, 80H), 1.52 (m, 8H), 3.00–3.02 (d, 1H), 3.39–3.44
(m, 4H), 3.49–3.51 (d, 4H), 3.56 (m, 4H), 3.83 (m, 6H),
3. Gokhale, P. C.; Zhang, C.; Newsome, J. T.; Pei, J.; Ahmad, I.;
Rahman, A.; Dritschilo, A.; Kasid, U. N. Clin. Cancer Res.
2002, 8, 3611–3621.
4. Drummond, D. C.; Meyer, O.; Hong, K.; Kirpotin, D. B.;
Papahadjopoulos, D. Pharmacol. Rev. 1999, 51, 691–744.
5. (a) Ramirez, F.; Ioannou, P. V.; Marecek, J. F.; Golding, B. T.;
Dodd, G. H. Synthesis 1976, 11, 769–770; (b) Duralski, A. A.;
Spooner, P. J. R.; Rankin, S. E.; Watts, A. Tetrahedron Lett.
1998, 39, 1607–1610; (c) Saunders, R. M.; Schwarz. J. Am.
Chem. Soc. 1966, 88, 3844–3847; (d) Mishina, I. M.;
Vasilenko, A. E.; Stepanov, A. E.; Shvets, V. I. Bioorg. Khim.
1985, 11, 992–994; (e) Stepanov, A. E.; Makarova, I. M.;
Shvets, V. I. Zh. Org. Khim. 1984, 20, 985–988.
6. (a) De haas, G. H.; Bonsen, P. P. M.; VanDeeen, L. L. M.
Biochim. Biophys. Acta 1966, 116, 114–124; (b) Inoue, K.;
Suhara, Y.; Nojima, S. Chem. Pharm. Bull. 1963, 1150–1156.
7. Murakami, K.; Molitor, E. J.; Liu, H. W. J. Org. Chem. 1999,
64, 648–651.

S. M. Ali et al. / Tetrahedron 62 (2006) 6990–6997
6997
8. Dreef, C. E.; Elie, C. J. J.; Hoogerhout, P.; Van Der Marel,
G. A.; Van Boom, J. H. Tetrahedron Lett. 1988, 29, 6513–
6516.
9. (a) Browne, J. E.; Driver, M. J.; Russel, J. C.; Sammes, P. G.
J. Chem. Soc., Perkin Trans. 1 2000, 653–657; (b) Gu, Q. M.;
Prestwich, G. D. J. Org. Chem. 1996, 61, 8642–8647.
10. Wilk, A.; Srinivasachar, K.; Beaucage, S. J. Org. Chem. 1997,
62, 6712–6713.
D. S.; Jesper, W. J. Org. Chem. 2000, 65, 5161–5166;
(c) Ulf, D.; Claudia, M. B. Bioorg. Med. Chem. Lett. 2000,
10, 1417–1420; (d) Evelyne, M.; Didier, G.; Michel, J.;
Anthony, R.; Jean, C. Tetrahedron 2000, 56, 8689–8701; (e)
Elisabeth, M.; Ewa, B.; Peter, S. Tetrahedron 2000, 56,
1475–1484; (f) Makoto, N.; Satoshi, S.; Akira, M. J. Org.
Chem. 2000, 65, 5016–5021; (g) Makiko, K.; Yoshihiti, U.;
Satoshi, S.; Akira, M. J. Am. Chem. Soc. 2000, 122, 2422–
2432; (h) Mark, T. T.; Mark, W. G. J. Org. Chem. 2000, 65,
5355–5359.
11. Watanabe, Y.; Inada, E.; Jinno, M.; Ozaki, S. Tetrahedron Lett.
1993, 67, 194–199.
12. Watanabe, Y.; Ishikawa, H. Tetrahedron Lett. 2000, 41, 8509–
8512.
13. Lindberg, J.; Ekeroth, J.; Konradsson, P. J. Org. Chem. 2002,
67, 194–199.
14. Jaeseung, K.; Jun, M. G.; Marc, M. G. Angew. Chem. 2003,
115, 6062–6065.
15. (a) Prestwich, G. D.; Marecek, J. F.; Mourey, R. J.; Thiebert,
A. B.; Ferris, C. D.; Danoff, S. K.; Snyder, S. H. J. J. Am.
Chem. Soc. 1991, 113, 1822–1825; (b) Chen, J.; Profit,
A. A.; Prestwich, G. D. J. Org. Chem. 1996, 61, 6305–6312;
(c) Prestwich, G. D. Acc. Chem. Res. 1996, 29, 503–513.
16. (a) Yoshihito, U.; Naoko, K.; Akira, M. Bioconjugate Chem.
2000, 11, 933–940; (b) Anders, E. H.; Alexei, A. K.; Mads,
17. (a) Chevallier, J.; Sakai, N.; Robert, F.; Kobayashi, J. G.;
Matile, S. Org. Lett. 2000, 13, 1859–1861; (b) Jiang, G.; Xu,
Y.; Falguleres, T.; Gruenberg, J.; Prestwich, G. D. Org. Lett.
2006, 7, 3837–3840; (c) Jiang, G.; Xu, Y.; Prestwich, G. D.
J. Org. Chem. 2006, 71, 934–939.
18. (a) Krishna, U. N.; Ahmad, M. U.; Ahmad, I. Tetrahedron Lett.
2004, 45, 2077–2079; (b) Krishna, U. M.; Ahmad, M. U.; Ali,
S. M.; Ahmad, I. Lipids 2004, 39, 595–600.
19. (a) Lin, Z.; Ahmad, M. U.; Ali, S. M.; Ahmad, I. Lipids 2004,
39, 285–290; (b) Lin, Z.; Ahmad, M. U.; Ali, S. M.; Ahmad, I.
Tetrahedron Lett. 2004, 45, 6923–6925.
20. Krishnudu, K.; Ahmad, M. U.; Ali, S. M.; Ahmad, I.
Tetrahedron Lett. 2004, 45, 2743–2746.