A new approach to the synthesis of lysophospholipids: Preparation of lysophosphatidic acid and lysophosphatidylcholine from p-nitrophenyl glycerate

Article abstract of DOI:10.1016/j.tetlet.2004.07.161

A new stereospecific synthesis of lysophosphatidic acid and lysophosphatidylcholine is reported. The sequence relies on p-nitrophenyl-D- glycerate as a chiral synthon, including chemoselective reduction of the active ester function without affecting other carboxylic ester groups present in the molecule.

Full text of DOI:10.1016/j.tetlet.2004.07.161

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 7371–7373
A new approach to the synthesis of lysophospholipids:
preparation of lysophosphatidic acid and
lysophosphatidylcholine from p-nitrophenyl glycerate
Renato Rosseto, Niloufar Bibak and Joseph Hajdu*
Department of Chemistry and Biochemistry, California State University, Northridge, CA 91330-8262, USA
Received 19 May 2004; revised 23 July 2004; accepted 26 July 2004
Available online 19 August 2004
Abstract—A new stereospecific synthesis of lysophosphatidic acid and lysophosphatidylcholine is reported. The sequence relies on
p-nitrophenyl-D-glycerate as a chiral synthon, including chemoselective reduction of the active ester function without affecting other
carboxylic ester groups present in the molecule.
Ó 2004 Elsevier Ltd. All rights reserved.
Lysophospholipids have recently become the focus of
special attention since it was discovered that in addition
to their role in phospholipid metabolism they function
as second messengers, exhibiting a broad range of bio-
logical activities in their own right.1–3 Lysophosphatidic
acid (1), released from activated cells such as platelets,
fibroblasts, and leukocytes, stimulates platelet aggrega-
tion,4 promotes smooth muscle contraction,5 contrib-
utes to modulation of blood pressure,6 and induces
cancer cell invasion.7 Lysophosphatidylcholine (2) has
been shown to exhibit regulatory activity of signaling
enzymes, including activation of p38, AP-1, and JNK
kinases8,9 as well as adenylyl cyclase.10 It inhibits plate-
let aggregation,11,12 and promotes migration of lympho-
cytes toward the site of inflammatory tissue by inducing
secretion of chemotactic agents from macrophages.13.
Despite their well-recognized biological importance, elu-
cidation of the mechanistic details involved in the enzy-
mological, cell-biological, and membrane-biophysical
activities of lysophospholipids remains to be accom-
plished, and it depends on availability of efficient syn-
thetic methods for preparation of structurally variable
lysophospholipid derivatives.
To date relatively few synthetic methods have been
developed for the preparation of lysophospholipids,
mainly due to difficulties associated with intramolecular
acyl group migration that results in a mixture of 1-acyl
and 2-acyl glycerophosphate derivatives.14,15 Recently
we have shown that such acyl migration can be pre-
vented by strategically chosen orthogonal protection of
the neighboring hydroxyl groups of the synthetic inter-
mediates.15 In the present communication we report a
new synthesis relying on p-nitrophenyl glycerate for
the preparation of lysophosphatidic acid and lysophos-
phatidylcholine. The sequence is based on chemoselec-
tive manipulation of the active ester function that
provides a rapid and efficient way for elaboration of
the desired substituted glycerol skeleton, requiring min-
imal use of protecting groups for construction of the
glycerophospholipid target.
The synthesis starts with preparation of the p-nitrophe-
nyl ester of glyceric acid (Scheme 1). Commercially
available 2,3-O-isopropylidene-D-glyceric acid methyl
ester (3) was converted to the corresponding carboxylic
acid by base catalyzed hydrolysis with NaOH in aque-
ous dioxane, followed by DCC promoted condensation
Keywords: Lysophosphatidic acid; Lysophosphatidylcholine synthesis;
p-Nitrophenyl glycerate; Chemoselective reduction.
*
Corrresponding author. Tel.: +1 818 677 3377; fax: +1 818 677
2912; e-mail: joseph.hajdu@csun.edu
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.07.161

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R. Rosseto et al. / Tetrahedron Letters 45 (2004) 7371–7373
Scheme 1. Reagents and conditions: (a) (i) 1.2M NaOH, dioxane–water (4:1), 0°C, rt, 45min; (ii) Dowex 50-X8, H+, dioxane–water (10:1), rt,
15min; (iii) p-NO2C6H4OH, DCC, CH2Cl2; (iv) 0.3N HCl, dioxane, rt, 4h; (b) palmitoyl chloride, MeCN–benzene (1:1), rt, 72h; (c) DHP, PPTS,
CHCl3; (d) NaBH4, glyme; (e) (i) iPr2NP(OCH2CH2CN)2, tetrazole, CHCl3–MeCN, (3:1); (ii) 30% aq H2O2–CH2Cl2; (iii) 0.2M DBU, toluene,
110°C, 16h; (f) (i) ethylene chlorophosphate, Et3N, benzene; (ii) (CH3)3N, MeCN, 65°C; (g) 0.15N HCl, dioxane–water (1:1).
with p-nitrophenol, and subsequent acidolytic cleavage
of the isopropylidene function to give the active ester
of D-glyceric acid (4) in an overall yield of 66%. Regio-
specific monoacylation of compound 4 at the primary
alcohol function was accomplished using two-fold molar
excess of palmitoyl chloride in a mixture of MeCN/benz-
ene (1:1) at room temperature for 72h. The solution
was diluted with glacial acetic acid, freeze-dried, and
the product 4 was chromatographed on a Sephadex
LH-20column using dichloromethane–ethyl acetate
(1:1) to give the pure sn-1-palmitoyl ester 5 as a white
desired headgroup at the incipient sn-3-position. We
applied it for the synthesis of 1 and 2 using readily avail-
able phosphorylation methods for elaboration of the
phosphomonoester and phosphodiester functions,
respectively.
For the preparation of lysophosphatidic acid, com-
pound 7 was phosphorylated by treatment with bis(b-
cyanoethyl)-N,N-diisopropylphosphoramidite18 in the
presence of tetrazole in MeCN–CHCl3 (1:3), at rt for
48h (58% yield), followed by oxidation of the phosphite
intermediate in a biphasic mixture of 30% aq H2O2/
dichloromethane (90%), and subsequent base catalyzed
elimination of the cyanoethyl groups with 2.5equiv
DBU in refluxing toluene overnight gave the phospho-
monoester 8 in 70% yield. Finally, acid catalyzed cleav-
age of the sn-2-tetrahydropyranyl group was carried out
in 0.15N HCl in dioxane–water (1:1, 86% yield). As has
been shown previously, under these conditions no acyl
migration from the primary to the secondary alcohol
1
powder in 62% yield. Absence of the H NMR signal
in the d 5.00–5.09 region clearly indicated that the sec-
ondary hydroxyl group remained unaffected in the
course of the reaction.15,16
Tetrahydropyranylation at the sn-2-glycerol position
was carried out with 1.2equiv 3,4-dihydropyran in
CHCl3, in the presence of pyridinium p-toluenesulfonate
as catalyst, at rt for 1h. Compound 6 was isolated by
extraction from a mixture of benzene–water, then
freeze-dried from benzene yielding an analytically pure
colorless oily product (92%). Selective reduction of the
sn-3-p-nitrophenyl ester function of 6 was achieved with
excess NaBH4 in 1,2-dimethoxyethane17 at rt for 1h.
The resulting mixture was passed through a short plug
of silica gel, eluted first with CHCl3 followed by EtOAc
to give the crude 1-palmitoyl-2-tetrahydropyranyl-sn-
glycerol 7. This product was further treated with a mix-
ture of benzene–water, then freeze-dried from benzene
to afford the analytically pure alcohol 7 in 62% yield
as a white powder. This glycerol derivative 7 turned
out to be a most useful intermediate for the synthesis
of lysophospholipids,15 allowing incorporation of the
1
position was detected. Indeed, the H NMR of product
1 shows baseline absorption in the d 5.00-5.09 range.16
For the synthesis of the target lysophosphatidylcholine,
compound 7 was allowed to react with 2-chloro-2-oxo-
1,3,2-dioxaphospholane/triethylamine in anhydrous
benzene,19 followed by treatment of the phosphorylated
intermediate with trimethylamine in acetonitrile at 65°C
(in a pressure bottle) for 24h. The resulting phospho-
choline 9 was then chromatographed on silica gel (chlo-
roform–methanol–water 65:25:4, 62% yield), and
subjected to acid hydrolysis to remove the sn-2-tetra-
hydropyranyl protecting group under the same condi-
tions as used for deprotection of the secondary alcohol

R. Rosseto et al. / Tetrahedron Letters 45 (2004) 7371–7373
7373
10. Yuan, Y.; Schoenwaelder, S. M.; Salem, H. H.; Jackson,
S. P. J. Biol. Chem. 1996, 271, 27090–27098.
function of lysophosphatidic acid to give the lysophos-
phatidylcholine 2 isolated in 98% yield.
11. Yuan, Y.; Jackson, S. P.; Newnham, H. H.; Mitchell,
C. A.; Salem, H. H. Blood 1995, 86, 4166–4174.
12. Marathe, G. K.; Silva, A. R.; de Castro Faria Neto, H. C.;
Tjoelker, L. W.; Prescott, S. M.; Zimmerman, G. A.;
McIntyre, T. M. J. Lipid Res. 2001, 42, 1430–1437.
13. Kuglyama, K.; Kerns, S.; Morrisett, J. D.; Roberts, R.;
Henry, P. D. Nature 1990, 344, 160–162.
14. (a) Chevallier, J.; Sakai, N.; Robert, F.; Kobayashi, T.;
Gruenberg, J.; Matile, S. Org. Lett. 2000, 2, 1859–1861;
(b) Wang, A.; Loo, R.; Chen, Z.; Dennis, E. A. J. Biol.
Chem. 1997, 272, 22030–22036.
15. Bibak, N.; Hajdu, J. Tetrahedron. Lett. 2003, 44,
5875–5877.
16. Srisiri, W.; Lee, Y. S.; OÕBrien, D. F. Tetrahedron Lett.
1995, 36, 5911–5914.
17. Using 1,2-dimethoxyethane as the solvent turned out to be
important, in MeCN–CH2Cl2 mixture the reduction was
slow, and the yield of the alcohol was low (ꢀ30%):
Takahashi, S.; Cohen, L. A. J. Org. Chem. 1970, 35,
1505–1508.
In summary, the synthesis presented here provides a
rapid and efficient method for the preparation of lyso-
phosphatidic acids and lysophosphatidylcholines, and
it should be applicable to the development of a series
of new synthetic lysophospholipid derivatives with the
desired target structures for biological and physico-
chemical studies. In addition, elaboration of the alcohol
function by selective sodium borohydride reduction of
the p-nitrophenyl ester function, in the presence of other
carboxylic ester groups, should provide a useful new
strategy not only for the synthesis of glycerol deriva-
tives, but also in carbohydrate chemistry, and in the syn-
thesis of natural products where selective manipulation
of multiple hydroxyl groups is required. Work toward
application of the method for preparation of functional-
ized lysophospholipids is currently underway in our
laboratory.20
18. We have used a base-labile variant of the original
phosphoramidite method in order to prevent cleavage of
the sn-2-tetrahydropyranyl group: Perich, J. W.; Johns,
R. B. Tetrahedron Lett. 1988, 29, 2369–2372.
Acknowledgements
This work was supported by the National Institutes of
Health, including grants S06 GM/HD48680 and T34
GM08395.
19. Roodsari, F. S.; Wu, D.; Pum, G. S.; Hajdu, J. J. Org.
Chem. 1999, 64, 7727–7737.
20. Selected data: 5: IR: (cmꢁ1) 3300, 1762, 1714, 1529, 1207,
1126; 1H NMR (CDCl3, 200MHz) d 0.87 (t, 3H, J = 6Hz),
1.24 (s br, 24H), 1.58 (m br, 2H), 2.36 (t, 2H, J = 7.4Hz),
4.52–4.69 (m, 3H), 7.32 (d, 2H, J = 9Hz), 8.29 (d, 2H,
9Hz); 13C NMR (CDCl3, 50MHz) d 14.06, 22.63, 24.82,
29.03, 29.18, 29.30, 29.39, 29.54, 29.60, 29.62, 31.87, 33.93,
64.94, 69.81, 122.16, 125.33, 145.74, 154.61, 169.95,
173.64; Rf (CHCl3/CH3CN 5:1) = 0.65. Anal. Calcd for
C25H39NO7: C 64.49, H 8.44, N 3.01. Found: C 64.85, H
8.49, N 2.88. FAB MS calcd for MNa+ (C25H39NO7Na)
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1
488.2624, found: 488.2611. 7: IR (CHCl3): 1732cmꢁ1; H
NMR (CDCl3) d: 0.86 (t, 3H, J = 6.8Hz), 1.24 (s, 24H),
1.50–1.58 (m, 6H), 1.59–1.61 (m, 2H), 2.26–2.31 (dt, 2H,
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