Phosphatidylcholine with cis-9,trans-11 and trans-10,cis-12 Conjugated Linoleic Acid Isomers: Synthesis and Cytotoxic Studies

Article abstract of DOI:10.1071/CH14606

Novel phosphatidylcholines and lysophosphatidylcholines with cis-9,trans-11 and trans-10,cis-12 conjugated linoleic acid (CLA) were synthesized in high yields (75-99%). The in vitro cytotoxic activities of these compounds against three human cancer cell lines (HL-60, MCF-7, and HT-29) were evaluated. The results revealed that there are differences in the activity between phosphatidylcholine with cis-9,trans-11 and trans-10,cis-12 CLA acyl groups. 1,2-Di(9Z,11E)-octadecadienoyl-sn-glycero-3-phosphocholine was the most potent cytotoxic agent among all tested CLA derivatives and its IC50 (concentration of a compound that inhibits the proliferation of 50% of the cancer cell population) was 29.4M against HL-60. Moreover, phosphatidylcholines with CLA acyls exhibited much lower cytotoxicity against non-cancer cells (Balb/3T3) than free CLA isomers.

Full text of DOI:10.1071/CH14606

CSIRO PUBLISHING
Aust. J. Chem. 2015, 68, 1065–1075
Full Paper
http://dx.doi.org/10.1071/CH14606
Phosphatidylcholine with cis-9,trans-11
and trans-10,cis-12 Conjugated Linoleic Acid
Isomers: Synthesis and Cytotoxic Studies
A
A
A
Natalia Niezgoda, Anna Gliszczyn´ska, Witold Gładkowski,
B
B
Katarzyna Kempin´ska, Joanna Wietrzyk,
and Czesław Wawrzen´czyk
A C
,
A
Department of Chemistry, Wrocław University of Environmental and Life Sciences,
Norwida 25, PL-50-375 Wrocław, Poland.
B
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy
of Sciences, Department of Experimental Oncology, Weigla 12, PL-53-114 Wrocław,
Poland.
C
Corresponding author. Email: czeslaw.wawrzenczyk@up.wroc.pl
Novel phosphatidylcholines and lysophosphatidylcholines with cis-9,trans-11 and trans-10,cis-12 conjugated linoleic
acid (CLA) were synthesized in high yields (75–99 %). The in vitro cytotoxic activities of these compounds against three
human cancer cell lines (HL-60, MCF-7, and HT-29) were evaluated. The results revealed that there are differences in
the activity between phosphatidylcholine with cis-9,trans-11 and trans-10,cis-12 CLA acyl groups. 1,2-Di(9Z,11E)-
octadecadienoyl-sn-glycero-3-phosphocholine was the most potent cytotoxic agent among all tested CLA derivatives and
its IC50 (concentration of a compound that inhibits the proliferation of 50 % of the cancer cell population) was 29.4 mM
against HL-60. Moreover, phosphatidylcholines with CLA acyls exhibited much lower cytotoxicity against non-cancer
cells (Balb/3T3) than free CLA isomers.
Manuscript received: 4 July 2014.
Manuscript accepted: 8 November 2014.
Published online: 29 January 2015.
and colon cancer (Caco-2, HT-29, MIP-101) cells.[13,15] Also
trans,trans isomers of CLA act strongly against breast cancer[16]
and osteosarcoma.[17] It is apparent from these reports that it is
important to test separately newly isolated or synthesized CLA
isomers or its derivatives.
Many chemotherapeutics such as cisplatin and 5-fluorouracil
are effective in cancer therapy, but some side effects can
occur during treatment.[18] A modern anticancer drug is
required to be highly bioactive and specific against cancer
cells only.[19] It should also be easily absorbed from the
intestinal tract when administered orally. CLA in the form of
a free fatty acid may have limited absorption capacity from
the intestine as suggested similarly for polyunsaturated fatty
acids in research carried out by Carineli et al.[20] CLA
coupled with a polar molecule such as phosphatidylcholine
(PC) should have favourable bioavailability in comparison
with CLA itself.
Recently, we presented work on the synthesis of PC with a
mixture of predominantly cis-9,trans-11 and trans-10,cis-12
CLA isomers in the sn-1 and sn-2 position.[21] We also showed
the results of a cytotoxic study on breast cancer cells and
promyelocytic leukaemia. In the current paper, we present an
extension of this earlier research. New PCs and lysophosphati-
dylcholines were prepared with the individual isomers cis-9,
trans-11 and trans-10,cis-12. The cytotoxic activity of the
synthesized compounds was also tested.
Introduction
For more than 25 years, conjugated linoleic acid (CLA) has been
of interest to researchers owing to its many biological activities.
It has been proved that it prevents cholesterol-induced athero-
sclerosis in animal models,[1] enhances growth promotion by
increasing body protein,[2,3] exhibits antiobesity properties
(reviewed by Whigham et al.),[4] and inhibits carcinogene-
sis.[5,6] More recent studies have shown that CLA can also
interact synergistically with other anticancer agents and
enhance their effects.[7,8] CLA (an unsaturated conjugated fatty
acid with two double bonds) can exist in several positional (from
C-7 up to C-14) and geometrical (trans, cis) isomers. The most
common is cis-9,trans-11, which occurs in the meat and milk of
ruminants.[9,10] Another well-known isomer with respect to
bioactivity is trans-10,cis-12. It is formed together with cis-9,
trans-11 as the second major product of the alkaline isomeri-
zation of linoleic acid.[11] It has been observed that a difference
in molecular structure of particular isomers can cause variation
in their activity. For instance, the cis-9,cis-11 isomer showed the
ability to inhibit more than 60 % of the breast cancer MCF-7 cell
population, whereas cis-9,trans-11 CLA inhibited only 20 %.[12]
The opposite effect was noticed in the colon cancer cell line
HT-29, where the highest cytotoxicity was observed for the
cis-9,trans-11 isomer.[13] The isomer that exhibits the widest
inhibitory effect is trans-10,cis-12. It strongly reduces prolif-
eration of breast cancer (MCF-7),[12] rat hepatoma (dRLh-84),[14]
Journal compilation Ó CSIRO 2015
www.publish.csiro.au/journals/ajc

1066
N. Niezgoda et al.
Results and Discussion
description of the reaction conditions has been given pre-
viously.[21] Pure isomers 1d and 1e used in the synthesis were
purified from the alkali-isomerized linoleic acid according to
methods we published elsewhere.[11,25] The PCs were obtained
in good yields of 96 % (2a), 75 % (2d), and 75 % (2e). The
isomer composition of CLA residues in 2d and 2e obtained by
gas chromatography (GC) was exactly the same as for the CLA
isomers 1d and 1e that were used in the synthesis. The cis-9,
trans-11 CLA isomer constituted 94.2 % of the acyl residues in
2d. A trace of trans-10,cis-12 (2.5 %) and 3.3 % of other isomers
(mainly trans,trans and cis,cis) were also present. The trans-10,
cis-12 isomer constituted 96.5 % of the acyl residues in 2e. The
cis-9,trans-11 isomer was presented only in 2.8 %. The content
of trans,trans and cis,cis isomers did not exceed 0.8 %.
The second group of PCs obtained contained different fatty
acid moieties in the sn-1 and sn-2 positions. The synthesis of this
type of phospholipids is a challenging task requiring regio-
selective incorporation of different fatty acid residues in two
positions of the GPC backbone. The literature contains several
useful methods for the synthesis of mixed-chain phosphatidyl-
cholines (MC-PC). Most of them were reviewed by D’Arrigo
et al.[26] Two of them drew our attention. The first method, based
on the tin-mediated synthesis of lysophosphatidylcholine
(Scheme 2b), involved the formation of a stannylene derivative,
and subsequent acylation with a stoichiometric amount of
acyl chloride to afford 1-acyl-2-hydroxy-sn-glycero-3-
phosphocholine (1-acyl LPC), followed by a second acylation
in the sn-2 position.[27,28] This method is useful for the synthesis
of 1-acyl LPC using medium-chain acyl chlorides, which are
commercially available. However, the synthesis of 1-acyl
LPC with conjugated linoleoyl residues requires an additional
step of acid chloride synthesis. We attempted to obtain trans-10,
cis-12 conjugated linoleoyl chloride using oxalyl chloride
(Scheme 2a), which tends to be a milder, more selective
reagent than thionyl chloride. The acid chloride was prepared
according to the method described by Mattson and Volpen-
hein.[29] One part of the fatty acid was mixed with 1.2 parts
of oxalyl chloride on a weight basis. The reaction was carried out
for 48 h. This method afforded the acid chloride in yield of 88 %
(according to 1H NMR). Subsequent synthesis of 1-(10E,12Z)-
octadecadienoyl-2-hydroxy-sn-glycero-3-phosphocholine via
the stannylene derivative was effective. Product formation in
80–85 % yield after 30 min of reaction was observed by
HPLC. Unfortunately, washing of 1-acyl LPC in water/alcohol
solution with heptane was an inefficient method of removing
reaction by-products. The water/alcohol extract contained
triethylamine chloride, which was difficult to separate from
A common procedure to obtain PC[22,23] with the same acyl
residue in the sn-1 and sn-2 position involves using the cadmium
salt of sn-glycero-3-phosphocholine (GPC) as a starting
material and was applied to synthesis of 1,2-di-(9Z,11E)-
octadecadienoyl-sn-glycero-3-phosphocholine (2d) and 1,2-di-
(10E,12Z)-octadecadienoyl-sn-glycero-3-phosphocholine (2e).
The synthesis of 1,2-di-(9Z,12Z)-octadecadienoyl-sn-glycero-
3-phosphocholine (2b) and 1,2-di(conjugated)linoleoyl-
sn-glycero-3-phosphocholine (2c) was described earlier.[21]
1,2-Octadecanoyl-sn-glycero-3-phosphocholine (2a)[24] was
also prepared to compare its cytotoxic activity with those of
2b–e. Fatty acids used for the synthesis of PCs and the structure
of one example of a synthesis product (2a) are shown in Fig. 1.
Acylation with cis-9,trans-11 (1d) and trans-10,cis-12 (1e)
CLA isomers ina biphasic reactionwas conducted inthe presence
of4-(N,N-dimethylamino)pyridine (DMAP) and a couplingagent
N,N0-dicyclohexylcarbodiimide (DCC) (Scheme 1). A detailed
sn-1
O
O
P
OϪ
Nϩ
O
O
O
H
O
PC (2a)
O
sn-2
O
stearic acid (SA, 1a)
OH
O
9
linoleic acid
OH
(c9,c12 LA, 1b)
OH
12
O
9
11
cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA, 1d)
12 10
OH
O
trans-10,cis-12 conjugated linoleic acid (t10,c12 CLA, 1e)
Fig. 1. Fatty acids used in the synthesis of phosphatidylcholines. Structure
of a synthesis product example.
O
P
O
Nϩ
O
O
P
OϪ
O
O
O
R
O
Nϩ
i
OϪ
O
H
ii
O
R
x CdCl2
ϩ
HO
O
OH
H
HO
PC (2a–e)
O
R
FA (1a–e)
GPC
R: (a) C17H35 (SA)
(b) C17H31 (c9,c12 LA)
(c) C17H31 (mCLA)
(d) C17H31 (c9,t11 CLA)
(e) C17H31 (t10,c12 CLA)
O
P
OϪ
Nϩ
O
R
O
O
HO
H
LPC (3a–e)
Scheme 1. Reagents and conditions: (i) Dimethylaminopyridine (DMAP), N,N0-dicyclohexylcarbodiimide (DCC),
anhydrous CH2Cl2, room temperature, 40 h, 2a–e (75–96 %); (ii) phospholipase A2 (PLA2), Tris-HCl (pH 8.5, 0.1 M,
0.75 mM CaCl2), sodium docusate (AOT), iso-octane, 408C, 30 min, 3a–e (89–99 %).

Phosphatidylcholine with c9,t11 and t10,c12 CLA
1067
lysophosphatidylcholine in both the crystallization process with
cold acetone as well as by column chromatography.
obtained in 80 % (5c), 80 % (5d), and 82 % (5e) yield by
acylation of 3c, 3d, and 3e with stearic acid (1a).
This led us to the conclusion that the aforementioned method
is not suitable for the synthesis and extraction of 1-acyl LPC
with long fatty acyl residues. C-18 fatty acyl residues have
higher hydrophobicity than medium- and short-chain LPCs,
resulting in large losses during extraction with non-polar
heptane or hexane. Therefore, we focussed on an alternative
route that consists of the enzymatic hydrolysis of 1,2-diacyl-sn-
glycero-3-phosphocholine in sodium docusate (AOT) mixed
reversed micelles catalyzed by phospholipase A2 (PLA2)[30]
(Scheme 1) and subsequent re-esterification with the appropri-
ate fatty acid using DCC and DMAP (Scheme 3).
1-Acyl LPC is a more common intermediate in PC synthesis
than 2-acyl LPC.[28,31,32] It is more stable in the reaction
mixture and does not significantly undergo acyl migration from
sn-1 to sn-2, which is favoured in commonly used organic
solvents.[33,34] Hydrolysis of 2a–e afforded 1-acyl LPCs 3a–e
in excellent yield (89–99 %) and in a short time (30 min). The
MC-PCs were prepared as two groups of compounds. First were
PCs with a stearoyl residue at sn-1 and conjugated linoleoyl
isomers at sn-2. Acylation of 3a by 1c, 1d or 1e gave three
products 4c, 4d, and 4e in 79, 75, and 73 % yield respectively.
The second group were PCs with conjugated linoleoyl isomer
residues in the sn-1 and stearoyl in the sn-2 positions. They were
Positional analysis of the mixed-chain PC was carried out to
verify fatty acid distribution in the mixed-chain PC obtained.
The analysis was based on the same method of PC hydrolysis
that was used during the preparation of 1-acyl LPC.[30] In the
present case, the hydrolysis was scaled down according to the
method described earlier.[21] The PLA2 used in this reaction is
a selective enzyme that hydrolyzes the ester bond only in the
sn-2 position, which allows the determination of the fatty acid
composition at each position separately. The positional analy-
sis of the acyl residues in 4c–e showed that the percentage of
CLA isomers in the sn-1 position was 7.4–12.1 %, whereas at
sn-2, it was 86.4–89.6 %. Similarly, CLA was present in both
the sn-1 (86.7–90.9 %) and the sn-2 (9.0–12.6 %) position of
5c–e CLA (Table 1). Our previous studies showed that no non-
enzymatic hydrolysis or migration processes took place during
the PLA2-catalyzed hydrolysis of PC in mixed reversed
micelles.[35] This indicates that partial acyl migration from
sn-1 to sn-2 occurred during the purification of 3a, 3c–d.
1-Acyl LPC is much more stable than 2-acyl LPC and the
equilibrium concentration ratio between these two compounds
is ,9 : 1.[35,36] Therefore, some traces of 2-acyl LPC always
occur in 1-acyl LPC preparations used in the synthesis of
4c–e, 5c–e.
(a)
O
O
i
R: (c) C17H31 (mCLA)
(d) C17H31 (c9,t11 CLA)
(e) C17H31 (t10,c12 CLA)
R
OH
R
Cl
RCOOH
RCOCI
O
O
P
OϪ
(b)
Nϩ
ϩ
R
Cl
O
P
OϪ
O
O
O
Nϩ
GPC
ii
H
O
RCOCI
Sn
O
HO
O
H
HO
GPC acetal
iii
O
O
P
OϪ
O
O
O
Nϩ
iv
P
OϪ
Nϩ
O
O
O
R
ϩ
C
17H35
OH
O
O
O
R
O
H
H
HO
FA (1a)
LPC
PC
O
C17H35
Scheme 2. (a) Synthesis of fatty acid chloride; and (b) synthesis of mixed-chain phosphatidylcholine via stannylene
derivatives. Reagent and conditions: (i) (COCl)2, hexane, room temperature (rt), 1 h; (ii) dibutyltin oxide (DBTO),
2-propanol, bp, 1 h; (iii) triethylamine (TEA), rt, 1 h; (iv) DMAP, DCC, anhydrous CH2Cl2, rt, 40 h.
O
O
O
P
OϪ
O
O
P
OϪ
Nϩ
Nϩ
i
ϩ
O
O
O
R
C17H35
OH
O
O
O
C17H35
O
H
H
HO
O
R
H
LPC (3a)
FA (1c–e)
PC (4c–e)
O
O
O
O
R
O
P
Nϩ
P
i
Nϩ
LPC (3c–e)
R: (c) C17H31 (mCLA) (d) C17H31 (c9,t11 CLA) (e) C17H31 (t10,c12 CLA)
O
R
O
O
C
17H35
OϪ
ϩ
OH
O
O
O
O
OϪ
HO
H
PC (5c–e)
FA (1a)
O
C17H35
Scheme 3. Reagents and conditions: (i) DMAP, DCC, anhydrous CH2Cl2, room temperature, 72 h, 4c–e (75–79 %); 5c–e
(80–82 %).

1068
N. Niezgoda et al.
1
All the synthesized compounds were characterized by H,
13C, 31P NMR, and high-resolution mass spectrometry (HRMS).
Four signals characteristic of the cis-9,trans-11 and trans-10,
cis-12 CLA moiety olefinic protons of 2c–e, 3c–e, 4c–d, and
5c–d were identified in the 1H NMR at d 6.28–6.29 for the inner
trans, 5.92–5.95 for the inner cis, 5.62–5.65 for the outer trans,
and 5.29–5.31 ppm for the outer cis olefinic protons. The carbon
atoms of the conjugated double bond systems in the cis-9,trans-
11 and trans-10,cis-12 isomers gave four signals in the
13C NMR spectrum of 2c–e, 3c–e, 4c–e, and 5c–e in the range
135–125 ppm. Detailed assignments are given in the experi-
mental section. Two signals from the carboxylic carbon of
the ester groups in the sn-2 (d 174.72–174.73) and sn-1
(d 173.33–173.35) positions were present in the 13C NMR
spectra of 1,2-diacyl-sn-glycero-3-phosphocholines. They were
shifted upfield in comparison with the corresponding carbon
atom signals in the CLA 13C NMR spectrum (d 179.72). Signals
from carboxylic carbons (d 174.06–174.13) in the sn-1 position
of 1-acyl-LPCs (3a–e) were shifted downfield compared with
the corresponding signals in PCs.
Cytotoxic Study
The cytotoxic activity of the synthesized PCs 2d–e, 4c–d, and
5c–d and LPCs 3c–e with CLA isomers residues was compared
with the activity of PCs containing commonly occurring stearic
(SA) and linoleic acid (LA) in their structures (2a, 2b, 3a, 3b)
and with the free fatty acids (1a–e) used for the synthesis. Cis-
platin was used as a positive control. Antitumour activities were
evaluated in vitro against three human cancer cell lines: human
promyelocytic leukaemia (HL-60), breast cancer (MCF-7), and
colon cancer (HT-29). The mouse fibroblast cell line Balb/3T3
was used in the experiment to investigate the effect of the tested
compounds on non-cancer cells. The results of the in vitro
cytotoxic tests were calculated as an IC50 (concentration of a
compound that inhibits the proliferation of 50 % of the cancer
cell population) (mg mLꢀ1) and converted to micromolar
(mmol mLꢀ1). As shown in Table 2, the fatty acids (1a–e)
showed medium cytotoxicity to both cancer and non-cancer
(Balb/3T3) cell lines.
Isomer trans-10,cis-12 CLA (1e) exhibited higher inhibitory
activity against HL-60 (35.8 mM), MCF-7 (110.1 mM), and HT-
29 (103.3 mM) than cis-9,trans-11 CLA (1d), linoleic acid (1b),
and stearic acid (1a). The lowest cytotoxic effect towards HL-60
and MCF-7 cell lines was observed for SA (1a). This suggests
that the cytotoxicity of the fatty acid (FA) is associated with the
presence of double bonds in the FA chain. Generally, activity is
higher if double bonds are conjugated. These results are consis-
tent with our previous studies[21] and those obtained by
others.[17,37]
Among all tested tumour cell lines, the HL-60 line was the
most sensitive to PC compounds (2a–e, 4c–e, 5c–e) (Fig. 2,
Table 3). Among the PCs with the same fatty acid in the sn-1 and
sn-2 position, PC with the cis-9,trans-11 CLA moiety (2d)
showed better activity against HL-60 (29.4 mM) and MCF-7
(45.8 mM) than PC with the trans-10,cis-12 CLA moiety (2e).
Surprisingly, the more potent trans-10,cis-12 isomer is less
active against HL-60 and MCF-7 as an acyl residue in the PC
molecules (2e) than the free acid (1e). Moreover, PCs with the
trans-10,cis-12 CLA isomer (2e, 4e, 5e) reduced proliferation of
HT-29 cells more strongly than PCs with cis-9,trans-11 CLA
moieties (2d, 4e, 5e) (Fig. 2).
Mixed-chain PCs (4c–e, 5c–e) were synthesized in order
to determine whether the position of the CLA isomer in the
PC molecule is related to their activity. All of the mixed-chain
PCs (4c–e, 5c–e) showed lower inhibitory activities against
cancer cells than the other tested compounds (Fig. 2, Table 3).
Table 1. Fatty acid composition of mixed-chain phosphatidylcholine
(4c–e and 5c–e)
SA, stearic acid; CLA, conjugated linoleic acid; mCLA, mixture of
conjugated linoleic acid isomers
Compound
Fatty acid
Composition [%]
sn-1
sn-2
4c
4d
4e
5c
5d
5e
SA
mCLAA
92.6
7.4
10.8
89.2
13.6
86.4
11.4
89.6
88.7
11.3
91.0
9.0
SA
c9,t11 CLAB
SA
t10,c12 CLAC
89.0
11.0
87.9
12.1
13.3
86.7
9.5
SA
mCLAA
SA
c9,t11 CLAB
90.9
10.6
89.4
SA
t10,c12 CLAC
87.3
12.6
AMixture of CLA isomers: 48.5 % cis-9,trans-11, 48.3 % trans-10,cis-12,
2.5 % other CLA isomers.
B94.2 % cis-9,trans-11, 5.8 % other CLA isomers.
C96.5 % trans-10,cis-12, 3.5 % other CLA isomers.
Table 2. Anti-proliferative in vitro activity of fatty acid on selected cell lines
CLA, conjugated linoleic acid
Compound label
Fatty acid
Compound ICA50 [mM]
HL-60
MCF-7
HT-29
Balb/3T3
1a
1b
1c
1d
1e
Stearic acid (SA)
120.4 ꢁ 9.6
102.6 ꢁ 13.4
65.7 ꢁ 19.3B
102.4 ꢁ 9.9
35.8 ꢁ 28.1
0.8 ꢁ 0.3B
.351.5
110.9 ꢁ 4.4
115.0 ꢁ 12.6
134.8 ꢁ 9.1
117.9 ꢁ 1.2
103.3 ꢁ 22.7
14.3 ꢁ 12.0
138.6 ꢁ 13.3
116.0 ꢁ 6.7B
125.4 ꢁ 3.7B
121.1 ꢁ 1.7
100.4 ꢁ 12.3
9.1 ꢁ 2.2C
Linoleic acid (LA)
cis-9,trans-11 þ trans-10,cis-12 CLA
cis-9,trans-11 CLA
trans-10,cis-12 CLA
Cisplatin
253.2 ꢁ 12.8B
124.9 ꢁ 2.1B
112.4 ꢁ 6.2
110.1 ꢁ 4.8
14.1 ꢁ 1.0B
AThe concentration of a compound that inhibits the proliferation of 50 % of the cancer cell population. Data are presented as mean ꢁ s.d. of three independent
experiments.
BData derived from separate experiment.[21]
CData derived from separate experiment.[38]

Phosphatidylcholine with c9,t11 and t10,c12 CLA
1069
The inhibitory activities of PC with CLA isomer residues at sn-2
and stearoyl at sn-1 (4c–e) and PC with CLA isomer residues at
sn-1 and stearoyl at sn-2 (5c–e) were not significantly different
to each other towards HL-60 and MCF-7. The HT-29 line
showed greater sensitivity to mixed-chain PC with trans-10,
cis-12 (4e, 4d) than cis-9,trans-11 CLA (5e, 5d) residues.
Proliferation inhibition by 4e and 5e was found to be 15 and
19 % respectively whereas 4d and 5d showed no inhibition for
HT-29 (Fig. 2). No significant difference in biological activity
between pairs 4c and 5c, 4d and 5d, and 4e and 5e of mixed-
chain PCs with CLA residues in sn-1 and sn-2 positions was
observed. Lysophosphatidylcholines 3a–c showed better cyto-
toxicity against HL-60 than PCs. 1-Acyl LPC with a stearoyl
residue (3a) showed unexpected activity (59.1–60.4 mM)
towards all cancer lines but also healthy Balb/3T3 cells. It is
noteworthy that all PC derivatives exhibited a much lower
cytotoxicity against healthy cells (Balb/3T3) than free FA
(Tables 2 and 3). The best activity among lysophosphatidylcho-
lines with conjugated linoleic acid was observed for the mixed
isomer LPC 3c and it was 58.0 (HL-60) and 111.5 mM (MCF-7).
Results obtained in this study suggest that there is no strict
relationship between the presence of the specific configuration
(cis-9,trans-11 or trans-10,cis-12) of the conjugated bond
system in the tested compounds and their cytotoxic activity.
As reported in scientific studies, particular CLA isomers
have different activities against various types of cancer
cells.[6,12–15,39–51] The mechanism of action may be complex
and could be attributed to alteration of lipid peroxidation, tissue
fatty acid composition, eicosanoid metabolism, gene expres-
sion, cell-cycle regulation, cell proliferation, and apoptosis
(reviewed by Kelley et al.).[52] It is possible that a particular
isomer in the form of the free acid affects cell metabolism in a
different way than in the form associated with a phospholipid.
This may be related to hydrolysis and release of the acyl residue
of only one of the CLA isomers from the phospholipid molecule
owing to the regioselectivity and substrate specificity of cellular
phospholipases or differences between the membrane transport
of the compounds. These could explain why the more potent
trans-10,cis-12 CLA isomer (HL-60, MCF-7) in the free form is
less active than cis-9,trans-11 in the PC form (Table 3).
HL-60
MCF-7
HT-29
Balb/3T3
100
90
80
70
60
50
40
30
20
10
0
2a2b2c2d 2e3a3b3c3d 3e4c4d4e5c5d5e 2a 2b 2c 2d 2e 3a 3b 3c 3d 3e 4c 4d 4e 5c 5d 5e 2a2b2c2d2e3a3b3c3d3e 4c 4d4e 5c5d5e 2a2b2c2d 2e3a3b3c3d 3e4c4d4e 5c5d 5e
Fig. 2. Cell proliferation inhibition (%). An SRB or MTT assay was performed after 72-h exposure to concentrations of 100 mg mLꢀ1 of the tested
compounds. Values are means ꢁ s.d., n ¼ 3. Resultsthat are significantlydifferent from eachother (P , 0.05): HL-60; 2a, b; 2a, c; 2a, d; 2a, e; 2b, c; 2b, e; 2c, d;
2d, e; 3a, b; 3b, c; 3b, e; MCF-7; 2a, d; 2b, c; 2b, d; 2c, d; 2c, e; 2d, e; 3a, c; 4d, 5c; HT-29: 2a, b; 2a, c; 2a, e; 2b, c; 2b, d; 2c, e; 2d, e; 3a, b; 3a, d; 3a, e; 3b, c;
3c, d; 3c, e; 4c, e; 4c, 5e; 4d, e; 4d, 5e; 4e, 5c; 4e, 5d; 5c, d; 5c, e; 5e, d; Balb/3T3; 1a, b; 1a, c; 1a, d; 1a, e; 1c, e; 2a, d; 2b, d; 2c, d; 2d, e; 3a, b; 3a, c; 3a, d; 3a, e.
Table 3. The anti-proliferative in vitro activity of synthesized phosphatidylcholines on selected cell lines
SA, stearic acid; LA, linoleic acid; CLA, conjugated linoleic acid; mCLA, mixture of c9,t11 and t10,c12 conjugated linoleic acid isomers
Compounds
Acyl residue
sn-2
Compounds’ ICA50 [mM]
sn-1
HL-60
MCF-7
HT-29
Balb/3T3
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4c
4d
4e
5c
5d
5e
SA
SA
.126.6
31.2 ꢁ 6.1
.127.9
.126.6
.127.9
.127.9
45.8 ꢁ 6.6
.127.9
59.8 ꢁ 2.3
.192.4
.192.4
.192.4
.192.4
.127.2
.127.2
.127.2
.127.2
.127.2
.127.2
.126.6
64.6 ꢁ 6.4
.127.9
.126.6
.127.9
.127.9
43.8 ꢁ 9.5
.127.86
59.1 ꢁ 4.6
.192.4
.192.4
.192.4
.192.4
.127.2
.127.2
.127.2
.127.2
.127.2
.127.2
LA
LA
mCLA
c9,t11 CLA
t10,c12 CLA
SA
mCLA
c9,t11 CLA
29.4 ꢁ 11.7
.127.9
.127.9
t10,c12 CLA
95.5 ꢁ 39.9
60.4 ꢁ 0.1
.192.4
–
60.4 ꢁ 2.0
129.7 ꢁ 47.7
58.0 ꢁ 2.3
96.1 ꢁ 33.5
91.0 ꢁ 73.0
.127.2
LA
–
mCLA
c9,t11 CLA
t10,c12 CLA
SA
–
111.5 ꢁ 56.5
.192.4
.192.4
–
–
mCLA
c9,t11 CLA
t10,c12 CLA
SA
.127.2
.127.2
SA
.127.2
.127.2
SA
.127.2
.127.2
mCLA
c9,t11 CLA
t10,c12 CLA
.127.2
.127.2
.127.2
SA
.127.2
.127.2
SA
AThe concentration of a compound that inhibits the proliferation of 50 % of the cancer cell population. Data are presented as mean ꢁ s.d. of three independent
experiments.

1070
N. Niezgoda et al.
Conclusion
acquisition were carried out using the Chromeleon 6.80 software
(Dionex Corporation). Analysis was carried out using a Betasil
DIOL 5-mm column (Thermo, 150 ꢂ 2.1 mm). The injection
volume was 15 mL in all of the experiments and the cooling
temperature for the samples was 208C. The column temperature
was maintained at 308C. The total time of analysis was 19 min.
In summary, new cis-9,trans-11 and trans-10,cis-12 PC and
lysophosphatidylcholine derivatives were synthesized in high
yields and the in vitro cytotoxic activities of these compounds
were investigated. Results obtained in this study suggest that
there is no strict relationship between the presence of the spe-
cific configuration (cis-9,trans-11 or trans-10,cis-12) of the
conjugated bond system in the tested compounds and their
cytotoxic activity. However, the results confirmed that there are
differences in activity between PC derivatives of cis-9,trans-11
and trans-10,cis-12 CLA. It seems that cytotoxic activity can be
connected with the spatial structure of CLA residues in PC,
which depends on the configuration of the double bonds. It is
possible that the particular isomer in the form of the free acid
affects cell metabolism in a different way than in the form
associated with a phospholipid.
The mobile phase had a constant flow of 1.5 mL minꢀ1
.
Solvent A (1 % HCOOH, 0.1 % triethylamine (TEA) in water),
solvent B (hexane), and solvent C (propan-2-ol) were used
in gradient mode starting with 3 : 40 : 57 (A : B : C (vol-%/
vol-%/vol-%)), at 4 min ¼ 10 : 40 : 50, at 9 min ¼ 10 : 40 : 50, at
9.1 min ¼ 3 : 40 : 57 and at 19 min ¼ 3 : 40 : 57.
The FA composition of the obtained PC was analysed by
GC on an Agilent 6890N apparatus with a flame ionization
detector (FID). The separation of FA methyl esters was achieved
using a 70 % cyanopropylpolysilphenylene–siloxane column
(TR FAME, 30 m ꢂ 0.25 mm ꢂ 0.25 mm). The initial oven tem-
perature was 1408C held for 3 min, which was then increased
to 1908C at a rate of 58C minꢀ1 and then to 2608C at a rate of
308C minꢀ1. This was then held for 3 min while the injector
temperature was 2508C. The FID temperature was set at 2808C.
Hydrogen was used as a carrier gas.
Experimental
General
All chemicals and reagents including stearic (1a) and linoleic
(1b) acids were purchased from Sigma–Aldrich Chemical
Co. The isomeric mixture of CLA (0.7 % LA; 48.5 % cis-9,
trans-11; 48.3 % trans-10,cis-12; 2.5 % other CLA isomers,
purity 97 %) (1c) and trans-10,cis-12 CLA (1e) (purity
96.5 %) was obtained according to the reported procedure.[11]
Cis-9,trans-11 CLA (purity 94.2 %) (1d) was prepared
according to the procedure described previously.[25] Phos-
pholipase A2 (Lecitase 10L) was a gift from Novozymes. The
enantiomerically pure GPC was purchased from Bachem and
converted to the cadmium chloride complex (GPC ꢂ CdCl2)
using the method described elsewhere.[21] 1,2-Di(conjugated)
linoleoyl-sn-glycero-3-phosphocholine (2c) (purity 98 %)
and 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (2b) were
also prepared according to a procedure described previ-
ously.[21] All of the solvents used in liquid chromatography
were of HPLC grade (Merck LiChrosolv Reagents) and were
purchased from Merck.
Positional analysis of 4c–e and 5c–e was carried out accord-
ing to the method described before.[21]
General Procedure for 1,2-Diacyl-sn-glycero-3-
phosphocholines Synthesis
1,2-diacyl-sn-glycero-3-phosphocholines 2a, 2d, and 2e were
prepared according to the procedure described previously.[21]
1,2-Dioctadecanoyl-sn-glycero-3-phosphocholines 2a
White solid (864 mg, 96 %), purity (according to HPLC)
99 %, traces of stearoyl LPC. HPLC Rt 6.69 min. The chemical
structure was confirmed by 1H NMR and 13C NMR. The spectral
data were in accordance with those reported in the literature.[24]
1,2-Di-(9Z,11E-octadecadienoyl)-sn-glycero-3-
phosphocholine 2d
TLC analyses were carried out on pre-coated silica gel 60
F254 plates (Merck). The compounds were detected using
either a 0.05 % primuline solution (8 : 2 acetone/H2O v/v)
followed by UV (365 nm) visualization, or a solution of 10 g
of Ce(SO4)2 and 20 g of phosphomolybdic acid in 1 L of 10 %
H2SO4 followed by heating. Column chromatography was
performed on silica gel (Kieselgel 60, 230–400 mesh (Merck)).
The chemical structures of the synthesized compounds were
confirmed by NMR spectroscopy and high-resolution electro-
spray ionization mass spectrometry (HR-ESI-MS). All of the
1H, 13C, and 31P NMR spectra were recorded using a Bruker
Avance DRX 300 spectrometer. Chemical shifts (1H and 13C)
are given in ppm downfield from tetramethylsilane (TMS) as
the internal standard. In 31P NMR spectra, chemical shifts were
referenced to 85 % H3PO4 as a standard. Coupling constant (J)
values are given in hertz. HR-ESI mass spectra were obtained
on a Bruker micrOTOF-Q.
Reaction progress and the purity of the obtained PC were
monitored by HPLC. HPLC was performed on an Ultimate 3000
Dionex chromatograph equipped with a DGP-3600A dual-pump
fluid control module, a TCC-3200 thermostatted column com-
partment, and a WPS-3000 autosampler. A Corona charged
aerosol detector (CAD) from ESA Biosciences was used, with
the following parameters: acquisition range: 100 pA, digital
filter set to none, N2 pressure: 0.24 MPa. The system and data
The title compound was obtained as a colourless greasy
solid (669 mg, 75 %), with 99 % purity (according to HPLC,
Rt 7.01 min), traces of 9Z,11E-octadecadienoyl LPC. dH
(300 MHz, CDCl3) 6.28 (ddd, J 15.0, 11.0, 1.1, 2H, H-11d),
5.93 (t, J 11.0, 2H, H-10d), 5.64 (dt, J 15.0, 7.0, 2H, H-12d),
5.30 (dt, J 15.0, 7.0, 2H, H-9d), 5.18 (m, 1H, H-2), 4.38 (dd, J
12.0, 2.8, 1H, one of the H-1), 4.26–4.18 (m, 2H, H-a), 4.13
(dd, J 12.0, 7.4, 1H, one of the H-1), 3.92 (dd, J 12.7, 5.9, 2H,
H-3), 3.84–3.74 (m, 2H, H-b), 3.34 (s, 9H, N(CH3)3), 2.34–
2.21 (m, 4H, H-2d), 2.18–2.0 (m, 8H, H-8d, H-13d), 1.64–1.44
(m, 4H, H-3d), 1.42–1.11 (m, 32H, H-4d-7d, H-14d-17d), 0.88
(t, J 6.9, 6H, H-18d). dC (75 MHz, CDCl3) 173.78 (C-1d in
sn-2), 173.40 (C-1d in sn-1), 134.53 (C-12d), 129.55 (C-9d),
128.58 (C-10d), 125.40 (C-11d), 70.29 (C-2), 70.15 (C-2),
66.31 (C-b), 66.23 (C-b), 63.43 (C-3), 63.36 (C-3), 62.50
(C-1), 58.85 (C-a), 58.78 (C-a), 53.92 (–N(CH3)3), 34.04
(C-2d in sn-2), 33.82 (C-2d in sn-1), 32.61 (C-13d), 31.53
(C-16d), 29.64–28.59 (C-4d–7d, C-14d–15d), 27.43 (C-8d)
24.65 (C-3d in sn-2), 24.63 (C-3d in sn-1), 22.35 (C-17d),
13.68 (C-18d). Note: signals labelled ‘d’ are from the cis-9,
trans-11 CLA moiety, those labelled ‘a,b’ are from the choline
moiety. dP (121 MHz, CDCl3) ꢀ0.42. m/z (HR-ESI) 782.5685;
calc. for C44H80NO9P: 782.5694 [M þ H]þ.

Phosphatidylcholine with c9,t11 and t10,c12 CLA
1071
1,2-Di-(10E,12Z-octadecadienoyl)-sn-glycero-3-
phosphocholine 2e
the stearoyl moiety, those labelled ‘a,b’ are from the choline
moiety. dP (121 MHz, 2 : 1 CDCl3/CD3OD v/v) ꢀ0.01. m/z
(HR-ESI) 524.3728; calc. for C26H54NO7P: 524.3711 [M þ H]þ.
The title compound was obtained as a colourless greasy solid
(669 mg, 75 %) with 98 % purity (according to HPLC, Rt
7.03 min), traces of 10E,12Z-octadecadienoyl LPC. dH
(300 MHz, CDCl3) 6.29 (ddd, J 15.0, 10.9, 1.1, 2H, H-11e),
5.93 (t, J 10.9, 2H, H-12e), 5.64 (dt, J 15.0, 7.0, 2H, H-10e), 5.30
(dt, J 10.9, 7.4, 2H, H-13e), 5.19 (m, 1H, H-2), 4.39 (dd, J 12.0,
2.8, 1H, one of the H-1), 4.36–4.25 (m, 2H, H-a), 4.12 (dd, J
12.0, 7.4, 1H, one of the H-1), 3.93 (dd, J 12.7, 5.9, 2H, H-3),
3.84–3.74 (m, 2H, H-b), 3.36 (s, 9H, N(CH3)3), 2.34–2.22 (m,
4H, H-2e), 2.20–2.00 (m, 8H, H-9e and H-14b), 1.66–1.49 (m,
4H, H-3e), 1.44–1.21 (m, 32H, H-4e-8e and H-15e-17e), 0.88 (t,
J 6.9, 6H, H-18e). dC (75 MHz, CDCl3) 134.65 (C-10e), 130.28
(C-13e), 128.72 (C-12e), 125.82 (C-11e), 70.69 (C-2), 70.60
(C-2), 66.56 (C-b), 66.48 (C-b), 63.57 (C-3), 63.51 (C-3), 63.15
(C-1), 59.50 (C-a), 59.44 (C-a), 54.55 (–N(CH3)3), 34.45 (C-2e
in sn-2), 34.26 (C-2e in sn-1), 33.04 (C-9e), 31.61 (C-16e),
29.76–29.08 (C-4e–8e and C-15e), 27.80 (C-14e) 25.10 (C-3e in
sn-2), 25.02 (C-3e in sn-1), 22.69 (C-17e), 14.19 (C-18e). Note:
signals labelled ‘e’ are from the trans-10,cis-12 CLA moiety,
those labelled ‘a,b’ are from the choline moiety. dP (121 MHz,
CDCl3) ꢀ0.15. m/z (HR-ESI) 782.5676; calc. for C44H80NO9P:
782.5694 [M þ H]þ.
1-(9Z,12Z)-Octadecadienoyl-2-hydroxy-sn-glycero-3-
phosphocholine 3b
The title compound was obtained as a colourless greasy solid
(313 mg, 95%) with 99% purity (according to HPLC, Rt
8.28min), traces of 1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-
3-phosphocholine. dH (300MHz, 2 : 1 CDCl3/CD3OD v/v) 5.19–
5.02 (m, 4H, H-9b, H-10b, H-12b, H13b), 4.07–3.97 (m, 2H,
H-a), 3.95–3.82 (m, 2H, H-1), 3.80–3.58 (m, 3H, H-2 and H-3),
3.40–3.33 (m, 2H, H-b), 3.15(s, 1H, -OH), 2.98(s, 9H, N(CH3)3),
2.53 (t, J 5.9, 2H, H-11b), 2.11 (t, J 7.6, 2H, H-2b), 1.89–1.74
(m, 4H, H-8b, H-14b), 1.44–1.31 (m, 2H, H-3b), 1.19–1.00
(m, 28H, H-4b–7b, H-15b–17b), 0.65 (t, J 6.9, 3H, H-18b).
dC (75 MHz, 2 : 1 CDCl3/CD3OD v/v) 174.06 (C-1b in sn-1),
129.79(C-12), 129.61 (C-10), 127.70(C-9), 127.56(C-13), 68.43
(C-2), 68.34 (C-2), 66.60 (C-b), 66.52 (C-b), 66.10 (C-3), 64.57
(C-1), 58.75 (C-a), 58.69 (C-a), 53.72, 53.68, 53.63 (–N(CH3)3),
33.71 (C-2a in sn-1), 31.18 (C-16a), 29.26 (C-15b), 29.09–28.32
(C-4b–7b, C-14b), 26.82 (C-8), 25.24 (C-11b), 24.51 (C-3b),
22.20 (C-17b), 13.51 (C-18b). Note: signals labelled ‘b’ are
from the linoleoyl moiety, those labelled ‘a,b’ are from the
choline moiety. dP (121MHz, 2 : 1 CDCl3/CD3OD v/v) ꢀ0.71.
m/z (HR-ESI) 518.3270; calc. for C26H50NO7P: 518.3241
[M þ H]þ.
General Procedure for 1,2-Diacyl-sn-glycero-3-
phosphocholines Enzymatic Hydrolysis
Hydrolysis of 1,2-diacyl-sn-glycero-3-phosphocholines 2a–e
was carried out according to the method described by Morgado
et al.[30] Two solutions, A: 0.635 mmol of 2a–e in 13.2 mL
isooctane, and B: 174 mg (0.393 mmol) of sodium docusate
(AOT) in 13.2 mL isooctane, were pre-incubated for ,0.5 h at
408C on a magnetic stirrer. Then, 262 mL of PLA2 (10000 U
mLꢀ1; 1 U is equivalent to the amount of enzyme producing
1 mmole of free fatty acid per minute at pH 8 and 408C) and the
same volume of Tris-HCl buffer (0.1 M, pH 8.5) with 0.75 M of
CaCl2 were then added to solution B and the reaction was started
by adding solution A. The progress of the enzymatic reactions
was monitored by TLC (65 : 25 : 4 CHCl3/CH3OH/H2O v/v/v).
The reaction was carried out for 30 min. The enzyme was sep-
arated from the reaction mixture using diatomaceous earth
(CeliteÒ 545) and the solvent was evaporated to dryness at 408C
under vacuum. 1-Acyl LPCs 3a–e were purified on a silica gel
column (65 : 25 : 4 CHCl3/CH3OH/H2O v/v/v). Product-
containing fractions of Rf 0.3 were collected and solvent evap-
orated under vacuum (458C).
1-(Conjugated)linoleoyl-2-hydroxy-sn-glycero-3-
phosphocholine 3c
The title compound was obtained as a colourless greasy solid
(294 mg, 89 %) with 99 % purity (according to HPLC, Rt
8.14 min), traces of 1,2-di-(conjugated)linoleoyl-sn-glycero-3-
phosphocholine. dH (300 MHz, CDCl3) 6.28 (m, 1H, H-11d or
H-11e), 5.92 (m, 1H, H-10d or H-12e), 5.62 (m, 1H, H-12d or
H-10e), 5.28 (m, 1H, H-9d or H-13e), 4.35–4.23 (m, 2H, H-a),
4.10–4.0 (m, 2H, H-1), 3.99–3.84 (m, 3H, H-2 and H-3), 3.82–
3.67 (m, 2H, H-b), 3.47 (s, 1H, –OH), 3.30 (s, 9H, N(CH3)3),
2.30 (t, J 7.6, 2H, H-2d or H-2e), 2.17–1.97 (m, 4H, H-8d, H-13d
or H-9e, H-14e), 1.64–1.47 (m, 2H, H-3d or H-3e), 1.41–1.19
(m, 16H, H-4d–7d, H-14d–17d or H-4e–8e, H-15e–17e), 0.88 (t,
J 6.8, 3H, H-18d or H-18e). dC (75 MHz, CDCl3) 174.12 (C-1d
and C-1e in sn-1), 134.92 (C-12d), 134.64 (C-10e), 130.26
(C-13e), 129.92 (C-9d), 128.89 (C-10d), 128.76 (C-12e),
125.84 (C-11e), 125.73 (C-11d), 68.84 (C-2), 68.74 (C-2),
67.29 (C-b), 67.23 (C-b), 66.37 (C-3), 66.29 (C-3), 65.68
(C-1), 59.65 (C-a), 54.48 (–N(CH3)3), 34.28 (C-2d and C-2e
in sn-1), 33.07 (C-13d and C-9e), 31.90 (C-16d), 31.63 (C-16e),
30.02–29.22 (C-4d–7d, C-14d–15d, C-4e–8e, and C-15e), 27.88
(C-8d), 27.82 (C-14e), 25.08 (C-3d and C-3e in sn-1), 22.77
(C-17d), 22.71 (C-17e), 14.26 (C-18d), 14.22 (C-18e). Note:
signals labelled ‘d’ are from the cis-9,trans-11 CLA moiety,
those labelled ‘e’ are from trans-10,cis-12 CLA moiety, those
labelled ‘a,b’ are from the choline moiety. dP (121 MHz,
CDCl3) ꢀ0.61. m/z (HR-ESI) 518.3265; calc. for C26H50NO7P:
518.3241 [M þ H]þ.
1-Octadecanoyl-2-hydroxy-sn-glycero-3-
phosphocholine 3a
The title compound was obtained as a white solid (329 mg,
99 %) with 99 % purity (according to HPLC, Rt 8.06 min),
traces of 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine. dH
(300 MHz, 2 : 1 CDCl3/CD3OD v/v) 4.07–3.98 (m, 2H, H-a),
3.96–3.83 (m, 2H, H-1), 3.80–3.58 (m, 3H, H-2 and H-3), 3.40–
3.33 (m, 2H, H-b), 3.15 (s, 1H, -OH), 2.99 (s, 9H, N(CH3)3),
2.11 (t, J 7.6, 2H, H-2a), 1.44–1.31 (m, 2H, H-3a), 1.11–0.99 (m,
28H, H-4a–17a), 0.64 (t, J 6.6, 3H, H-18a). dC (75 MHz, 2 : 1
CDCl3/CD3OD v/v) 174.13 (C-1a in sn-1), 68.44 (C-2), 68.35
(C-2), 66.60 (C-b), 66.56 (C-b), 66.12 (C-3), 64.57 (C-1), 58.70
(C-a), 58.69 (C-a), 53.54, 53.69, 53.64 (–N(CH3)3), 33.74 (C-2a
in sn-1), 31.59 (C-16a), 28.50–28.56 (C-7a–14a), 24.54 (C-3a),
22.33 (C-17a), 13.55 (C-18a). Note: signals labelled ‘a’ are from
1-(9Z,11E)-Octadecadienoyl-2-hydroxy-sn-glycero-3-
phosphocholine 3d
The title compound was obtained as a colourless greasy
solid (310 mg, 94 %) with 99 % purity (according to HPLC,

1072
N. Niezgoda et al.
Rt 8.15 min), traces of 1,2-di-(9Z,11Z-octadecadienoyl)-sn-
glycero-3-phosphocholine. dH (300 MHz, CDCl3) 6.28 (ddd, J
15.0, 11.0, 1.1, 1H, H-11d), 5.93 (t, J 11.0, 1H, H-10d), 5.64 (dt,
J 15.0, 7.0, 1H, H-12d), 5.28 (dt, J 11.0, 7.5, 1H, H-9d), 4.37–
4.23 (m, 2H, H-a), 4.12–4.03 (m, 2H, H-1), 3.99–3.86 (m, 3H,
H-2 and H-3), 3.79–3.70 (m, 2H, H-b), 3.47 (m, 1H, –OH), 3.30
(s, 9H, N(CH3)3), 2.31 (t, J 7.6, 2H, H-2d), 2.19–1.97 (m, 4H, H-
8d, H-13d), 1.64–1.47 (m, 2H, H-3d), 1.41–1.17 (m, 16H, H-4d–
7d, H-14d–17d), 0.88 (t, J 6.9, 3H, H-18d). dC (75 MHz, CDCl3)
174.10 (C-1d in sn-1), 134.93 (C-12d), 129.93 (C-9d), 128.83
(C-10d), 125.73 (C-11d), 68.84 (C-2), 68.76 (C-2), 67.29 (C-b),
67.25 (C-b), 66.38 (C-3), 66.29 (C-3), 65.39 (C-1), 59.64 (C-a),
54.48 (–N(CH3)3), 34.28 (C-2d in sn-1), 33.07 (C-13d), 31.90
(C-16d), 30.11–29.24 (C-4d–7d, C-14d–15d), 27.88 (C-8d),
25.08 (C-3d in sn-1), 22.77 (C-17d), 14.26 (C-18d). Note:
signals labelled ‘d’ are from the cis-9,trans-11 CLA moiety;
those labelled ‘a,b’ are from the choline moiety. dP (121 MHz,
CDCl3) ꢀ0.37. m/z (HR-ESI) 518.3232; calc. for C26H50NO7P:
518.3241 [M þ H]þ.
v/v/v). Product-containing fractions of Rf 0.4 were collected and
solvent evaporated under vacuum (458C).
1-Octadecanoyl-2-(conjugated)linoleoyl-sn-glycero-3-
phosphocholine 4c
The title compound was obtained as a colourless greasy solid
(119 mg, 79 %) with 98 % purity (according to HPLC, Rt
6.79 min), traces of 1-octadecanoyl LPC. dH (300 MHz, CDCl3)
6.29 (m, 1H, H-11d or H-11e), 5.93 (m, 1H, H-10d or H-12e),
5.64 (m, 1H, H-12d or H-10e), 5.30 (m, 1H, H-9d or H-13e), 5.19
(m, 1H, H-2), 4.43–4.21 (m, 2H, H-a), 4.39 (dd, J 12.3, 2.9, 1H,
one of the H-1), 4.43–4.21 (m, 2H, H-a), 4.12 (dd, J 12.0, 7.2,
1H, one of the H-1), 4.00–3.85 (m, 2H, H-3), 3.84–3.73 (m, 2H,
H-b), 3.36 (s, 9H, N(CH3)3), 2.35–2.22 (m, 4H, H-2d or H-2e
and H-2a), 2.19–1.98 (m, 4H, H-8d and H-13d or H-9e and
H-14e), 1.65–1.48 (m, 4H, H-3d or H-3e and H-3a), 1.40–1.13
(m, 44H, H-4d–7d, H-14d–17d or H-4e–8e, H-15e–17e, and
H-4a–17a), 0.93–0.81 (t, J 6.7, 6H, H-18a and H-18d or H-18e).
dC (75 MHz, CDCl3) 173.73 (C-1d or C-1e in sn-2), 173.35
(C-1a in sn-1), 134.95(C-12d), 134.67 (C-10e), 130.30 (C-13e),
129.91 (C-9d), 128.86 (C-10d), 128.72 (C-12e), 125.83 (C-11e),
125.71 (C-11d), 70.74 (C-2), 70.64 (C-2), 66.52 (C-b), 66.49
(C-b), 63.63 (C-3), 63.56 (C-3), 63.15 (C-1), 59.53 (C-a), 59.47
(C-a), 54.66 (–N(CH3)3), 34.47 (C-2d and C-2e in sn-2), 34.29
(C-2a in sn-1), 32.54 (C-13d and C-9e), 32.08 (C-16a), 31.90
(C-16d), 31.63 (C-16e), 30.14–29.09 (C-4a–15a, C-4d–7d,
C-14d–15d, C-4e–8e, C-15e), 27.82 (C-8d and C-14e), 25.11
(C-3a), 25.05 (C-3d or C-3e), 22.84 (C-17a), 22.77 (C-17d),
22.71 (C-17e), 14.27 (C-16a and C-18d or C-18e). Note: signals
labelled ‘d’ are from the cis-9,trans-11 CLA moiety, those
labelled ‘e’ are from trans-10,cis-12 CLA moiety, those labelled
‘a’ are from stearoyl moiety and those labelled ‘a,b’ are from
the choline moiety. dP (121 MHz, CDCl3) 16.48. m/z (HR-ESI)
784.5874; calc. for C44H84NO8P: 784.5851 [M þ H]þ.
1-(10E,12Z)-Octadecadienoyl-2-hydroxy-sn-glycero-3-
phosphocholine 3e
The title compound was obtained as a colourless greasy solid
(307 mg, 93 %) with 99 % purity (according to HPLC, Rt
8.16 min), traces of 1,2-di-(10E,12Z-octadecadienoyl)-sn-
glycero-3-phosphocholine. dH (300 MHz, 2 : 1 CDCl3/CD3OD
v/v) 6.03 (ddd, J 15.0, 10.9, 1.1, 1H, H-11e), 5.67 (t, J 10.9, 1H,
H-12e), 5.38 (dt, J 15.0 7.0, 1H, H-10e), 5.03 (dt, J 10.9, 7.4, 1H,
H-13e), 4.05–3.96 (m, 2H, H-a), 3.94–3.81 (m, 2H, H-1), 3.78–
3.56 (m, 3H, H-2 and H-3), 3.38–3.31 (m, 2H, H-b), 3.11
(m, 1H, –OH), 2.96 (s, 9H, N(CH3)3), 2.09 (t, J 7.6, 2H,
H-2e), 2.95–1.75 (m, 4H, H-9e and H-14e), 1.42–1.29 (m, 2H,
H-3e), 1.19–0.97 (m, 16H, H-4e–8e and H-15e–17e), 0.63 (t, J
6.9, 3H, H-18e). dC (75 MHz, 2 : 1 CDCl3/CD3OD v/v) 174.34
(C-1e in sn-1), 134.37 (C-10e), 129.87 (C-13e), 128.57 (C-12e),
125.64 (C-11e), 68.57 (C-2), 68.58 (C-2), 66.83 (C-b), 66.76
(C-b), 66.34 (C-3), 64.94 (C-1), 59.02 (C-a), 58.96 (C-a), 53.94,
53.89, 53.85 (–N(CH3)3), 33.95 (C-2e in sn-1), 33.75 (C-9e),
31.36 (C-16e), 29.54–28.79 (C-4e-8e and C-15e), 27.50
(C-14e), 24.76 (C-3e in sn-1), 22.43 (C-17e), 13.71 (C-18e).
Note: signals labelled ‘e’ are from the trans-10,cis-12 CLA
moiety, those labelled ‘a,b’ are from the choline moiety. dP
(121 MHz, 2 : 1 CDCl3/CD3OD v/v) 0.69. m/z (HR-ESI)
518.3261; calc. for C26H50NO7P: 518.3241 [M þ H]þ.
1-Octadecanoyl-2-(9Z,11E)-octadecadienoyl-sn-
glycero-3-phosphocholine 4d
The title compound was obtained as a colourless greasy solid
(113 mg, 75 %) with 98 % purity (according to HPLC, Rt
6.74 min), traces of 1-octadecanoyl LPC. dH (300 MHz, CDCl3)
6.27 (ddd, J 15.0, 11.0, 1.1, 1H, H-11d), 5.93 (t, J 11.0, 1H,
H-10d), 5.64 (dt, J 15.0, 7.0, 1H, H-12d), 5.29 (dt, J 15.0, 7.0,
1H, H-9d), 5.18 (m, 1H, H-2), 4.38 (dd, 12.0, 2.8, 1H, one of the
H-1), 4.34–4.22 (m, 2H, H-a), 4.11 (dd, J 12.0, 7.4, 1H, one of
the H-1), 3.97–3.85 (m, 2H, H-3), 3.83–3.73 (m, 2H, H-b), 3.34
(s, 9H, N(CH3)3), 2.34–2.23 (m, 4H, H-2d and H-2a), 2.20–2.03
(m, 4H, H-8d and H-13d), 1.65–1.48 (m, 4H, H-3d and H-3a),
1.43–1.18 (m, 44H, H-4d–7d, H-14d–17d, and H-4a–17a),
0.92–0.81 (m, 6H, H-18a and H-18d). dC (75 MHz, CDCl3)
173.72 (C-1d in sn-2), 173.34 (C-1a in sn-1), 134.93 (C-12d),
129.94 (C-9d), 128.86 (C-10d), 128.70 (C-11d), 70.69 (C-2),
70.59 (C-2), 66.55 (C-b), 66.48 (C-b), 63.61 (C-3), 63.54 (C-3),
63.14 (C-1), 59.51 (C-a), 59.46 (C-a), 54.56 (–N(CH3)3), 34.45
(C-2d in sn-2), 34.28 (C-2a in sn-1), 33.05 (C-13d), 32.07
(C-16a), 31.89 (C-16d), 30.17–28.87 (C-4a–15a, C-4d–7d,
C-14d–15d), 27.84 (C-8d), 25.10 (C-3a), 25.05 (C-3d), 22.83
(C-17a), 22.76 (C-17d),14.27 (C-16a and C-18d). Note: signals
labelled ‘d’ are from the cis-9,trans-11 CLA moiety, those
labelled ‘a’ are from the stearoyl moiety and those labelled
‘a,b’ are from the choline moiety. dP (121 MHz, CDCl3)
16.48. m/z (HR-ESI) 784.5830; calc. for C44H84NO8P: 784.5851
[M þ H]þ.
General Procedure for 1-Octadecanoyl-2-acyl-sn-
glycero-3-phosphocholine Synthesis
Compounds 4c–e were prepared from 1-stearoyl-2-hydroxy-
sn-glycero-3-phosphocholine (3a) (100 mg, 0.191 mmol) by
adding 106 mg (0.381 mmol) of a mixture of CLA isomers (1c),
cis-9,trans-11 (1d) or trans-10,cis-12 CLA (1e) dissolved in
5.7 mL of anhydrous CH2Cl2, DMAP (53.8 mg, 0.191 mmol),
and finally a solution of DCC (119 mg, 0.419 mmol) in 1.9 mL
of CH2Cl2. The reaction was carried out for 48 h under a nitrogen
atmosphere in the dark and at ambient temperature. The pre-
cipitate that was formed in the reaction mixture was removed
using a Shott funnel. Ion-exchange resin (Dowex 50W X8, Hþ
form) was added to the filtrate and the mixture was stirred for
30 min to dislodge DMAP. The Dowex resin was filtered off and
the solvent was evaporated under vacuum. The crude PC was
purified on a silica gel column (65 : 25 : 4 CHCl3/CH3OH/H2O

Phosphatidylcholine with c9,t11 and t10,c12 CLA
1073
1-Octadecanoyl-2-(10E,12Z)-octadecadienoyl-sn-
glycero-3-phosphocholine 4e
(C-4a–15a, C-4d–7d, C-14d–15d, C-4e–8e, C-15e), 27.81 (C-8d
and C-14e), 25.13 (C-3a), 25.02 (C-3d or C-3e), 22.83 (C-17a),
22.76 (C-17d), 22.70 (C-17e),14.25 (C-16a and C-18d or
C-18e). Note: signals labelled ‘d’ are from the cis-9,trans-11
CLA moiety, those labelled ‘e’ are from the trans-10,cis-12
CLA moiety, those labelled ‘a’ are from the stearoyl moiety and
those labelled ‘a,b’ are from the choline moiety. dP (121 MHz,
CDCl3) ꢀ0.17. m/z (HR-ESI) 784.5859; calc. for C44H84NO8P:
784.5851 [M þ H]þ.
The title compound was obtained as a colourless greasy solid
(110 mg, 73 %) with 99 % purity (according to HPLC, Rt
6.72 min), traces of 1-octadecanoyl LPC. dH (300 MHz, CDCl3)
6.28 (ddd, J 15.0, 10.9, 1.1, 1H, H-11e), 5.93 (t, J 10.9, 1H,
H-12e), 5.64 (dt, J 15.0 7.0, 1H, H-10e), 5.29 (dt, J 10.9, 7.4, 1H,
H-13e), 5.19 (m, 1H, H-2), 4.39 (dd, J 12.0, 2.8, 1H, one of the
H-1), 4.34–4.25 (m, 2H, H-a), 4.12 (dd, J 12.0, 7.4, 1H, one of
the H-1), 3.98–3.86 (m, 2H, H-3), 3.83–3.75 (m, 2H, H-b), 3.35
(s, 9H, N(CH3)3), 2.35–2.22 (m, 4H, H-2e and H-2a), 2.20–2.03
(m, 4H, H-9e and H-14e), 1.65–1.48 (m, 4H, H-3e and H-3a),
1.46–1.18 (m, 44H, H-4e–8e, H-15e–17e, and H-4a–17a), 0.97–
0.81 (m, 6H, H-18a and H-18e). dC (75 MHz, CDCl3) 173.73
(C-1e in sn-2), 173.35 (C-1a in sn-1), 134.68 (C-10e), 130.31
(C-13e), 128.73 (C-12e), 125.84 (C-11e), 70.77 (C-2), 70.67
(C-2), 66.46 (C-b), 66.50 (C-b), 63.64 (C-3), 63.58 (C-3), 63.16
(C-1), 59.54 (C-a), 59.48 (C-a), 54.56 (–N(CH3)3), 34.46 (C-2e
in sn-2), 34.29 (C-2a in sn-1), 32.55 (C-9e), 32.09 (C-16a),
31.63 (C-16e), 30.15–28.97 (C-4a–15a, C-4e–8e, C-15e), 27.83
(C-14e), 25.11 (C-3a), 25.05 (C-3e), 22.84 (C-17a), 22.71
(C-17e),14.27 (C-16a and C-18e). Note: signals labelled ‘e’
are from the trans-10,cis-12 CLA moiety, those labelled ‘a’ are
from the stearoyl moiety and those labelled ‘a,b’ are from the
choline moiety. dP (121 MHz, CDCl3) ꢀ0.20. m/z (HR-ESI)
784.5829; calc. for C44H84NO8P: 784.5851 [M þ H]þ.
1-(9Z,11E)-Octadecadienoyl-2-octadecanoyl-sn-
glycero-3-phosphocholine 5d
The title compound was obtained as a colourless greasy solid
(121 mg, 80 %) with 98 % purity (according to HPLC, Rt
6.74 min), traces of 1-(9Z,11E)-octadecadienoyl LPC. dH
(300 MHz, CDCl3) 6.27 (ddd, J 15.0, 11.0, 1.1, 1H, H-11d),
5.93 (t, J 11.0, 1H, H-10d), 5.64 (dt, J 15.0, 7.0, 1H, H-12d), 5.29
(dt, J 15.0, 7.0, 1H, H-9d), 5.18 (m, 1H, H-2), 4.39 (dd, 12.0, 2.8,
1H, one of the H-1), 4.36–4.26 (m, 2H, H-a), 4.12 (dd, J 12.0,
7.4, 1H, one of the H-1), 3.97–3.86 (m, 2H, H-3), 3.82–3.73 (m,
2H, H-b), 3.36 (s, 9H, N(CH3)3), 2.35–2.21 (m, 4H, H-2d and
H-2a), 2.19–2.03 (m, 4H, H-8d and H-13d), 1.65–1.48 (m, 4H,
H-3d and H-3a), 1.43–1.18 (m, 44H, H-4d–7d, H-14d–17d, and
H-4a–17a), 0.92–0.81 (m, 6H, H-18a and H-18d). dC (75 MHz,
CDCl3) 173.69 (C-1a in sn-2), 173.38 (C-1d in sn-1), 134.94
(C-12d), 129.97 (C-9d), 128.84 (C-10d), 125.69 (C-11d), 70.67
(C-2), 70.58 (C-2), 66.57 (C-b), 66.54 (C-b), 63.60 (C-3), 63.58
(C-3), 63.14 (C-1), 59.50 (C-a), 59.46 (C-a), 54.60 (ꢀN(CH3)3),
34.47 (C-2a in sn-2), 34.25 (C-2d in sn-1), 33.05 (C-13d), 32.07
(C-16a), 31.89 (C-16d), 30.20–28.93 (C-4a–15a, C-4d–7d,
C-14d–15d), 27.83 (C-8d), 25.13 (C-3a), 25.01 (C-3d), 22.84
(C-17a), 22.76 (C-17d), 14.26 (C-16a and C-18d). Note: signals
labelled ‘d’ are from the cis-9,trans-11 CLA moiety, those
labelled ‘a’ are from the stearoyl moiety and those labelled
‘a,b’ are from the choline moiety. dP (121 MHz, CDCl3) ꢀ0.21.
m/z (HR-ESI) 784.5830; calc. for C44H84NO8P: 784.5851
[M þ H]þ.
General Procedure for 1-Acyl-2-octadecanoyl-sn-
glycero-3-phosphocholine Synthesis
Compounds 5c, 5d, 5e were prepared from 3c, 3d, 3e (100 mg
0.192 mmol) respectively, by adding 109 mg (0.384 mmol) of
stearic acid (1a) dissolved in 5.8 mL of anhydrous CH2Cl2,
DMAP (54.3 mg, 0.192 mmol), and finally a solution of DCC
(120 mg, 0.423 mmol) in 1.9 mL of CH2Cl2. The reaction was
carried out for 48 h under a nitrogen atmosphere in the dark and
at ambient temperature. The product was extracted according to
the method described for compounds 4c–d.
1-(Conjugated)linoleoyl-2-octadecanoyl-sn-
glycero-3-phosphocholine 5c
1-(10E,12Z)-Octadecadienoyl-2-octadecanoyl-sn-
glycero-3-phosphocholine 5e
The title compound was obtained as a colourless greasy solid
(121 mg, 80 %) with 99 % purity (according to HPLC, Rt
6.73 min), traces of 1-(conjugated)lineoyl LPC. dH (300 MHz,
CDCl3) 6.28 (m, 1H, H-11d and H-11e), 5.95 (m, 1H, H-10d and
H-12e), 5.65 (m, 1H, H-12d and H-10e), 5.31 (m, 1H, H-9d and
H-13e), 5.19 (m, 1H, H-2), 4.39 (dd, J 12.3, 2.9, 1H, one of the
H-1), 4.37–4.26 (m, 2H, H-a), 4.11 (dd, J 12.0, 7.2, 1H, one of
the H-1), 4.00–3.86 (m, 2H, H-3), 3.84–3.75 (m, 2H, H-b), 3.36
(s, 9H, N(CH3)3), 2.35–2.22 (m, 4H, H-2d or H-2e and H-2a),
2.21–1.99 (m, 4H, H-8d and H-13d or H-9e and H-14e),
1.65–1.48 (m, 4H, H-3d or H-3e and H-3a), 1.44–1.18 (m,
44H, H-4d–7d, H-14d–17d or H-4e–8e, H-15e–17e and H-4a–
17a), 0.94–0.82 (m, 6H, H-18a and H-18d or H-18e). dC
(75 MHz, CDCl3) 173.69 (C-1a in sn-2), 173.37 (C-1d or C-1e
in sn-1), 134.93 (C-12d), 134.67 (C-10e), 130.28 (C-13e),
129.96 (C-9d), 128.85 (C-10d), 128.71 (C-12e), 125.81
(C-11e), 125.70 (C-11d), 70.69 (C-2), 70.60 (C-2), 66.58
(C-b), 66.52 (C-b), 63.59 (C-3), 63.54 (C-3), 63.15 (C-1),
59.48 (C-a), 59.43 (C-a), 54.59 (–N(CH3)3), 34.48 (C-2a in
sn-2), 34.25 (C-2d and C-2e in sn-1), 33.05 (C-13d and C-9e),
32.07 (C-16a), 31.89 (C-16d), 31.62 (C-16e), 30.11–28.95
The title compound was obtained as a colourless greasy solid
(124 mg, 82 %) with 99 % purity (according to HPLC, Rt
6.73 min), traces of 1-(10E,12Z)-octadecadienoyl LPC. dH
(300 MHz, CDCl3) 6.28 (ddd, J 15.0, 10.9, 1.1, 1H, H-11e),
5.93 (t, J 10.9, 1H, H-12e), 5.64 (dt, J 15.0 7.0, 1H, H-10e), 5.29
(dt, J 10.9, 7.4, 1H, H-13e), 5.19 (m, 1H, H-2), 4.39 (dd, J 12.0,
2.8, 1H, one of the H-1), 4.36–4.23 (m, 2H, H-a), 4.11 (dd, J
12.0, 7.4, 1H, one of the H-1), 4.00–3.86 (m, 2H, H-3), 3.85–
3.73 (m, 2H, H-b), 3.35 (s, 9H, N(CH3)3), 2.35–2.22 (m, 4H, H-
2e and H-2a), 2.20–2.02 (m, 4H, H-9e and H-14e), 1.65–1.48
(m, 4H, H-3e and H-3a), 1.46–1.18 (m, 44H, H-4e–8e, H-15e–
17e, and H-4a–17a), 0.97–0.81 (m, 6H, H-18a and H-18e). dC
(75 MHz, CDCl3) 173.69 (C-1a in sn-2), 173.36 (C-1e in sn-1),
134.67 (C-10e), 130.28 (C-13e), 128.72 (C-12e), 125.81
(C-11e), 70.69 (C-2), 70.60 (C-2), 66.56 (C-b), 66.52 (C-b),
63.59 (C-3), 63.55 (C-3), 63.14 (C-1), 59.48 (C-a), 59.43 (C-a),
54.60 (–N(CH3)3), 34.48 (C-2a in sn-2), 34.26 (C-2e in sn-1),
33.04 (C-9e), 32.07 (C-16a), 31.62 (C-16e), 30.09–28.87 (C-4a–
15a, C-4e–8e, C-15e), 27.81 (C-14e), 25.13 (C-3a), 25.03
(C-3e), 22.83 (C-17a), 22.70 (C-17e),14.25 (C-16a and
C-18e). Note: signals labelled ‘e’ are from the trans-10,cis-12

1074
N. Niezgoda et al.
CLA moiety, those labelled ‘a’ are from the stearoyl moiety and
those labelled ‘a,b’ are from the choline moiety. dP (121 MHz,
CDCl3) ꢀ0.24. m/z (HR-ESI) 784.5877; calc. for C44H84NO8P:
784.5851 [M þ H]þ.
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Supplementary Material
1H and 13C NMR spectra for compounds 2d, 2e, 3a–e, 4c–e,
and 5c–e are available on the Journal’s website.
Acknowledgements
This project was financed by National Science Centre Project no. 2013/09/
D/NZ9/02457.
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