Synthesis of enantiopure 2-Carba-cyclic phosphatidic acid and effects of its chirality on biological functions

Article abstract of DOI:10.1016/j.bbalip.2011.01.003

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator, which has a quite unique cyclic phosphate ring at sn-2 and sn-3 positions of the glycerol backbone. We have designed and chemically synthesized several metabolically stabilized derivatives of cPA. 2-Carba-cPA (2ccPA) is one of the synthesized compounds in which the phosphate oxygen was replaced with a methylene group at the sn-2 position, and it showed much more potent biological activities than natural cPA. Here, we developed a new method of 2ccPA enantiomeric synthesis. And we examined the effects of 2ccPA enantiomers on autotaxin (ATX) activity, cancer cell invasion and nociceptive reflex. As well as racemic-2ccPA, both enantiomers showed inhibitory effects on ATX activity, cancer cell invasion and nociceptive reflex. As their effects were not significantly different from each other, the chirality of 2ccPA may not be critical for these biological functions of 2ccPA.

Full text of DOI:10.1016/j.bbalip.2011.01.003

Biochimica et Biophysica Acta 1811 (2011) 271–277
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbalip
Synthesis of enantiopure 2-carba-cyclic phosphatidic acid and effects of its chirality
on biological functions
Emi Nozaki a, Mari Gotoh a, Harumi Hotta b, Shuwa Hanazawa a,c
,
Susumu Kobayashi c, Kimiko Murakami-Murofushi a,
⁎
a
Graduate School of Humanities and Sciences, Department of Life Science, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan
Department of Autonomic Neuroscience, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan
Faculty of Pharmaceutical Sciences, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator, which has a quite unique cyclic
phosphate ring at sn-2 and sn-3 positions of the glycerol backbone. We have designed and chemically
synthesized several metabolically stabilized derivatives of cPA. 2-Carba-cPA (2ccPA) is one of the synthesized
compounds in which the phosphate oxygen was replaced with a methylene group at the sn-2 position, and it
showed much more potent biological activities than natural cPA. Here, we developed a new method of 2ccPA
enantiomeric synthesis. And we examined the effects of 2ccPA enantiomers on autotaxin (ATX) activity,
cancer cell invasion and nociceptive reflex. As well as racemic-2ccPA, both enantiomers showed inhibitory
effects on ATX activity, cancer cell invasion and nociceptive reflex. As their effects were not significantly
different from each other, the chirality of 2ccPA may not be critical for these biological functions of 2ccPA.
© 2011 Elsevier B.V. All rights reserved.
Received 20 November 2010
Received in revised form 29 December 2010
Accepted 13 January 2011
Available online 28 January 2011
Keywords:
Enantiopure 2-carba-cyclic phosphatidic acid
Stereoselectivity
Chirality
Autotaxin
Cancer cell invasion
Nociceptive reflex
1. Introduction
As the cyclic phosphate ring of cPA would be opened by hydrolysis
resulted in the formation of LPA [11], we have synthesized several
Cyclic phosphatidic acid (cPA) is a naturally occurring phospho-
lipid mediator. This bioactive lipid was originally isolated and
identified from myxoamoebae of a true slime mold, Physarum
polycephalum [1]. At present, cPA has been found in a wide range of
organisms, from slime molds to humans [2,3]. The chemical formula of
cPA is similar to lysophosphatidic acid (LPA), but cPA has a quite
unique structure with a cyclic phosphate ring at sn-2 and sn-3
positions of the glycerol backbone, and a chiral carbon atom is
assigned an R designation [4]. These features of cPA may provide the
difference of biological functions of cPA and LPA. For example, LPA
stimulates cell proliferation and induces cancer cell invasion and
metastasis [5]. The intraplantar injection of LPA into the hind limb of
mice induces nociceptive flexor responses [6], and intrathecal
injection of LPA induces neuropathic pain in mice [7]. In contrast,
cPA inhibits autotaxin (ATX) activity, cancer cell invasion and
metastasis [8,9]. And recently, we found that cPA suppressed
nociceptive responses by primary afferent C-fiber [10].
metabolically stabilized carba derivatives of cPA (ccPA) [11,12].
2-Carba-cPA (2ccPA) is the compound in which one of the phosphate
oxygen is replaced with a methylene group at the sn-2 position. We
have revealed that 2ccPA well conserved numerous biological
functions of cPA, and the inhibition of cancer cell invasion and
metastasis [11] and nociceptive reflex suppression [10] by 2ccPA were
much more potent than those of natural cPA. These results suggested
that 2ccPA could be used as a therapeutic compound for cancer and
pain. Meanwhile, these data were obtained by using a racemic-2ccPA,
due to the difficulty of enantiopure 2ccPA synthesis. As it is well
known that each enantiomer shows same or different biological
activities in many cases, the determination of biological activities of
each enantiopure 2ccPA is strongly required for further 2ccPA studies.
In this report, we newly developed the method of 2ccPA
enantiomer synthesis. And we clarified the effects of each enantiomer
on ATX activity, cancer cell invasion and nociceptive reflex.
2. Materials and methods
2.1. Synthesis of (R)-2ccPA (Fig. 1a) and (S)-2ccPA (Fig. 1b)
Abbreviations: cPA, Cyclic phosphatidic acid; LPA, Lysophosphatidic acid; ATX,
Autotaxin; 2ccPA, 2-carba-cyclic phosphatidic acid
(2R,3E)-2-(Hydroxymethyl)-5-methylhex-3-enyl acetate (2) [13].
To a solution of (3E)-2-(hydroxymethyl)-5-methylhex-3-enol
(1, 36.9 g) in vinyl acetate (500 ml) was added molecular sieves 3A
⁎
Corresponding author. Tel./fax: +81 3 5978 5362.
E-mail address: murofushi.kimiko@ocha.ac.jp (K. Murakami-Murofushi).
1388-1981/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbalip.2011.01.003

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E. Nozaki et al. / Biochimica et Biophysica Acta 1811 (2011) 271–277
Fig. 1. a. synthesis of (R)-2ccPA (13). Reagents and conditions: (a) PPL, MS 3A, Vinyl acetate, rt, 24 h, 52%; (b) I2, PPh3, imidazole, THF, rt, 12 h, 98%; (c) HP(O)(OMe)2, Cs2CO3, TBAI,
DMF, rt, 24 h, 66%; (d) K2CO3, MeOH, −20 °C, 2 h, 5 64% and 6 8%; (e) BOMCl, iPr2NEt, CH2Cl2, rt, 4.5 h, 94%; (f) O3, MeOH, CHCl3, −78 °C, 3.5 h, then sodium borohydride, rt, 1 h, 80%;
(g) Et3N, toluene, 80 °C, 36 h, 39%; (h) H2, Pd(OH)2, THF, rt, 6 days, 64%; (i) palmitoleic chloride, Et3N, 4-DMAP, rt, 19 h, 75%; (j) TMSBr, CH2Cl2, −20 °C, 75%; (k) 0.01 N NaOH aq,
extraction, 43%. (R=(CH2)7CH=CH(CH2)5CH3). b. Synthesis of (S)-2ccPA (13a). Reagents and conditions: (a) O3, MeOH, CHCl3, −78 °C, 1 h, then PPh3, NaBH4, 0 °C, 1 h, 88%.
(b) These reagents and conditions follow the same procedures for 9–13. (R=(CH2)7CH=CH(CH2)5CH3).
(18.5 g) and PPL (porcine pancreas lipase, 18.5 g) at room temper-
ature. The reaction mixture was stirred for 24 h and then filtered
through Celite and concentrated in vacuo. The residue was purified by
chromatography on silica gel (hexane/ethyl acetate=20/1 to 1/1) to
afford 2 (24.6 g, 52%) as a colorless oil. Enantiomeric excess was
determined to be N99% ee by chiral HPLC (DAICEL OD, hexane:
i-PrOH=50:1, flow rate 1.0 mL/min, monitoring 220 nm, Rt;
12.41 min for 2, and 15.96 min for ent-2).
solution and extracted with ethyl acetate. The combined organic layer
was washed with brine, dried over Na2SO4, filtered and concentrated
in vacuo. The residue was purified by chromatography on silica gel
(hexane) to afford 3 (149 g, 98%) as a yellow oil.
1H NMR (600 MHz, CDCl3) δH: 5.56 (dd, 1H, J=15.6, 6.8 Hz), 5.18
(ddd, 1H, J=15.6, 8.2, 1.2 Hz), 4.14 (dd, 1H, J=11.0, 5.6 Hz), 3.99 (1H,
dd, J=11.0, 7.2 Hz), 3.20–3.25 (m, 2H), 2.45 (m, 1H), 2.28 (m, 1H),
2.06 (s, 3H), 0.99(d, 3H, J=6.8 Hz), 0.98 (d, 3H, J=6.8 Hz); 13C NMR
(100 MHz, CDCl3) δc: 9.3, 20.9, 22.3, 22.3, 31.2, 42.8, 66.6, 124.8, 144.8,
170.8; [α]D24 +4.51 (c 1.04, CHCl3); HRMS (ESI) calcd for C10H17IO2Na
[M+Na]+ 319.0171, found 319.0170.
1H NMR (600 MHz, CDCl3) δH: 5.61 (dd, 1H, J=15.6, 6.6 Hz), 5.24
(ddd, 1H, J=15.6, 8.2, 1.2 Hz), 4.19 (dd, 1H, J=11.2, 5.6 Hz), 4.07 (1H,
dd, J=11.2, 7.2 Hz), 3.46–3.70 (m, 2H), 2.52 (m, 1H), 2.29 (m, 1H),
2.07 (s, 3H), 0.98 (d, 6H, J=6.6 Hz); [α]D23 +27.8 (c 2.35, CHCl3); HRMS
(ESI) calcd for C10H18O3Na [M+Na]+ 209.1153, found 209.1154.
(2 S,3E)-2-(Iodomethyl)-5-methylhex-3-enyl acetate (3).
To a solution of 2 (95.4 g) in cold THF (500 ml) was added
triphenylphosphine (161 g), imidazole (41.8 g) and iodine (156 g).
The reaction mixture was stirred at room temperature. After 12 h, the
reaction mixture was quenched with saturated Na2S2O3 aqueous
(2 S,3E)-Dimethyl 2-(acetoxymethyl)-5-methylhex-3-enylpho-
sphonate (4).
To a solution of dimethyl phosphite (13 ml), Cs2CO3 (49.5 g) and
tetrabutylammonium iodide (5.4 g) in DMF (100 ml) was added a
solution of 3 (21.4 g) in DMF (50 ml), and stirred for 24 h at room
temperature. The reaction mixture was quenched with water (the
solution was became clear), and extracted with ethyl acetate. The

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273
combined organic layer was washed with water and brine and dried
over Na2SO4, filtered and concentrated in vacuo. The residue was
purified by chromatography on silica gel (hexane/ethyl acetate=2/1)
to afford 4 (13.3 g, 66%) as a colorless oil.
1H NMR (600 MHz, CDCl3) δH: 7.28–7.48 (m, 5H), 4.76 (s, 2H), 4.60
(s, 2H), 3.75 (d, 3H, J=10.8 Hz), 3.75 (d, 3H, J=10.8 Hz), 3.69–3.73 (m,
2H), 3.64 (d, 2H, J=5.7 Hz), 2.97 (br, 1H), 2.20–2.27 (m, 1H), 1.82–1.96
(m, 2H); 13C NMR (100 MHz, CDCl3) δc: 23.3, 24.7, 36.5, 52.5, 64.2, 64.3,
69.8, 69.9, 94.9, 127.8, 127.8, 128.5, 128.5, 137.7; [α]D23 +1.4 (c 1.00,
CHCl3); HRMS (ESI) calcd for C14H23O6PNa [M+Na]+ 341.1131, found
341.1129.
1H NMR (600 MHz, CDCl3) δH: 5.55 (dd, 1H, J=15.4, 6.8 Hz), 5.23
(ddd, 1H, J=15.4, 8.6, 1.0 Hz), 3.90–4.15 (m, 2H), 3.73 (d, 3H,
J=1.1 Hz), 3.71 (d, 3H, J=1.1 Hz), 2.76 (m, 1H), 2.26 (m, 1H), 2.05
(s, 3H), 1.93–2.00 (m, 1H), 1.74–1.81 (m, 1H), 0.97 (d, 6H, J=6.8 Hz);
13C NMR (100 MHz, CDCl3) δc: 20.9, 22.3, 22.3, 27.1, 30.9, 36.6, 52.2,
52.2, 67.2, 125.7, 125.8, 170.8; [α]D24 +5.49 (c 1.01, CHCl3); HRMS
(ESI) calcd for C12H23O5PNa [M+Na]+ 301.1180, found 301.1180.
(2 S,3E)-Dimethyl 2-(hydroxymethyl)-5-methylhex-3-enylpho-
sphonate (5) and (E)-6-methylpent-4-ene-2-methoxy-2λ5-[1,2]oxa-
phospholane 2-oxide (6).
(R)-4-Benzyloxymethoxymethyl-2-methoxy-[1,2]oxaphospholane
2-oxide (9).
To a stirred solution of 8 (12.4 g) in toluene (80 ml) was added
triethylamine (6.6 ml) at 80 °C. After 36 h, the reaction mixture was
concentrated in vacuo. The residue was purified by chromatography
on silica gel (methanol/chloroform=1/50) to afford 9 (4.42 g, 39%) as
a colorless oil.
To a solution of 4 (13.3 g) in MeOH was added K2CO3 (9.9 g) at
−20 °C, and stirred for 2 h. The reaction mixture was quenched with
saturated NH4Cl aqueous solution and extracted with chloroform. The
combined organic layer was washed with brine and dried over
Na2SO4, filtered and concentrated in vacuo. The residue was purified
by chromatography on silica gel (ethyl acetate) to afford 5 (7.25 g,
64%) as a colorless oil and 6 (0.75 g, 8%) as a colorless oil.
1H NMR (600 MHz, CDCl3) δH: 7.29–7.38 (m, 5H), 4.75 (s, 2H), 4.56
(s, 2H), 4.16–4.33 (m, 1H), 3.82–4.01 (m, 1H), 3.79 (d, 1.5H,
J=6.6 Hz), 3.77 (d, 1.5H, J=6.6 Hz), 3.55–3.63 (m, 2H), 2.76–2.90
(m, 1H), 1.95–2.05 (m, 1H), 1.69–1.76 (m, 2H); 13C NMR (100 MHz,
CDCl3) δc: 21.1, 37.1, 52.4, 67.7, 68.9, 69.6, 94.6, 127.6, 127.6, 128.3,
129.5, 129.5, 137.4; IR (neat): 2952, 2904, 1454, 1270, 1053, 996, 859,
824, 742, 701, 581 cm-1; HRMS (ESI) calcd for C13H19O5PNa [M+Na]+
309.0862, found 309.0876.
Compound 5: 1H NMR (600 MHz, CDCl3) δH: 5.55 (dd, 1H, J=15.4,
6.6 Hz), 5.26 (ddd, 1H, J=15.4, 8.2, 0.8 Hz), 3.74 (dd, 6H, J=11.0,
6.0 Hz), 3.54–3.59 (m, 2H), 2.66 (brs, 1H), 2.57–2.66 (m, 1H), 2.23–
2.31 (m, 1H), 1.82–1.95 (m, 1H), 1.74–1.81 (m, 1H), 0.98 (d, 3H,
J=6.8 Hz), 0.97 (d, 3H, J=6.8 Hz); 13C NMR (100 MHz, CDCl3) δc:
22.4, 22.4, 27.1, 30.9, 39.9, 52.3, 52.4, 66.3, 126.7, 126.9; [α]D23 +4.5
(c 0.99, CHCl3); HRMS (ESI) calcd for C10H21O4PNa [M+Na]+
259.1075, found 259.1082.
(R)-(2-Methoxy-2-oxo-2λ5-[1,2]oxaphospholan-4-yl)-methanol
(10).
To a solution of 9 (7.87 g) in THF (140 ml) was added 20% Pd
(OH)2/C at room temperature. The reaction mixture was stirred 6 days
under hydrogen atmosphere and then filtered through Celite and
concentrated in vacuo. The residue was purified by chromatography
on silica gel (methanol/chloroform=1/10) to afford 10 (2.91 g, 64%)
as a colorless oil.
Compound 6: 1H NMR (600 MHz, CDCl3) δH: 5.56 (dd, 1H, J=15.4,
6.6 Hz), 5.22 (m, 1H), 4.10–4.28 (m, 1H), 3.79 (ddd, 3H, J=11.0, 2.0,
0.6 Hz), 3.60–3.82 (m, 1H), 3.04–3.22 (m, 1H), 2.22–2.30 (m, 1H),
2.01–2.17 (m, 1H), 1.60–1.73 (m, 1H), 0.97 (d, 6H, J=6.8 Hz); HRMS
(ESI) calcd for C9H17O3PNa [M+Na]+ 227.0807, found 227.0814.
(2 S,3E)-Dimethyl 2-(benzyloxymethoxymethyl)-5-methylhex-3-
enylphosphonate (7).
To a solution of 5 (7.17 g) in dichloromethane (60 ml) was added
diisopropylethylamine (7.2 ml) at 0 °C under argon atmosphere.
Benzylchloromethyl ether (5.8 ml) was added to the solution and,
the reaction mixture was allowed to warm up to room temperature
and stirred for 4.5 h. The reaction mixture was quenched with
saturated NH4Cl aqueous solution and extracted with ethyl acetate.
The combined organic layer was washed with brine and dried over
Na2SO4, filtered and concentrated in vacuo. The residue was purified
by chromatography on silica gel (hexane/ethyl acetate=3/1) to
afford 7 (10.2 g, 94%) as a colorless oil.
1H NMR (600 MHz, CDCl3) δH: 4.17–4.36 (m, 1H), 3.89–4.11
(m, 1H), 3.80 (d, 1.5H, J=3.6 Hz), 3.78 (d, 1.5H, J=3.6 Hz), 3.67–3.73
(m, 2H), 2.70–2.85 (m, 1H), 1.96–2.06 (m, 1H), 1.71–1.90 (m, 2H); 13
C
NMR (100 MHz, CDCl3) δc: 20.9, 39.2, 52.6, 62.3, 69.1; IR (neat): 3387,
2955, 1464, 1414, 1258, 1049, 989, 862, 824 cm-1; HRMS (ESI) calcd
for C5H11O4PNa [M+Na]+ 189.0293, found 189.0282.
(R)-Palmitoleic acid 2-methoxy-2-oxo-2λ5-[1,2]oxaphospholan-
4-ylmethyl ester (11).
To a stirred solution of 10 (439 mg) in dichloromethane (6.6 mL)
was added palmitoleic chloride (936 mg), triethylamine (0.5 ml) and
4-DMAP (64.5 mg) at 0 °C. The reaction mixture was allowed to warm
up to room temperature and stirred for 19 h. The reaction mixture
was quenched with brine and extracted with ethyl acetate. The
combined organic layer was washed with brine and dried over
Na2SO4, filtered and concentrated in vacuo. The residue was purified
by chromatography on silica gel (hexane/ethyl acetate=1/2 to 1/4) to
afford 11(792 mg, 75%) as a colorless oil.
1H NMR (600 MHz, CDCl3) δH: 7.28–7.36 (m, 5H), 5.36 (dd, 1H,
J=15.4, 6.4 Hz), 5.31 (ddd, 1H, J=15.6, 8.2, 1.2 Hz), 4.75 (s, 2H), 4.59
(s, 2H), 3.70 (d, 6H, J=10.8 Hz), 3.57 (ddd, 1H, J=9.4, 6.8, 2.0 Hz),
3.48 (dd, 1H, J=9.4, 6.8 Hz), 2.68–2.73 (m, 1H), 2.24–2.29 (m, 1H),
2.06–2.13 (m, 1H), 1.72–1.76 (m, 1H), 0.98 (d, 3H, J=6.8 Hz), 0.97
(d, 3H, J=6.8 Hz); 13C NMR (100 MHz, CDCl3) δc: 22.3, 22.4, 26.4,
27.8, 30.9, 37.4, 52.1, 69.4, 71.6, 94.6, 126.7, 126.8, 127.7, 127.8, 128.4,
128.4, 137.8, 139.6; [α]D23 +21.0 (c 1.03, CHCl3); HRMS (ESI) calcd for
1H NMR (600 MHz, CDCl3) δH: 5.26–5.33 (m, 2H), 4.23–4.29 (m,
0.5H), 4.02–4.18 (m, 2H), 3.90–3.95 (m, 0.5H), 3.77–3.80 (m, 1H), 3.76
(dd, 3H, J=11.2, 3.6 Hz), 3.67–3.73 (m, 2H), 2.80–2.91 (m, 1H), 2.27 (t,
2H, J=7.2 Hz), 2.00–2.07(m, 1H), 1.94–1.99 (m, 4H), 1.62–1.71 (m, 1H),
1.56–1.58 (m, 2H), 1.28–1.30 (br, 16H), 0.88 (t, 3H, J=6.8 Hz); 13C NMR
(100 MHz, CDCl3) δc: 14.1, 20.7, 22.3, 22.6, 24.8, 27.1, 27.2, 29.0, 29.1,
29.1, 29.6, 29.7, 31.7, 34.0, 36.3, 52.8, 64.0, 68.5, 129.7, 130.0, 173.4; IR
(neat): 2926, 2854, 1739, 1465, 1272, 1175, 1049, 1005, 860, 823 cm-1;
HRMS (ESI) calcd for C21H39O5P [M+] 402.2535, found 402.2541.
(R)-Palmitoleic acid 2-hydroxy-2-oxo-2λ5-[1,2]oxaphospholan-
4-ylmethyl ester (12).
C
18H29O5PNa [M+Na]+ 379.1650, found 379.1660.
(S)-(2-Benzyloxymethoxymethyl-3-hydroxypropyl)-phosphonic
acid dimethyl ester (8).
To a stirred solution of 7 (9.37 g) in dichloromethane (90 ml) and
methanol (50 ml) was bubbled O3 at −78 °C until the color of solution
turned blue. After 3.5 h, the reaction mixture was treated sodium
borohydride (6.0 g), allowed room temperature and stirred for 1 h. The
reaction mixture was quenched with saturated NH4Cl aqueous solution
and extracted with dichloromethane. The combined organic layer was
dried over Na2SO4, filtered and concentrated in vacuo. The residue was
purified by chromatography on silica gel (methanol/chloroform=1/30
to 1/20) to afford 8 (6.72 g, 80%) as a colorless oil.
To a stirred solution of 11 (959 mg) in dichloromethane (12 ml) was
added bromotriethylsilane (0.95 ml) at −20 °C. After 1 h, the reaction
mixture was allowed to warm up to 0 °C and stirred for 1 h. The reaction
mixture was concentrated in vacuo. The residue was purified by
chromatography on silica gel (methanol/chloroform=1/10) to afford
11(792 mg, 75%) as a colorless oil.
1H NMR (500 MHz, CDCl3+1 drop CD3OD) δH: 5.31–5.38 (m, 2H),
3.91–4.31 (m, 4H), 2.85–2.95 (m, 1H), 2.31 (t, 2H, J=7.6 Hz), 2.05–2.12

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E. Nozaki et al. / Biochimica et Biophysica Acta 1811 (2011) 271–277
(m, 1H), 1.99–2.03 (m, 4H), 1.70–1.78 (m, 1H), 1.59–1.63 (m, 2H),
1.26–1.30 (br, 16H), 0.88 (t, 3H, J=6.8 Hz); 13C NMR (100 MHz, CDCl3)
δc: 1.0, 14.1, 22.6, 24.9, 27.1, 27.2, 28.9, 29.0, 29.1, 29.2, 29.3, 29.7, 29.8,
31.7, 34.1, 59.7, 61.3, 129.7, 129.9, 174.0; 31P NMR (400 MHz, CD3OD)
δP: 48.75.
(R)-2ccPA (13).
A solution of 12 (900 mg) in diethyl ether (250 ml) was extracted
with 0.01 mol/L sodium hydroxide aqueous solution 6 times (total
volume, 250 ml). The aqueous layer was freeze-dried and the residue
was purified by chromatography on silica gel (methanol/chloroform/
water=80/400/1). The purified compound was dissolved water and
freeze-dried to afford 13 (428 mg, 43%) as a white powder.
1H NMR (500 MHz, CDCl3 +1 drop CD3OD) δH: 5.30–5.40 (m, 2H),
3.96–4.34 (m, 3H), 3.66–3.78 (m, 1H), 2.73–2.87 (m, 1H), 2.29 (t, 2H,
J=7.4 Hz), 1.98–2.05 (m, 4H), 1.77–1.87 (m, 1H), 1.57–1.65 (m, 2H),
1.37–1.48 (m, 1H), 1.24–1.37 (br, 16H), 0.87 (t, 3H, J=6.8 Hz) ; 13C
NMR (100 MHz, CDCl3) δc: 14.4, 23.7, 26.0, 28.1, 28.1, 30.0, 30.1, 30.2,
30.2, 30.8, 30.8, 32.9, 34.9, 38.6, 38.6, 66.6, 67.8, 130.8, 130.9, 175.3;
31P NMR (400 MHz, CD3OD) δP: 42.64; [α]D23 -12.3 (c 0.71, CHCl3).
(S)-4-Benzyloxymethoxymethyl-2-methoxy-[1,2]oxaphospho-
lane 2-oxide (10a).
Fig. 2. Effects of chirality of 2ccPA on ATX activity. The catalytic activity of ATX in CCM
was examined in the presence of (R)-2ccPA 16:1 (closed circle), (S)-2ccPA 16:1 (open
circle) and racemic-2ccPA 16:1 (closed square). Each data point represents the mean
S.E. of triplicate wells and is representative of at least three independent experiments
with similar results. Each data was calculated as percent inhibition in comparison
respective controls of 100% without any compounds.
To a stirred solution of 6 (114.7 mg) in dichloromethane (2.8 ml)
and methanol (4.7 ml) was bubbled O3 at −78 °C until the color of
solution turned blue. After 1 h, the reaction mixture was treated
triphenylphosphine (162.0 mg) and allowed to 0 °C. The reaction
mixture was treated sodium borohydride (63.7 mg) and stirred for
1 h. The reaction mixture was quenched with acetic acid (0.1 ml) and
concentrated in vacuo. The residue was purified by chromatography
on silica gel (methanol/chloroform=1/30) to afford 10a (81.8 mg,
88%) as a colorless oil.
KCl, 140 mM NaCl, 50 mM Tris–HCl, pH 8.0). The fluorescence
intensity (em/ex: 485/530 nm) of each well was measured by Cyto
Flour series 4000 micro plate reader (Applied Biosystems, Foster City,
CA) at 0 time and after 2 h incubation at 37 °C. Data were normalized
to vehicle control after subtraction of 0 time value and expressed as
percent inhibition activity of compounds. All data were reported as
mean S.E. of triplicate wells.
1H NMR (600 MHz, CDCl3) δH: 5.56 (dd, 1H, J =15.4, 6.8 Hz), 5.22
(m, 1H), 4.10–4.28 (m, 1H), 3.79 (ddd, 3H, J=11.2, 2.0, 0.6 Hz), 3.60–
3.82 (m, 1H), 3.04–3.22 (m, 1H), 2.22–2.30 (m, 1H), 2.01–2.17 (m,
1H), 1.60–1.73 (m, 1H), 0.97 (d, 6H, J=6.8 Hz); HRMS (ESI) calcd for
C5H11O4PNa [M+Na]+ 189.0293, found 189.0282.
2.5. Invasion assay
Invasion of the cells across 8 μm pore size membrane was assessed
using Chemo Tx System (Neuro Probe) according to the manufacturer's
protocol. Briefly, cells were stained with calcein-AM (Dojindo, Kuma-
moto, Japan), and the cell suspension was centrifuged and washed with
PBS. After washing, the cells were resuspended in DMEM containing
0.1% BSA (4×105 cells/ml). The 200 μM lysophosphatidylcholine (LPC)
(S)-2ccPA (13a).
13a was prepared from 10a following the same procedures for
10–13.
[α]D23 +13.6 (c 0.72, CHCl3).
2.2. Culture of MDA-MB-231
Human breast cancer MDA-MB-231 cells (a generous gift from Dr.
Gabor Tigyi, University of Tennessee) were cultured in Dulbecco's
modified Eagle medium (DMEM; Nissui, Tokyo, Japan) supplemented
with 10% fetal bovine serum (FBS; Biowest, FL). And cells were
cultured at 37 °C in a humidified incubator containing 5% CO2.
2.3. Collection and concentration of conditioned medium (CCM)
MDA-MB-231 cells were grown until 90% confluence. The cells
were washed 3 times with phosphate buffered saline (PBS). The cells
were incubated for 12 h in serum-free DMEM, and then the
conditioned medium was centrifuged at 200×g for 5 min to remove
cellular debris. Subsequently, the conditioned medium was concen-
trated to 10-fold on a 100 kDa cutoff filter (Millipore, Cork, Ireland).
2.4. ATX inhibition assay
ATX inhibition activity of compounds ((R)-2ccPA 16:1, (S)-2ccPA
16:1 and racemic-2ccPA 16:1) was measured using FS-3 (Echelon
Biosciences, Inc., UT), which fluoresces upon cleavage by ATX. FBS or
CCM from MDA-MB-231 cells was used as the source of ATX. FBS or
CCM and various concentrations of compounds in 10 μM fatty acid
free bovine serum albumin (BSA; Sigma-Aldrich, MO) were mixed
with 1 μM of FS-3 in assay buffer (1 mM CaCl2, 1 mM MgCl2, 5 mM
Fig. 3. Effects of chirality of 2ccPA on MDA-MB-231 cell invasion. The effect of (R)-2ccPA
16:1, (S)-2ccPA 16:1 and racemic-2ccPA 16:1 on MDA-MB-231 cell in vitro invasion
was examined by Boyden-chamber modified assay. The MDA-MB-231 cells were
seeded on upper chamber and 200 μM of LPC and 3 μM of compounds were added into
lower well. 100% represents maximal migration activity in the presence of 200 μM LPC.
Each column represents the mean S.E. of triplicate wells and is representative of at
least three independent experiments with similar results. *Pb0.05, **Pb0.01,
significantly different from control by 1 way ANOVA.

E. Nozaki et al. / Biochimica et Biophysica Acta 1811 (2011) 271–277
275
comparison to respective controls without any compounds, and are
presented as the mean and S.E. of triplicate wells.
2.6. Recording of spinal somato-somatic reflex
Male Wistar rats (n=4; weighing 320–360 g) were anesthetized
by pentobarbital (50 mg/kg, i.p.). A juglar vein was catheterized for i.
v. administration of compounds ((R)-2ccPA 16:1 or (S)-2ccPA 16:1,
0.2 mg/kg). A juglar artery was catheterized to record arterial blood
pressure and heart rate (Rekuchicoda WT-685 G, Nihon Kohden,
Tokyo, Japan). The ventilation was monitored with a gas analyzer
(Respirator SN-480-7, Sinano seisakuzyo, Tokyo, Japan) and adjusted
to maintain an end-tidal CO2 level at about 3.0%. Body temperature
was kept about 37.5 °C using an automatically regulated heating pad
and lamp (Animal blanket controller ATB-1100, Nihon koden, Tokyo,
Japan). The spinal cord was completely transected at the upper
thoracic level.
With the rat in a supine position, a branch of the right saphenous
nerve innervating thigh skin was cut at level of thigh and covered with
warm paraffin oil. The central cut end segment of the nerve was
placed on bipolar platinum iridium wire electrodes for electrical
stimulation. Single square pulse stimuli of 10 V with 0.5 ms duration
were delivered every 3 s by a digital electrical stimulator (Nihon
koden, Tokyo, Japan). Electromyogram (EMG) was recorded from the
right leg muscles by inserting silver electrodes using the AC
preamplifier (Bioelectric amplifier MEG-1200, Nihon koden, Tokyo,
Japan, time constant set at 0.01 s). The amplified EMG signals were
digitized (micro 1401, Cambridge Electronic Design, Cambridge, UK)
and rectified for later processing (Spike 2 version 5, Cambridge
Electronic Design, Cambridge, UK). With single shocks, reflex
responses of EMG activity were averaged for approximately 50 trials
at 3 intervals. The size of the reflex response was measured as the area
under the evoked response and expressed as % of the control size
preceding drug injection. All data were reported as mean S.E.
2.7. Statistical analysis
All values are given as mean S.E. The data were statistically
analyzed by Student's t-test, one way ANOVA and nonlinear regres-
sion. A P-value of b0.05 was considered to be statistically significant.
3. Results
3.1. Effects of chirality of 2ccPA on ATX activity
Fig. 4. Effects of chirality of 2ccPA on somato-somatic C-reflexes. In the top of figure, the
specimen record of A- and C-reflexes (average of 50 trials) was elicited by single
electrical stimulation of a saphenous nerve using stimulus strength of 10 V. In the
bottom of figure, the graph summarizes the size of the C-reflex 0–12.5 min after i.v.
injection of compounds at a dose of 0.2 mg/kg (n=4). The mean size (area under the
evoked responses) of the C-reflex 0–15 min before each injection was expressed as
100%. All subsequent reflexes were expressed as percentages of the control values. Each
column represents the mean S.E. ***Pb0.005, significantly different from control by 1
way ANOVA.
ATX activity was measured using FS-3 which fluoresces upon
cleavage by ATX. The catalytic activity of ATX in CCM from MDA-MB-
231 cells was significantly inhibited by (R)-2ccPA 16:1, (S)-2ccPA 16:1
and racemic-2ccPA 16:1 (Fig. 2). In case of (R)-2ccPA 16:1, it inhibited
ATX activity as low concentration as 10 nM. At 10 μM, an inhibitory
effect of (R)-2ccPA 16:1 on ATX arose over 60%. And, the catalytic
activity of ATX in FBS was also significantly inhibited by (R)-2ccPA 16:1,
(S)-2ccPA 16:1 and racemic-2ccPA 16:1 (data not shown). The
inhibitory activity of (R)-2ccPA was similar to (S)-2ccPA and racemic-
2ccPA, indicating that the chirality of 2ccPA did not affect the degree of
ATX inhibition. Additionally, it has been reported that no stereoselective
differences were found between (R)-3ccPA and (S)-3ccPA toward the
inhibition of ATX [14].
and 3 μM compounds ((R)-2ccPA 16:1, (S)-2ccPA 16:1 and racemic-
2ccPA 16:1) in DMEM containing 0.1% BSA were added to lower well.
And 25 μl of cell suspension were added to each upper filter well. Plates
were incubated for 8 h at 37 °C, and the medium was carefully removed
from the upper filter. Then the upper filter was carefully washed with
PBS containing 2 mM EDTA, and gently wiped with a cotton swab to
remove the non-invaded cells. Then the filter was separated from the
microplate, and measured the fluorescence (em/ex: 485/530 nm) of
invaded cells on the bottom of each well by fluorescence plate reader
(Cyto Flour series 4000). Data were calculated as percent inhibition in
3.2. Effects of chirality of 2ccPA 16:1 on MDA-MB-231 cell invasion
After 8 h incubation of invasion assay plate at 37 °C, MDA-MB-231
cells were invaded to bottom membrane through 8 μm pore. It was due
to the hydrolysis of LPC in bottom well by secreted ATX from the cells
of upper well, which caused formation of the bioactive lipid LPA. In the
presence of 2ccPAs, invasion of MDA-MB-231 cells was inhibited as

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E. Nozaki et al. / Biochimica et Biophysica Acta 1811 (2011) 271–277
shown in Fig. 3. The inhibitory activity of (R)-2ccPA was almost the
same as that of (S)-2ccPA and racemic-2ccPA. Similar to the ATX
inhibition, the chirality of 2ccPA did not affect the inhibitory activity of
cancer cell invasion.
ATX activity, and as a result, neuropathic pain may be inhibited. On the
other hand, it has been clarified that 2ccPA suppressed nociceptive
reflex via C-fiber [10]. While the mechanisms of 2ccPA suppressing pain
are still unknown, we did not detect significance to anti-nociceptive
effects by (R)-2ccPA and (S)-2ccPA.
3.3. Effects of chirality of 2ccPA on somato-somatic C-reflexes in
anesthetized rats
Until now, eight kinds of LPA receptor have been reported. Among
these receptors, LPA1 and LPA3 are suggested to be involved in cancer
cell invasion [22-24] and LPA-induced neuropathic pain [25-27]. It has
been reported that LPA1 and LPA3 did not have stereospecificity for LPA
[28]. Recently, it has been reported that (S)-3ccPA was slightly more
efficacious in the activation on LPA5 compared to the (R)-3ccPA [14]. It
needs to investigate stereoselectivity of LPA receptors toward to
2ccPA. In the case of 2ccPA, it has been reported that it can activate
LPA1, LPA3 and LPA5 receptors, but the ability is less than that of LPA
[29]. In this study, however, we did not detect the significant difference
between 2ccPA enantiomers to the inhibition of ATX activity, cancer
cell invasion and C-fiber suppression. Therefore it is suggested that
target molecules, which related to 2ccPA on inhibition of cancer cell
invasion and pain relief, did not have stereoselectivity for each
enantiomers.
Single shock stimulation of the myelinated A- and unmyelinated
C-afferent fibers of the saphenous nerve (at 10 V with 0.5 ms pulse
duration) produced two distinct A- and C-somatic reflex components
in the hindlimb EMG, a short latency (about 10 ms) A-reflex and a
long latency (about 40 ms) C-reflex (the top of Fig. 4). Injection of
0.2 mg/kg, i.v. of compounds ((R)-2ccPA 16:1 or (S)-2ccPA 16:1)
depressed the C-reflex, but not A-reflex. In most experiments, 2ccPA
induced depression of the C-reflex components started several min
after the injection and reached their maxima in less than 12 min, and
then gradually returned to the control within 30 min.
The bottom of Fig. 4 summarized the effects of i.v. injection of
compounds ((R)-2ccPA 16:1 and (S)-2ccPA 16:1) on C-reflex tested in 4
rats. The responses were measured 0–12.5 min after i.v. injection of
compounds after reaching the maximum effects. The size of C-reflex
response was measured as the area under the evoked response. The
averaged responses expressed as % of the control size preceding drug
injection. After injection of (R)-2ccPA 16:1, the C-reflex reached 86 3%
of the control. After injection of (S)-2ccPA 16:1, the C-reflex reached 84
3% of the control. These results indicated that the chirality of 2ccPA did
not affect their inhibitory activities of C-reflexes suppression.
In conclusion, our study shows the method of enantiopure
synthesis of 2ccPA, and it suggests that the chirality of 2ccPA is not
involved in 2ccPA biological functions such as ATX inhibition, cancer
cell invasion and anti-nociceptive effects. Based on these findings,
racemic 2ccPA is considered to be utilized as an effective compound
for therapeutic applications for cancer and pain.
Acknowledgements
4. Discussion
We thank Dr. Nobuhiro Watanabe (Department of Autonomic
Neuroscience, Tokyo Metropolitan Geriatric Hospital and Institute OF
Gerontology) and Dr. Yasutaka Kakiuchi (Department of Biology,
Ochanomizu University) for technical advices and valuable discussions.
Cyclic-PA is a natural occurring bioactive lipid, and the chiral
carbon atom is assigned an R designation. We have already reported
that 2ccPA and 3ccPA have inhibitory activities on ATX, cancer cell
invasion and metastasis, as well as cPA. However, these data were
obtained by using a racemic-2ccPA and (S)-3ccPA , those were easy to
synthesize chemically [11]. As it is well known that each enantiomer
shows same or different biological activities, the biological activities of
each enantiopure cPA derivatives are required to be demonstrated.
In this study, we focus on 2ccPA to investigate the effects of
enantiopure derivatives on some biological functions, because of
2ccPA was shown to be the most potent ATX inhibitor [12]. Here, we
newly developed the method of 2ccPA enantiomer synthesis. And we
clarified the effects of each enantiomer on ATX activity, cancer cell
invasion and nociceptive reflex.
References
[1] K. Murakami-Murofushi, M. Shioda, K. Kaji, S. Yoshida, H. Murofushi, Inhibition of
eukaryotic DNA polymerase α with a novel lysophosphatidic acid (PHYLPA)
isolated from myxoamoebae of Physarum polycephalum, J. Biol. Chem. 267 (1992)
21512–21517.
[2] Y. Takahashi, Y. Shimada, M. Shioda, S. Yoshida, H. Murofushi, K. Murakami-
Murofushi, Isolation of a new species of Physarum lysophosphatidic acid, PHYLPA,
and its effect on DNA polymerase activity, Cell Struct. Funct. 18 (1993) 135–138.
[3] T. Kobayashi, R. Tanaka-Ishii, R. Taguchi, H. Ikezawa, K. Murakami-Murofushi,
Existence of a bioactive lipid, cyclic phosphatidic acid, bound to human serum
albumin, Life Sci. 65 (1999) 2185–2191.
[4] S. Kobayashi, R. Tokunoha, M. Shibasaki, R. Shinagawa, K. Murakami-Murofushi,
Synthesis of 1-O- acylglycerol 2, 3-cyclic phosphate: determination of the
ATX has lysophospholipase D (lyso PLD) activity, which produces
LPA from lysophosphatidylcholine (LPC) by hydrolysis [15,16]. Many
cancer cells secrete ATX [17,18], and it causes the synthesis of LPA
[5,17,18]. In consequence, LPA stimulates cancer cell invasion and
metastasis [18]. Therefore, it is considered that 2ccPA suppressed
synthesis of LPA via inhibition of ATX activity, and as a result, cancer
cell invasion was inhibited. In this study, we did not detect significant
difference between (R)-2ccPA and (S)-2ccPA on the inhibitory
activities on ATX and cancer cell invasion.
It has been reported that LPA induces neuropathic pain [6,7]. The
neuropathic pain is suggested to develop aberrant myelination
(splitting, detachment and loss of myelin), and LPA decreases in gene
expression and protein production of the myelin-associated proteins,
and as a result, demyelination was induced [19,20]. Moreover, LPA
induces the loss of C-fiber responses and the down-regulation of spinal
substance P signal [21]. It induces that sprouting A-fiber of LPA-induced
demyelination to C-fiber or ephaptic crosstalk between different types
of fibers, and in consequence, abnormal sensory perceptions might be
caused by gettingcrossatthe level of sensory fibers or atthe level of pain
transmission in the spinal dorsal horn [19]. From these findings, it is
suggested that 2ccPA may suppress the synthesis of LPA by inhibition of
absolute structure of PHYLPA,
Tetrahedron Lett. 34 (1993) 4047–4050.
[5] G.B. Mills, W.H. Moolennar, The emerging role of lysophosphatidic acid in cancer,
Nat. Rev. Cancer 3 (2003) 582–591.
[6] K. Renback, M. Inoue, H. Ueda, Lysophosphatidic acid-induced, pertussis toxin-
a specific inhibitor of DNA polymerase α,
sensitive nociception through
a substance P release from peripheral nerve
endings in mice, Neurosci. Lett. 270 (1999) 59–61.
[7] M. Inoue, M.H. Rashid, R. Fujita, J.J.A. Contos, J. Chun, H. Ueda, Initiation of
neuropathic pain requires lysophosphatidic acid receptor signaling, Nat. Med. 10
(2004) 712–718.
[8] K. Murakami-Murofushi, A. Uchiyama, Y. Fujiwara, T. Kobayashi, S. Kobayashi, M.
Mukai, H. Murofushi, G. Tigyi, Biological functions of a novel lipid mediator, cyclic
phosphatidic acid, Biocim. Biophys. Acta 1582 (2002) 1–7.
[9] Y. Fujiwara, Cyclic phosphatidic acid-A unique bioactive phospholipid, Biochim.
Biophys. Acta 1781 (2008) 519–524.
[10] Y. Kakiuchi, J. Nagai, M. Gotoh, H. Hotta, H. Murofushi, T. Ogawa, H. Ueda, K.
Murakami-Murofushi, Antinociceptive effect of cyclic phosphatidic acid and its
derivative in animal models of acute and chronic pain (submitted).
[11] A. Uchiyama, M. Mukai, Y. Fujiwara, S. Kobayashi, N. Kawai, H. Murofushi, M. Inoue, S.
Enoki, Y. Tanaka, T. Niki, T. Kobayashi, G. Tigyi, K. Murakami-Murofushi, Inhibition of
transcellular tumor cell migration and metastasis by novel carba-derivatives of cyclic
phosphatidic acid, Biochim. Biophys. Acta 1771 (2007) 103–112.
[12] D.L. Baker, Y. Fujiwara, K.R. Pigg, R. Tsukahara, S. Kobayashi, H. Murofushi, A.
Uchiyama, K. Murakami-Murofushi, E. Koh, R.W. Bandle, H. Byun, R. Bittman, D.
Fan, M. Murph, G.B. Mills, G. Tigyi, Carba analogs of cyclic phosphatidic acid are
selective inhibitors of autotaxin and cancer cell invasion and metastasis, J. Biol.
Chem. 281 (2006) 22786–22793.

E. Nozaki et al. / Biochimica et Biophysica Acta 1811 (2011) 271–277
277
[13] G. Guanti, L. Banfi, E. Narisano, Chemoenzymic preparation of asymmetrized tris
(hydroxymethyl)methane (THYM*) and of asymmetrized bis(hydroxymethyl)
acetaldehyde (BHYMA*) as new highly versatile chiral building blocks, J. Org.
Chem. 57 (1992) 1540–1554.
[14] R. Gupte, A. Siddam, Y. Lu, W. Li, Y. Fujiwara, N. Panupinthu, T. Pham, D.L. Baker, A.L.
Parrill, M. Gotoh, K. Murakami-Murofushi, S. Kobayashi, G.B. Mills, G. Tigyi, D.D.
Miller, Synthesis and pharmacological evaluation of the stereoisomers of 3-carba
cyclic-phosphatidic acid, Bioorg. Med. Chem. Lett. 20 (2010) 7525–7528.
[15] M. Umezu-Goto, Y. Kishi, A. Taira, K. Hama, N. Dohmae, K. Takio, T. Yamori, G.B.
Mills, K. Inoue, J. Aoki, ando H. Arai, Autotaxin has lysophospholipase D activity
leading to tumor cell growth and motility by lysophosphatidic acid production, J.
Cell Biol. 158 (2002) 227–233.
[16] A. Tokumura, E. Majima, Y. Kariya, K. Tominaga, K. Kogure, K. Yasuda, K. Fukuzawa,
Identification of human plasma lysophospholipase D, a lysophosphatidic acid-
producing enzyme, as autotaxin, a multifunctional phosphodiesterase, J. Biol. Chem.
277 (2002) 39436–39442.
[17] Y. Kishi, S. Okudaira, M. Tanaka, K. Hama, D. Shida, J. Kitayama, T. Yamori, J. Aoki, T.
Fujimaki, H. Arai, Autotaxin is overexpressed in glioblastoma multiforme and
contributes to cell motility of glioblastoma by converting lysophosphatidylcholine
to lysophosphatidic acid, J. Biol. Chem. 281 (2006) 17492–17500.
[18] G. Tigyi, Aiming drug discovery at lysophosphatidic acid targets, Br. J. Pharmacol.
161 (2010) 241–270.
[21] M. Inoue, A. Yamaguchi, M. Kawakami, J. Chun, H. Ueda, Loss of spinal substance P
pain transmission under the condition of LPA1 receptor-mediated neuropathic
pain, Mol. Pain 2 (2006) 25.
[22] P. Wang, X. Wu, W. Chen, J. Liu, X. Wang, The lysophosphatidic acid (LPA)
receptors their expression and significance in epithelial ovarian neoplasms,
Gynecol. Oncol. 104 (2007) 714–720.
[23] J. Aoki, A. Inoue, S. Okudaira, Two pathways for lysophosphatidic acid production,
Biochim. Biophys. Acta 1781 (2008) 513–518.
[24] E. Gendaszewska-Darmach, Lysophosphatidic acids, cyclic phosphatidic acids and
autotaxin as promising targets in therapies of cancer and other diseases, Acta
Biochim. Pol. 55 (2008) 227–240.
[25] J. Allard, S. Barron, S. Trottier, P. Cervera, C. Daumas-Duport, E. Leguern, A. Brice, J.C.
Schwartz, P. Sokoloff, Edg-2 in myelin-forming cells: isoforms, genomic mapping,
and exclusion in Charcot-Marie-Tooth disease, Glia 26 (1999) 176–185.
[26] J.A. Weiner, J. Chun, Schwann cell survival mediated by the signaling phospholipid
lysophosphatidic acid, Proc. Natl. Acad. Sci. USA 96 (1999) 5233–5238.
[27] L. Ma, H. Uchida, J. Nagai, M. Inoue, J. Chun, J. Aoki, H. Ueda, Lysophosphatidic acid-3
receptor-mediated feed-forward production of lysophosphatidic acid: an initiator of
nerve injury-induced neuropathic pain, Mol. Pain 5 (2009) 6.
[28] K. Yokoyama, D.L. Baker, T. Virag, K. Liliom, H. Byun, G. Tigyi, R. Bittman,
Stereochemical properties of lysophosphatidic acid receptor activation and
metabolism, Biochim. Biophys. Acta 1582 (2002) 295–308.
[19] H. Ueda, Molecular mechanisms of neuropathic pain-phenotypic switch and
initiation mechanisms, Pharmacol. Ther. 109 (2006) 57–77.
[20] R. Fujita, N. Kiguchi, H. Ueda, LPA-mediated demyelination in ex vivo culture of
dorsal root, Neurochem. Int. 50 (2007) 351–355.
[29] J.R. Williams, A.L. Khandoga, P. Goyal, J.I. Fells, D.H. Perygin, W. Siess, A.L. Parrill, G.
Tigyi, Y. Fujiwara, Unique ligand selectivity of the GPR92/LPA5 lysophosphatidate
receptor indicates role in human platelet activation, J. Biol. Chem. 284 (2009)
17304–17319.