The chemical synthesis and preliminary biological studies of phosphodiester and phosphorothioate analogues of 2-methoxy-lysophosphatidylethanolamine

Article abstract of DOI:10.1016/j.bmcl.2016.05.075

The chemical synthesis of phosphorothioate/phosphodiester analogues of 2-methoxy-lysophosphatidylethanolamine has been described. For the preparation of phosphorothioate derivatives oxathiaphospholane approach has been employed. The phosphodiester compounds were prepared by OXONE oxidation of corresponding phosphorothioates. Each lysophospholipid analogue was synthesized as a series of four compounds, bearing different fatty acid residues both saturated (14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1→2 acyl migration. The cytotoxicity of newly synthesized 2-methoxy-lysophosphatidylethanolamine derivatives was evaluated with resazurin-based method in prostate cancer PC3 cell line. The highest reduction of cell viability was noted for LPE analogues containing myristoyl acyl chain.

Full text of DOI:10.1016/j.bmcl.2016.05.075

Accepted Manuscript
The chemical synthesis and preliminary biological studies of phosphodiester
and phosphorothioate analogues of 2-methoxy-lysophosphatidylethanolamine
Agata Sowińska, Przemysław Rytczak, Edyta Gendaszewska-Darmach, Anna
Drzazga, Maria Koziołkiewicz, Andrzej Okruszek
PII:
S0960-894X(16)30573-X
http://dx.doi.org/10.1016/j.bmcl.2016.05.075
BMCL 23934
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 March 2016
25 May 2016
26 May 2016
Please cite this article as: Sowińska, A., Rytczak, P., Gendaszewska-Darmach, E., Drzazga, A., Koziołkiewicz, M.,
Okruszek, A., The chemical synthesis and preliminary biological studies of phosphodiester and phosphorothioate
analogues of 2-methoxy-lysophosphatidylethanolamine, Bioorganic & Medicinal Chemistry Letters (2016), doi:
http://dx.doi.org/10.1016/j.bmcl.2016.05.075
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The chemical synthesis and preliminary biological
studies of phosphodiester and phosphorothioate
analogues of 2-methoxy-lysophosphatidylethanolamine
Agata Sowińska, Przemysław Rytczak, Edyta Gendaszewska-Darmach,
Anna Drzazga, Maria Koziołkiewicz, Andrzej Okruszek*
Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz
University of Technology, Stefanowskiego 4/10, 90-924 Lodz, Poland.
The chemical synthesis of phosphorothioate/phosphodiester analogues of 2-methoxy-
lysophosphatidylethanolamine has been described. For the preparation of phosphorothioate
derivatives oxathiaphospholane approach has been employed. The phosphodiester
compounds were prepared by OXONE® oxidation of corresponding phosphorothioates.
Each lysophospholipid analogue was synthesized as a series of four compounds, bearing
different fatty acid residues both saturated (14:0, 16:0, 18:0) and unsaturated (18:1). The
methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of
prepared analogues by preventing 1->2 acyl migration. The cytotoxicity of newly
synthesized 2-methoxy-lysophosphatidylethanolamine derivatives was evaluated with
resazurin-based method in prostate cancer PC3 cell line. The highest reduction of cell
viability was noted for LPE analogues containing myristoyl acyl chain.
-----------------------------------------------------------------------------------------------------------------
* Corresponding author.
E-mail address: andrzej.okruszek@p.lodz.pl (A. Okruszek).
For many years lysophospholipids (LPLs) have been considered as membrane
components functioning as mediators in the pathways of the synthesis of various
phospholipids and as structural components embedding proteins into cell membranes.
However, the rapidly expanding chemistry and biology of lipids revealed that
lysophospholipids also act as intercellular lipid mediators and can be regarded as hormone-
like signaling molecules.1 Primary representatives of this broad family are lysophosphatidic
acid (LPA), sphingosine-1-phosphate (S1P), and lysophosphatidylcholine (LPC). While LPA,
S1P or even LPC have been studied in detail, the actions of other LPLs such as
lysophosphatidylethanolamine (LPE) have not been investigated to such a degree.
Natural 1-O-acyl-sn-glycerol-3-O-phosphoethanolamine (lysophosphatidylethanolamine,
LPE, 1) has a zwitterionic structure (see Fig. 1) and exists in optically active form (R
enantiomer), as a mixture of several compounds, possessing fatty acids of different length
and saturation status (16:0, 18:0, 18:1,18:2, 20:1, 20:3, 20:4, 22:6). LPE accumulates in
human serum reaching concentration of several hundreds ng/mL.2
1

H
H
H
O
+
O
O
O
O
N
R
C O HO
H
P
Figure 1. The structure of natural R-lysophosphatidylethanolamine (LPE, 1).
Substituents R-C(O)- refer to various fatty acid residues present in lysophospholipids.
Informations concerning LPE as a bioactive lipid are rather sparse as compared to other
phospholipids. 16:0 LPE together with taurocholic acid, and 22:5 LPC have been defined as
“marker metabolites” that can be used to distinguish the different stages of
hepatocarcinogenesis.3 Regarding carcinogenesis, LPE was found to induce intracellular
calcium mobilization in OVCAR-3 and SK-OV3 human ovarian cancer cells as well as
chemotactic migration and cellular invasion in SK-OV3 cells.4
Lysophosphatidylethanolamine can also increase intracellular Ca2+ concentration as well as
cellular proliferation and migration in MDA-MB-231 breast cancer cells.5 Furthermore, LPE
from mushroom Grifola frondosa extract inhibited serum deprivation-induced apoptosis and
induced increase in intracellular Ca2+ concentration and neuronal differentiation in PC-12
neuronal cells.6
In our previous publications we have described the chemical synthesis of sulfur
analogues of LPA, cPA and LPC, in which either one or two nonbridging phosphate oxygen
atoms are substituted by sulfur to give phosphorothioate or phosphorodithioate analogues,
respectively.7-9 In addition, for LPA and LPC analogues also 2-hydroxyl function was
modified by its methylation. In many studies regarding lysophospholipids an attention of
researchers was paid mainly to S1P, LPA, cPA and LPC containing oleoyl or palmitoyl fatty
acid residues as representatives of two types LPL with unsaturated or saturated carbon
chains. Our recent studies have emphasized the significance of the sulfur and acyl chain
length on the biological activity of LPC10. These results as well as intriguing biological
properties of natural LPE prompted us to undertake studies on the synthesis of its
analogues possessing different acyl groups and modified both within phosphate group and
at carbon 2 of glycerol. On the basis of our earlier studies performed on the synthesis of
phosphorothioate derivatives of nucleotides and oligonucleotides11 we expected that
lysophospholipid phosphorothioate analogues should have similar physicochemical
properties as natural lysophospholipids, yet should be more resistant towards hydrolytic
enzymes.12 In order to prevent possible 1->2 acyl migration in LPE analogues, the oxygen
atom at position 2 of glycerol was protected by methylation.7-9,13,14 In fact this modification
should also increase the metabolic stability of both phosphorothioate and phosphodiester
analogues of LPE. The conditions of chemical synthesis allow us also to obtain LPE
analogues as homogenic compounds, each containing only one fatty acid residue. Thus, 1-
acyl-2-methoxy-LPE phosphorothioateanalogues (4a-d) were prepared as a series of four
2

different compounds, bearing the residues of the following fatty acids: a) myristic (14:0), b)
palmitic (16:0), c) stearic (18:0),d) oleic (18:1).
The diversity of biological functions and activity of lysophosphatidylcholine allowed us to
assume that LPE phosphorothioate/phosphodiester derivatives 4 and 5 may also have
interesting biological or even therapeutic properties. For preliminary biological studies the
LPE phosphorothioates (4a-d) and phosphodiesters (5a-d) were synthesized starting from
racemic glycidol.
The chemical synthesis of 1-acyl-2-methoxyglycerols (2a-d), that were crucial building
blocks for the preparation of 4a-d was performed exactly as described in our previous
papers.7-9 Thus, all four aforementioned fatty acids were reacted with commercially
available racemic glycidol in the presence of catalytic amounts of n-tributylamine into 1-
acylglycerols according to described procedure.15 In the following step 1-acylglycerols were
regioselectively silylated with t-butyldimethylsilyl chloride in the position 3 of glycerol
(primary hydroxyl group).16 The silyl ethers were then methylated at the central oxygen of
glycerol with trimethylsilyldiazomethane in the presence of 40% fluoroboric acid.17 The 2-
methoxy ethers were desilylated with tetra-n-butylammoniumfluoride in tetrahydrofuran,16
yielding desired glyceride building blocks 2a-d. All aforementioned products were isolated
and purified by silica gel flash column chromatography and characterized by spectroscopic
methods as described earlier.7-9
The introduction of one nonbridging sulfur atom into lysophosphatitydylethanolamine
molecule was performed using oxathiaphospholane approach.18 This method was
successfully employed for the synthesis of phosphorothioate nucleotide and oligonucleotide
analogues. Its application for the synthesis of phosphorothioate analogues of 1-acyl-2-
methoxy-lysophosphatidylethanolamine 4a-b is shown on Scheme 1.
O
O
O
S
O C R
O C R
O C R
1.
iPr2N
P
O
S-Ethylthiotetrazole
2. S8/CH2Cl2
OMe
OMe
OH
OMe
HOCH2CH2NH2
DBU
_
S
O
O
S
P
P
+
OCH2CH2NH3
O
S
O
2a-d
3a-d
4a-d
Scheme 1. The synthesis of LPE 1-acyl-2-methoxy-phosphorothioates (4a-d)
Acyl groups: a – myristoyl, b – palmitoyl, c – stearoyl, d – oleoyl; yield 56-83%
For the synthesis of phosphorothioate analogues racemic 1-acyl-2-methoxyglycerols
2a-d were reacted in anh. dichloromethane solution with equimolar amount of 2-N,N-
diisopropylamino-1,3,2-oxathiaphospholane in the presence of S-ethylthiotetrazole, and
then with elemental sulfur, to give 3-(2-thio-1,3,2-oxathiaphospholane) lipid derivatives 3a-d,
each showing in 31P NMR two signals at δ ca 105 ppm. Such chemical shifts were earlier
described as characteristic for various 2-alkoxy-2-thio-1,3,2-oxathiaphospholane
derivatives.18 Compounds 3a-d were isolated by silica gel flash column chromatography as
pale-yellow oils and characterized by spectroscopic methods. Due to appearance of two
3

signals in 31P NMR spectra it was concluded that each of 3a-d is a mixture of two
stereoisomers, that however could not be separated by flash chromatography or TLC. Each
of oxathiaphospholane derivatives 3a-d was then treated in anh. dichloromethane with 3
molar equivalents of ethanolamine and 2 molar equivalents of 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU). The ring opening condensation reaction occurred exclusively at hydroxyl
group of ethanolamine19 and was accompanied by spontaneous elimination of episulfide.18
Crude products were purified by silica gel flash column chromatography in
chloroform/methanol/water gradient to give LPE 1-acyl-2-methoxy-phosphorothioates (4a-d)
as white solids in 56-83% yield. Each product was obtained as a mixture of two
stereoisomers that was indicated by the presence of two signals in 31P NMR at δ ca 59
ppm. Such chemical shifts are characteristic for dialkyl phosphorothioates.11,18 The purified
products 4a-d were characterized by spectroscopic methods (1H and 31P NMR, MALDI TOF
MS). They were soluble in chloroform, methanol and water, and did not decompose when
stored at -20oC. The details are given in Table 1 and in supplementary material.
Table 1. Yields, 31P NMR and MS data of LPE 1-acyl-2-methoxy-phosphorothioates 4a-d
Compound 31P NMR (CDCl3) MALDI TOF MS (M-) Calculated MW
Yield
[%]
83
61
56
[ppm]
[m/z]
454,4
482,4
510,3
508,5
[Da]
4a
4b
4c
4d
59,85; 59,92
59,93; 60,01
59,19; 59,26
58,42; 58,50
455,61
483,66
511,71
509,70
82
The presence of two signals in 31P NMR spectra of each 4a-d (and also 3a-d) can be
attributed to the presence of two centers of asymmetry (at C2 and at phosphorus), leading
to the formation of two pairs of diastereoisomers. Unfortunately, these stereoisomers could
not be separated by either column or thin layer chromatography (TLC).
For the synthesis of phosphodiester LPE analogues each of racemic 1-acyl-2-methoxy-
LPE phosphorothioates 4a-d containing one of the aforementioned fatty acid residues were
reacted with an excess of OXONE® in methanol/acetate buffer (pH 5.6) by stirring for 18 h
at 38oC (see Scheme 2). The commercially available OXONE® reagent is a mixture of
potassium salts (2KHSO5xKHSO4xK2SO4) containing potassium peroxymonosulphate with
known oxidizing properties. Its application for the oxidation of various phosphorothioates
into corresponding phosphates was recently described in the literature.20
O
O
O C R
O C R
OMe
OMe
OXONE
acetate buffer
methanol
_
S
_
O
O
O
P
P
+
+
OCH2CH2NH3
OCH2CH2NH3
O
O
4a-d
5a-d
4

Scheme 2. The synthesis of LPE 1-acyl-2-methoxy-phosphodiesters (5a-d)
Acyl groups: a – myristoyl, b – palmitoyl, c – stearoyl, d – oleoyl; yield 12-22%
Crude products were purified by silica gel flash column chromatography to give 1-acyl-
2-methoxy-LPE phosphodiesters 5a-d as white solids in 12-22% yield. Compounds 5a-d
showed in 31P NMR single signals at δ ca 0 ppm, characteristic for dialkyl
phosphodiesters.21 The purified products 5a-d were characterized by spectroscopic
methods (1H and 31P NMR, LSI MS). They were soluble in chloroform, methanol and water,
and were found to be stable on storage at -20oC. The details are given in Table 2 and in
supplementary material. The products were obtained as enantiomeric mixtures.
Table 2. Yields, 31P NMR and MS data of 1-acyl-2-methoxy-LPE phosphodiesters 5a-d
Compound 31P NMR (CDCl3)
[ppm]
LSI MS (M-)
[m/z]
Calculated MW
[Da]
Yield
[%]
13
12
18
5a
5b
5c
5d
0,61
0,68
0,70
0,02
438,3
466,4
494,4
492,5
439,61
467,66
495,71
493,70
22
The chemical synthesis of both phosphodiester21,22 and phosphorothioate23 derivatives
of phosphatidylethanolamine, containing two fatty acid residues at positions 1 and 2 of
glycerol, has been described in literature with application of corresponding 3-
oksazaphospholidine intermediates. However, this methodology appeared to be ineffective
for the synthesis of aforementioned 1-acyl-2-methoxyl phosphodiester and
phosphorothioate analogues of lysophosphatidylethanolamine (data not shown).
All newly synthesized compounds 4a-d and 5a-d were subjected to biological
characterization for their in vitro cytotoxic activity in human prostate PC3 cancer cell line
(PC3), which is known to express LPA1 receptor24 utilized not only by LPA but also by
LPE.5 The cells (5,000 per well) were seeded onto 96-well plates and cultured for 24 and 48
h, followed by treatment with modified LPE derivatives. Viability of treated PC3 cells was
examined using a resazurin-based PrestoBlue cell viability reagent. The cytotoxicity data
are presented in Figures 2 and 3. All of the tested compounds, both at 5 μM and at 10 μM
concentration did not significantly inhibit PC3 viability. The exception was 10 μM 1-
myristoyl-2-methoxy-LPE (5a) reducing the number of viable cells to 68.2% and 59.8% after
24- and 48 hour treatment, respectively (Fig. 2).
5

A
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
*
*
*
*
5a
5b
5c
5d
B
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
*
*
*
*
*
5a
5b
5c
25 µM
5d
5 µM
10 µM
50 µM
Figure 2. The effect of 1-acyl-2-methoxy-LPC phosphodiester analogues 5a-d on the
viability of prostate cancer PC3 cells. Viability was assessed using PrestoBlue kit after 24 h
(A) and 48 h (B) of incubation. Data represent the means ± SD from at least three
independent experiments; *P < 0.05, versus unstimulated control cells (100 %)
Compound 5a appeared to be the most toxic also at higher concentration used (25 μM and
50 μM), reducing the cellular viability to 22.7% and 6.3% as compared to untreated control
cells after 48 hours. 1-Palmitoyl-2-methoxy-LPE (5b) appeared to be the second among
tested LPLs with regard to its cytotoxicity. After 48 h incubation 5b reduced the number of
cells to 78.8%, 49.5% and 24.9% when applied at the 10 μM, 25 μM and 50 μM
6

concentration, respectively. Significant cytotoxic effect was also observed for other species
without sulfur (5c and 5d), however the effect was lower than in the case of 5a and 5b.
It is noteworthy that all phosphorothioate analogues appeared to be less toxic as compared
to their phosphodiester counterparts (Fig. 3).
A
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
*
4a
4b
4c
4d
B
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
*
*
*
4a
4b
4c
4d
5 µM
10 µM
25 µM
50 µM
Figure 3. The effect of 1-acyl-2-methoxy-LPC phosphorothioate analogues 4a-d on the
viability of prostate cancer PC3 cells. Viability was assessed using PrestoBlue kit after 24 h
(A) and 48 h (B) of incubation. Data represent the means ± SD from at least three
independent experiments; *P < 0.05, versus unstimulated control cells (100 %)
7

1-Oleoyl-2-methoxy-lysophosphatidylethanolamine phosphorothioate (4d) and 1-stearoyl-2-
methoxy-lysophosphatidylethanolamine phosphorothioate (4c) did not exhibit any
cytotoxicity even at the 50 μM concentration. Other sulfur-containing compounds decreased
the number of viable cells after 48 h to 59.8% and 43.8% in the case of 1-mirystoyl-2-
methoxy-lysophosphatidylethanolamine phosphorothioate (4a) and to 79.1% and 70.0% for
1-palmitoyl-2-methoxy-lysophosphatidylethanolamine phosphorothioate (4b) used at the 25
μM and 50 μM concentration, respectively.
In summary, almost all tested compounds showed low toxicity against PC3 prostate
cancer cells when used at the 5 μM and 10 μM concentration. On the other hand, only 1-
stearoyl-2-methoxy-lysophosphatidylethanolamine phosphorothioate (4c) and 1-oleoyl-2-
methoxy-lysophosphatidylethanolamine phosphorothioate (4d) appeared to be non-toxic
even at the 50 μM concentration. Surprisingly, unlike in case of LPCs, the highest reduction
of cell viability was noted for LPE analogues containing myristoyl acyl chain. The myristoyl
LPC analogues were found by us to be much less toxic than corresponding palmitoyl
derivatives.9,10 Similar effect was also observed in neutrophils, where stimulation with 1-
palmitoyl-LPC induced calcium flux with modest membrane permeabilization.25
The new phosphorothioate analogues of 1-acyl-2-methoxy-LPE were prepared
employing oxathiaphospholane chemistry.18 It was found that their oxidation with OXONE®
can provide corresponding LPE phosphodiester analogues. For each analogue a series of
four compounds was synthesized, containing different fatty acid residues with both
saturated (14:0, 16:0, 18:0) and unsaturated (18:1) hydrocarbon chains. For preliminary
biological studies, the LPE analogues were prepared from racemic glycidol. Thus, LPE
phosphorothioate analogues were obtained as a mixture of diastereoisomers and
phosphodiester analogues were enantiomers. The attempts to synthesize the most active
myristoyl compounds in stereodefined form at C2 carbon will be performed in the next step
of research starting from enantiomers of glycidol.
Recent studies on chemistry and biology of lysophospholipids revealed that these
compounds are important intercellular lipid mediators and can be regarded as signaling
molecules.1 However, further development of research focused on molecular functions of
LPL and their receptors requires search of highly specific agonists and antagonists. Our
methods allow to synthesize wide range of new, glycerol-based LPL analogues with unique
structures and properties different from their native counterparts. These compounds were
found to be valuable tools in the studies on the LPL mechanism of action.8-10
The main goal of our previous studies has been the chemical synthesis of LPL
analogues (LPA, cPA, LPC) modified by the presence of methoxyl group in sn-2 position of
glycerol skeleton (LPA, LPC) and presence or absence of phosphorothioate function within
hydrophilic head group.2b,7-10 In the course of those studies, followed by preliminary
biological investigations, we have noted that important factor influencing biological activities
of cPA and LPC analogues is acyl chain length and geometry. However, one should take
into account that the concept of research regarding this issue has been neglected in many
cases; the best documented studies on activities of cPA or LPC species concern only 18:1
(oleoyl) and 16:0 (palmitoyl) derivatives. Significant cytotoxicity was observed only in the
case of 16:0 LPC analogues, while other derivatives appeared to be much less toxic.10 We
suggested that this effect might be caused by some nonspecific interactions of palmitoyl
8

LPC with cell membrane and its partial permeabilization.10 In earlier studies of other authors
I was observed that LPC 16:0 was more active in ROS generation than other LPC with
different length of fatty chain.26 Moreover, oleoyl LPC with unsaturated acyl chain (18:1)
couldn’t produce reactive oxygen species.26
Taking into account these observations we decided to synthesize LPE analogues with
four different acyl chains: 14:0, 16:0, 18:0 and 18:1.They were supposed to exhibit the
similar cytotoxic profile as LPC due to high structure similarity. Unexpectedly, the highest
reduction of PC3 cell viability was observed for LPE analogues containing myristoyl acyl
chain unlike in the case LPC where palmitoyl-containing derivative was found to be most
toxic. For comparison, the cytotoxicity data of corresponding 1-acyl-2-methoxy-LPC
analogues (phosphodiesters and phosphorothioates) obtained in the same cell system are
presented in supplementary material. These results do not confirm abovementioned
nonspecific damage of cell membrane and indicate an existence of unknown factor(s) that
discriminate LPE and LPC containing different acyl chains.
It was demonstrated that unsaturation of the acyl chain makes the compound less-toxic
(in the case of phosphodiester LPE analogues) or non-toxic (in the case of
phosphorothioate LPE analogues) as compared to the cytotoxicity of other LPE analogues.
Although specific LPE receptors have not been identified, Im et al. suggested that
membrane LPA1 receptor can be activated not only by LPA but also by LPE. 4,5b On the
other hand, some results of his group indicate no involvement of LPA1 receptor in LPE-
induced cellular response.4,5b We suggest that highest cytotoxicity of myristoyl LPE
derivatives as compared to other LPE analogues cannot be mediated by the single LPA1
receptor. A recently resolved crystal structure of LPA1 indicates that the receptor can
recognize a diverse set of ligands with different lengths and geometry of acyl chains rather
than being specific to the commonly used oleoyl LPA (18:1) and probably cannot
discriminate between myristoyl-, palmitoyl- and oleoyl-LPE.27 Therefore, factors
discriminating structurally related LPE remain to be identified.
Cytotoxicity of sulfur-containing LPE analogues 4a-d appeared to be lower than those
observed for corresponding phosphodiester compounds 5a-d. Similar effects have been
observed in our earlier studies for the corresponding LPC analogues.9,10 This unexpected
result should be further analyzed by molecular modeling methods and/or physicochemical
approach.
Acknowledgement
This work was supported by a grant from the Polish National Science Centre (grant no.
2011/01/B/ST5/06383).
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Graphical abstract
The Chemical Synthesis and Preliminary Biological Studies
of Phosphodiester and Phosphorothioate Analogues
of 2-Methoxy-lysophosphatidylethanolamine
Agata Sowińska, Przemysław Rytczak, Edyta Gendaszewska-Darmach,
Anna Drzazga, Maria Koziołkiewicz and Andrzej Okruszek
+
O
O
OCH2CH
2NH3
R
C O
OMe
P
X
O
Phosphodiesters (X=O); Phosphorothioates (X=S)
2-OMe-LPE phosphorothioates (myristoyl, palmitoyl, stearoyl and oleoyl derivatives)
were prepared by oxathiaphospholane method; their oxidation with OXONE® led to
corresponding phosphodiester analogues. The products were tested for their cytotoxicity
toward prostate cancer PC3 cell line.
11