Synthesis of novel phosphatidylcholine lipids with fatty acid chains bearing aromatic units. Generation of oxidatively stable, fluid phospholipid membranes

Article abstract of DOI:10.1016/S0040-4039(02)00758-X

The first examples of diacylphospholipid analogs, which contain aromatic units at various depths of their hydrocarbon chains, have been synthesized. The membranes produced from the sonicated aqueous suspensions of these newly synthesized lipids are oxidatively stable and possess long shelf life. These membranes maintain fluid character at ambient temperature making them ideal for membrane protein reconstitution studies.

Full text of DOI:10.1016/S0040-4039(02)00758-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 4203–4206
Synthesis of novel phosphatidylcholine lipids with fatty acid
chains bearing aromatic units. Generation of oxidatively stable,
fluid phospholipid membranes
Santanu Bhattacharya* and Marappan Subramanian
Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India
Received 5 February 2002; revised 9 April 2002; accepted 17 April 2002
Abstract—The first examples of diacylphospholipid analogs, which contain aromatic units at various depths of their hydrocarbon
chains, have been synthesized. The membranes produced from the sonicated aqueous suspensions of these newly synthesized lipids
are oxidatively stable and possess long shelf life. These membranes maintain fluid character at ambient temperature making them
ideal for membrane protein reconstitution studies. © 2002 Elsevier Science Ltd. All rights reserved.
Glycerophospholipids are important structural compo-
nents of the bilayer membranes that constitute the
cellular organelles.1 Their lipophilic parts are typically
composed of fatty acid chains, which are linked to the
glycerol backbones via ester or ether functionalities.2
Due to their importance in the investigations of the
structure and function of biological membranes, a num-
ber of phospholipid analogues have been synthesized
incorporating modified lipophilic chains. These include
ones containing sulfur-substituted fatty acids,3a vicinal-
chain tethered macrocyclic phospholipids,3b–d chains
incorporating fluorenyl residues,3e or chains with mono-
acetylenic residues,3f or with terminal acryloyl groups,3g
and phospholipids with methyl-substituted chains,3h,i
among others. In addition, some phospholipid ana-
logues exhibit significant antifungal,4a antitumor,4b
antihypertensive,4c anti-inflammatory4d properties.
Consequently, there have been many attempts to find
novel routes to the synthesis of various phospholipid
derivatives.5
Phospholipids bearing unsaturated fatty acid chains are
particularly useful in the reconstitution of membrane
proteins in vitro.6 However, in order to keep the
protein functional, it is necessary to maintain the mem-
brane in its functionally fluid state with significant
dynamic disorder. Lipophilic chains with olefinic unsat-
uration help maintain the membrane in a fluidized
state, which is necessary for optimal membrane bound
protein activity and its function of signal transduction
* Corresponding author and Swarnajayanti Fellow (DST); e-mail: sb@orgchem.iisc.ernet.in
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00758-X

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S. Bhattacharya, M. Subramanian / Tetrahedron Letters 43 (2002) 4203–4206
across bilayers. Phospholipids containing naturally
occurring unsaturated fatty acids bearing cis-double
bonds such as oleic acid (cis-9-octadecenoic acid),
linoleic acid (cis,cis-9,12-octadecadienoic acid), or
arachidonic acid (cis,cis,cis,cis-5,8,11,14-eicosatetra-
enoic acid) are generally used for membrane protein
reconstitution. However, the vesicular membranes gen-
erated from such phospholipids, suffer from easy lipid
peroxidation7 limiting their potential for protein recon-
stitution studies. In addition, these lipids are photo-
chemically sensitive, restricting the shelf life and
applicability of the resulting vesicular formulations
even further.
hexanophenone in 58% yield. This upon reduction
under Huang–Minlon conditions gave n-hexyl benzene,
6, which was isolated in ꢀ66% yield as a colorless
liquid, upon distillation under reduced pressure. The
n-hexyl benzene was then subjected to acylation with
methyl adipoyl chloride in the presence of anhydrous
AlCl3 in dry carbon disulfide under refluxing conditions
to afford 7 in ꢀ55% yield. The ketoester, 7 was then
converted to 6-(4-n-hexylphenyl)hexanoic acid, 8 in
ꢀ60% yield first upon reduction (under reflux) with
NH2NH2/KOH in ethylene glycol followed by workup
and acidification. The synthesis of 12-phenyl-dode-
canoic acid, 5 also began with acylation of benzene
with methyl 12-chloro-12-oxo dodecanoate in the pres-
ence of anhydrous AlCl3. Work up afforded the keto
acid, 4 in 58% yield. Then the keto group in 4 was
selectively reduced again under Huang–Minlon condi-
tions to furnish 5 in ꢀ70% yield. For the synthesis of
11-(4-methylphenyl)undecanoic acid, 9, toluene was
subjected to alkylation using ethyl 11-bromounde-
canoate under Friedel–Crafts conditions. Workup fol-
lowed by saponification and acidification afforded 9 in
ꢀ57% isolated yield.
One way to alleviate such difficulties is to design phos-
pholipids with chains bearing oxidatively stable unsatu-
ration. Unlike their olefinic counterparts, aromatic
systems possess far greater chemical stability, especially
para-substituted aromatic rings (e.g. in 1 and 2) which
are generally metabolically stable to biological oxida-
tion. As a part of our ongoing chemical biology
program8 focused on optimizing lipid lead structures
that would offer suitable membranes for reconstitution
of membrane proteins at ambient conditions, we
became interested in achieving syntheses of a series of
phospholipids which incorporates aromatic units in
their hydrophobic segments. Incorporation of such aro-
matic units into hydrocarbon chains leads to sponta-
neous fluidization of the resulting membranes. In this
paper we describe a convenient synthesis of three novel
phospholipid analogues (1–3), in which unsaturation in
the form of aromatic units forms part of the acyl chains
at various locations along the chains.
The key starting material for the synthesis of phospho-
lipid, free sn-glycerophosphocholine was obtained9
from natural lecithin, which was first extracted from
egg yolk and then subjected to hydrolysis using 0.1 M
Bu4N+·OH− in MeOH to afford free sn-glycerophos-
phocholine [(R)-GPC] after chromatographic purifica-
tion. The resulting material was then converted to a
complex with CdCl2 (GPC·CdCl2) to render it soluble
in CHCl3. Finally the syntheses of the desired phospho-
lipids from the newly synthesized fatty acids were
accomplished by full acylation of GPC·CdCl2 with 2.2
mol of the respective anhydride of the appropriate fatty
acid containing aromatic moieties in ethanol-free dry
CHCl3 in the presence of 4-dimethylaminopyridine
(DMAP). This is summarized in Scheme 2. All the
The synthesis of the target phospholipids began with
the preparation of fatty acids with aromatic units incor-
porated at specified locations of the hydrocarbon chain
(Scheme 1). Friedel–Crafts acylation of benzene with
n-hexanoyl chloride in the presence of anhydrous
AlCl3, under refluxing conditions for ꢀ10 h afforded
Scheme 1. Reagents, conditions and yields: (a) MeO2C(CH2)10COCl, anhyd. AlCl3, in benzene, 4°C (1 h)“rt (12 h) workup in dil.
ice-cold HCl, 58%; (b) NH2NH2–KOH, ethylene glycol, reflux, 4 h; (c) H3O+, ice-water, 70%; (d) caproyl chloride, anhyd. AlCl3,
reflux, 10 h, 58%; (e) NH2NH2–KOH, ethylene glycol, reflux, 4 h, 66%; (f) MeO2C(CH2)4COCl, anhyd. AlCl3, CS2, reflux, 12 h,
55%; (g) NH2NH2–KOH, ethylene glycol, reflux, 4 h, 66%; (h) H3O+, ice-water, 90%; (i) ethyl 11-bromoundecanoate, anhyd.
AlCl3, rt, 4 h; HCl-ice water, 60%; (j) 10% KOH–MeOH, reflux, 6 h; H3O+–ice water, 95%.

S. Bhattacharya, M. Subramanian / Tetrahedron Letters 43 (2002) 4203–4206
4205
Scheme 2. Reagents, conditions and yields: (a) i. RCO2H (1 equiv.) Et3N, −20°C, 1 h; add ClCO2Et–THF, stir at −20°C, 2 h, then
at rt, 1 h, ii cool to −20°C, add a solution of RCO2H (1 equiv.)/Et3N in THF −20°C, 1 h; stir overnight at rt, (isolated yields:
90–95%); (b) DMAP, GPC·CdCl2, CHCl3, stir at rt, 48 h (isolated yields: 72, 55, 63% for 1–3, respectively).
numbered intermediates and final products were char-
3. (a) Li, R.; Pascal, R. A. J. Org. Chem. 1993, 58, 1952; (b)
Ladika, M.; Fisk, T. E.; Wu, W. W.; Jons, S. D. J. Am.
Chem. Soc. 1994, 116, 12093; (c) Menger, F. M.; Chen,
X. Y. Tetrahedron Lett. 1996, 37, 323; (d) Patwardhan,
A. P.; Thompson, D. H. Org. Lett. 1999, 1, 241; (e)
Starck, J.-P.; Nakatani, Y.; Ourisson, G. Tetrahedron
1995, 51, 2629; (f) Ru¨rup, J.; Mannova, M.; Brezesinski,
G.; Schmid, R. D. Chem. Phys. Lipids 1994, 70, 187; (g)
Sisiri, W.; Lee, Y.-S.; O’Brien, D. F. Tetrahedron Lett.
1995, 36, 8945; (h) Menger, F. M.; Wood, M. G.; Zhou,
Q. Z.; Hopkins, H. P.; Fumero, J. J. Am. Chem. Soc.
1988, 110, 6804; (i) Morr, M.; Fortkamp, J.; Ru¨he, S.
Angew Chem., Int. Ed. Engl. 1997, 36, 2460.
4. (a) Lu, Q.; Ubillas, R. P.; Zhou, Y.; Dubenko, L. G.;
Dener, J. M.; Litvak, J.; Phuan, P.-W.; Flores, M.; Ye,
Z.; Gerber, E. Y.; Truong, T.; Bierer, D. E. J. Nat. Prod.
1999, 62, 824; (b) Hilgard, P.; Klenner, T.; Stekar, J.;
Unger, C. Cancer Chemother. Pharmacol. 1993, 32, 90; (c)
Wissner, A.; Schaub, R. E.; Sum, P. E.; Kohler, C. A.;
Goldstein, B. M. J. Med. Chem. 1986, 29, 328; (d) Shen,
T. Y. J. Med. Chem. 1981, 24, 1.
1
acterized by IR, H, 13C NMR spectroscopy and ele-
mental analysis.10
Brief sonic dispersal, or vortex mixing (10 min) above
50°C, of the thin film prepared from each of the above
phospholipid in HEPES (5 mM) buffer containing 1
mM EDTA at pH 7.4 afforded stable, translucent
aqueous suspensions. The existence of multi-walled
vesicular microstructures (MLVs) was evident from
negative stain transmission electron microscopy studies
(not shown) with each of these samples. Differential
scanning calorimetric examination of these membranes
spanning 5–90°C did not show the presence of any peak
due to main-chain melting transition. Inclusion of any
of these lipids with dipalmitoyl phosphatidylcholine
(DPPC), however, reduced the main chain melting tran-
sition of the DPPC by several degrees depending on the
mol% of the synthetic phospholipids incorporated.
Taken together it is evident that these newly synthe-
sized phospholipids retain a fluid like melted state
(liquid-crystalline like state) at ambient temperatures in
their membranes.
5. (a) Martin, S. F.; Josey, J. A.; Wong, Y.-L.; Dean, D. W.
J. Org. Chem. 1994, 59, 4805; (b) Erukulla, R. K.; Byun,
H. S.; Bittman, R. Tetrahedron Lett. 1994, 35, 5783; (c)
Garigapati, V. R.; Roberts, M. F. Tetrahedron Lett. 1993,
34, 769; (d) Kazi, A. B.; Hajdu, J. Tetrahedron Lett. 1992,
33, 2291; (e) Herbert, N.; Beck, A.; Lennox, R. B.; Just,
G. J. Org. Chem. 1992, 57, 1777.
In summary, a convenient, general and adaptable
method has been developed for the synthesis of three
novel phospholipid analogues bearing acyl chains with
unsaturations, which form the first examples of this
class of compounds that are oxidatively stable. Such
synthetic phospholipids will become valuable tools for
both standard and innovative experiments in the fields
of membrane receptor research, enzymology and
bioseparation techniques. Work is now underway in
our laboratory toward the protein reconstitution stud-
ies with these stable phospholipid membranes.
6. Bhattacharya, S.; Marti, T.; Otto, H.; Heyn, M. P.;
Khorana, H. G. J. Biol. Chem. 1992, 267, 6757.
7. Gilbertson, S. M.; Porter, N. A. J. Am. Chem. Soc. 2000,
122, 4032.
8. For representative examples of other lipid design, see: (a)
Bhattacharya, S.; De, S. Chem. Eur. J. 1999, 5, 2335; (b)
Bhattacharya, S.; Dileep, P. V. Tetrahedron Lett. 1999,
40, 8167; (c) Bhattacharya S.; De, S.; Subramanian, M. J.
Org. Chem. 1998, 63, 7640; (d) Bhattacharya, S.; Ghosh,
S.; Easwaran, K. R. K. J. Org. Chem. 1998, 63, 9232; (e)
Bhattacharya, S.; Acharya, S. N. G. Langmuir 2000, 16,
87; (f) Bhattacharya, S.; De, S. Chem. Commun. 1995,
651.
Acknowledgements
This work was supported by the Swarnajayanti Fellow-
ship Grant of the Department of Science and Technol-
ogy, Government of India.
9. For details, see: Lipid Biochem. Prep.; Bergelson, L. D.,
Ed.; Elsevier: Amsterdam, 1980; Chapter II.3, p. 125.
10. All the intermediates and final compounds were charac-
terized by IR, 1H, 13C NMR and elemental analysis.
1
References
Selected spectral data for compounds 1–3: (1) H NMR
(400 MHz, CDCl3) l (ppm) 0.88 (t, 6H, 2×-CH3
(br m, 16H, 2×(-CH2)4), 1.58 (br m, 12H, 2×(-CH2 3
2.29 (m, 4H, 2×-OC(O)CH2) 2.54 (m, 8H, 2×(-CH2
C6H4CH2 ) ), 3.74 (t, 2H,
-) 3.32 (s, 9H, 3×N+(CH3 3
-CH2 OC(O)), 4.11 (m,
N+(CH3)3), 3.92 (m, 2H, -CH-CH2
6
), 1.29
1. See: Voet, D.; Voet, J. G. Biochemistry, 2nd ed.; Wiley:
New York, 1995; p. 280.
2. For reviews, see: Phospholipid Handbook; Cevc, G., Ed.;
Marcel Dekker: New York, 1993.
6
6
) ),
6 -
6
6
6
6
6

4206
S. Bhattacharya, M. Subramanian / Tetrahedron Letters 43 (2002) 4203–4206
1H, -CH-CHa
Me3+-CH-CHaHb
(m, 8H, 2×-C6H4
6
HbOP(O)), 4.28 (m, 3H, OCH2
OP(O)), 5.19 (m, 1H, -CH-CH2O), 7.05
-). 13C NMR (67.5 MHz, CDCl3) l
6
CH2N+
63.44, 66.48, 70.57, 115.23, 124.67, 125.4, 126.32, 126.51,
128.12, 128.92, 129.22, 173.21, 173.57. IR (cm−1): 1730
(ester CꢀO), 1230 (PꢀO), 810 (P-O). Anal. calcd for
C44H72NO8P: C, 68.27, H, 9.38, N, 1.81. Found: C,
67.9, H, 9.71, N, 1.6. (3) 1H NMR (400 MHz, CDCl3)
6
6
6
(ppm) 13.85, 22.38, 24.55, 26.67, 30.96, 31.31, 31.53,
33.86, 34.03, 35.13, 35.37, 54.19, 59.22, 62.83, 63.39,
66.23, 70.29, 70.43, 127.98, 128.05, 139.34, 139.99,
172.97, 173.26. IR (cm−1): 1735 (ester CꢀO), 1460, 1230
(PꢀO), 820 (P-O). Anal. calcd for C44H72NO8P.H2O: C,
66.72, H, 9.42, N, 1.77. Found: C, 66.4, H, 9.72, N,
l (ppm) 1.25 (br m, 28H, 2×(-CH
2×(-CH2)2), 2.29 (m, 4H, 2×-OC(O)CH2
2×-CH ) ), 3.76 (br t, 2H,
2-C6H5), 3.33 (s, 9H, 3×N+(CH3 3
-CH OC(O)), 4.12
2N+(CH3)3), 3.93 (m, 2H, -CH-CH2
(m, 1H, -CH-CHaHbOP(O)), 4.30 (m, 1H, -CH-
CHaHbOP(O)), 4.39 (m, 2H, OCH2
CH2N+Me3), 5.19
(m, 1H, -CH-CH2O) 7.17–7.25 (mixture of m, 10H, aro-
6
2)7), 1.59 (m, 8H,
6
6
), 2.58 (m, 4H,
6
6
6
6
1
6
1.54. (2) H NMR (400 MHz, CDCl3) l (ppm) 1.18 (br
6
6
m, 24H, 2×(-CH2 6
(mixture of br m, 14H, 2×-OC(O)CH2
C6H4-CH3 ) ), 3.65 (t, 2H,
), 3.22 (s, 9H, 3×N+(CH3 3
-CH2 OC(O)), 4.05
N+(CH3)3), 3.83 (m, 2H, -CH-CH2
(m, 1H, -CH-CHaHbOP(O)), 4.29 (m, 1H, -CH-
CHaHbOP(O)), 4.31 (m, 2H, OCH2
CH2N+Me3), 5.1 (m,
1H, -CH-CH2O), 6.98 (m, 8H, 2×-C6H4
-). 13C NMR
6
) ), 1.5 (br m, 8H, 2×(-CH2 2
6 ) ), 2.4
) and 2×(-CH6 2-
6
6
matic protons). 13C NMR (50 MHz, CDCl3) l (ppm)
24.9, 24.99, 29.24, 29.37, 29.64, 31.47, 34.16, 34.31,
35.97, 50.29, 54.31, 59.43, 63.02, 63.56, 63.62, 65.32,
66.37, 70.44, 70.56, 125.56, 128.21, 128.35, 142.86,
173.46, 173.76. IR (cm−1): 1740 (ester CꢀO), 1400, 1220
(PꢀO), 820 (P-O). Anal. calcd for C44H72NO8P·0.5H2O:
C, 67.49; H, 9.4; N, 1.79. Found: C, 67.23; H, 9.76; N,
1.49%.
6
6
6
6
6
6
6
6
6
(67.5 MHz, CDCl3) l (ppm) 20.96, 21.47, 22.28, 24.88,
26.19, 27.63, 29.31, 29.49, 31.53, 34.11, 34.29, 35.5,
35.91, 36.89, 38.41, 50.5, 53.86, 54.46, 59.25, 62.96,