Total syntheses of bioactive oxidized ethanolamine phospholipids

Article abstract of DOI:10.1021/ol034729z

Matrix presented Truncated ethanolamine phospholipids containing aldehyde functionality, e.g. OVPE, and the corresponding acids, are generated by oxidative cleavage of polyunsaturated phospholipids. To confirm their identities and facilitate studies of the chemistry and biological actions of these analogues of biologically active phosphatidylcholines, e.g. OVPC, total syntheses were developed. An efficient general strategy was used that features selective N-protection of 2-lysophosphatidylethanolamine, and generation of the target compounds by mild deprotection of stable precursors.

Full text of DOI:10.1021/ol034729z

ORGANIC
LETTERS
2003
Vol. 5, No. 16
2797-2799
Total Syntheses of Bioactive Oxidized
Ethanolamine Phospholipids
Bogdan G. Gugiu and Robert G. Salomon*
Department of Chemistry, Case Western ReserVe UniVersity,
CleVeland, Ohio 44106-7078
rgs@po.cwru.edu
Received May 1, 2003
ABSTRACT
Truncated ethanolamine phospholipids containing aldehyde functionality, e.g. OVPE, and the corresponding acids, are generated by oxidative
cleavage of polyunsaturated phospholipids. To confirm their identities and facilitate studies of the chemistry and biological actions of these
analogues of biologically active phosphatidylcholines, e.g. OVPC, total syntheses were developed. An efficient general strategy was used that
features selective N-protection of 2-lysophosphatidylethanolamine, and generation of the target compounds by mild deprotection of stable
precursors.
Because they exhibit biological activities with apparent
pathological significance, interest in the chemistry and
biology of oxidized phospholipids (oxPL) is growing.
Oxidative cleavage of the arachidonic acid ester of 2-lyso-
phosphatidylcholine (PC) generates the oxovaleryl ester
OVPC (1).1 This aldehydic PC and the corresponding
carboxylic acid inhibit lipopolysaccharide-induced E-selectin
expression by human aortic endothelial cells and activate
endothelial cells to bind monocytes.2 Truncated γ-oxygenated
R,â-unsaturated aldehydic 2 and carboxylic oxPLs are ligands
for the macrophage scavenger receptor CD36 that mediates
endocytosis of oxidized low-density lipoprotein (LDL) by
macrophage cells.3
e.g., generating Schiff bases and carboxyalkylpyrroles 34
whose levels are elevated in cardiovascular5 and retinal6
diseases.
Primarily owing to the availability of authentic samples
by total syntheses,7,8 studies on oxPL have focused on
phosphatidylcholines.1,3 Phosphatidylethanolamines (PEs)
(2) Subbanagounder, G.; Leitinger, N.; Schwenke, D. C.; Wong, J. W.;
Lee, H.; Rizza, C.; Watson, A. D.; Faull, K. F.; Fogelman, A. M.; Berliner,
J. A. Arterioscler. Thromb. Vasc. Biol. 2000, 2248-2254.
(3) Podrez, E. A.; Batyreva, E.; Shen, Z.; Zhang, R.; Deng, Y.; Sun,
M.; Gugiu, B. G.; Finton, P. J.; Shen, L.; Febbraio, M.; Hayn, M.;
Silverstein, R. L.; Hoff, H. F.; Salomon, R. G.; Hazen, S. L. J. Biol. Chem.
2002.
(4) Gu, X.; Sun, M.; Gugiu, B.; Hazen, S.; Crabb, J. W.; Salomon, R.
G. J. Org. Chem. 2003, 68, 3749-3761.
(5) Kaur, K.; Salomon, R. G.; O’Neil, J.; Hoff, H. F. Chem. Res. Toxicol.
1997, 10, 1387-96.
The aldehydes 1 and 2 also covalently modify proteins
by reactions with primary amino groups of lysyl residues,
(6) Crabb, J. W.; Miyagi, M.; Gu, X.; Shadrach, K.; West, K. A.;
Sakaguchi, H.; Kamei, M.; Hasan, A.; Yan, L.; Rayborn, M. E.; Salomon,
R. G.; Hollyfield, J. G. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 14682-7.
(7) (a) Deng, Y.; Salomon, R. G. J. Org. Chem. 1998, 63, 7789-7794.
(b) Deng, Y.; Salomon, R. G. J. Org. Chem. 2000, 65, 6663-6665.
(1) Watson, A. D.; Leitinger, N.; Navab, M.; Faull, K. F.; Ho¨rkko¨, S. J.;
Palinski, W.; Schwenke, D.; Salomon, R. G.; Sha, W.; Subbanagounder,
G.; Fogelman, A. M.; Berliner, J. A. J. Biol. Chem. 1997, 272, 13597-
13607.
10.1021/ol034729z CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/09/2003

constitute an important fraction of LDL phospholipids and
are major components of certain brain and retinal mem-
branes.9 To facilitate studies of the generation, chemistry,
and biology of oxidized PEs we now report efficient methods
for their preparation. The reactivity of the primary amino
group of PEs toward aldehyde functionality10 prompted us
to design syntheses that avoided aldehydic intermediates or
generated them in the final step from stable precursors
suitable for storage.
a
Scheme 1
PE analogues 4a-c of OVPC (1) and homologues are
expected to be generated by oxidative cleavage of docosa-
hexaenoic, arachidonic, and linoleic acid esters of 2-lyso-
phophatidylethanolamine. Syntheses were devised for 4a-c
as well as the corresponding carboxylic acids 5a-c and the
oxPE analogues 6a-c of the most avid oxPC ligands for
the CD36 receptor.3
a Reagents and conditions: (a) p-NPTeoc, Na2CO3 1 M, 48 h.
(b) DCC, DMAP CHCl3, CH2dCH(CH2)nCO2H, 72 h, n ) 2, 3, 7.
(c) O3, MeOH, -60 °C, then Me2S, MeOH -10 °C to room
temperature. (d) CH(OCH3)3, Montmorillonite K10, rt. (e) 1 M
TBAF/ THF, 72 h, rt. (f) TFA 50%:CHCl3 (1:2), 0 °C, 90 min.
Our strategy was to protect the amine moiety of the
commercially available 2-lysoPE and couple the latter with
the appropriate acids. Initially we examined the use of
t-BOC.11 This was satisfactory for preparing the acids 5a-
c. However, 2-trimethylsilanylethoxycarbonyl (Teoc), used
with success for amino acids,12,13 is a more suitable protecting
group because deprotection can be accomplished under mild
conditions with tetrabutylammonium fluoride. The present
report documents the first application of the Teoc group to
protect the primary amino group in 2-lyso-phosphatidyl-
ethanolamines.
Of reagents available for the protection of amines with
Teoc,13 we found p-nitrophenyl trimethylsilanylethyl carbon-
ate (p-NPTeoc) to be most effective. With this reagent14 we
were able to introduce the Teoc protecting group selectively
on the amine moiety under mild conditions. As shown in
Scheme 1, protection of 2-lysoPE (HO-PE) with p-NPTeoc
followed by coupling with the appropriate commercially
available acids gave the Teoc N-protected alkenoylphospho-
lipids 4-pentenoyl- (8a), 5-hexenoyl- (8b), and 9-decenoyl-
(8c) phosphatidylethanolamine. Ozonolysis at -60 °C fol-
lowed by reduction in methanol with Me2S of the intermedi-
ate ozonides15 produced the Teoc N-protected aldehydes
4-oxobutyroyl- (9a), 5-oxovaleroyl- (9b), and 9-oxononanoyl-
(9c) phosphatidylethanolamine.
In an analogous fashion (Scheme 2) the t-BOC N-protected
alkenoylphospholipids (13a-c) were prepared, as well as
the corresponding aldehydes (14a-c).
Attempts to directly deprotect12,16 the Teoc derivatives
9a-c or the t-BOC derivatives 14a-c, using a variety of
conditions summarized in Scheme 1 and Table 1 in the
Supporting Information, failed to provide the desired phos-
phatidylethanolamine aldehydes 4a-c. Masking of the Teoc
N-protected aldehyde-phospholipids 9a-c (Scheme 1) as
dimethylacetals, using Montmorillonite K10 and trimethyl-
orthoformate, and deprotection of the ethanolamine moiety
in 10a-c, using tetrabutylammonium fluoride (TBAF) in
THF at room temperature, delivers the stable precursors
11a-c. The corresponding oxidized lipids (OBPE (4a),
OVPE (4b) and ONPE (4c)) can be conveniently generated
from 11a-c, as needed, under mild conditions by stirring
in a heterogeneous mixture of chloroform and 50% TFA (2:
1) at 0 °C.17
(8) Sun, M.; Deng, Y.; Batyreva, E.; Sha, W.; Salomon, R. G. J. Org.
Chem. 2002.
(9) Ansell, G. B.; Hawthorne, J. N.; Dawson, R. M. C. Form and function
of Phospholipids; Elsevier Scientific Publishing Company: New York, 1973;
Vol. 3.
(10) (a) Liu, J.; Itagaki, Y.; Ben-Shabat, S.; Nakanishi, K.; Sparrow, J.
R. J. Biol. Chem. 2000, 275, 29354-60. (b) Bhuyan, K. C.; Master, R. W.;
Coles, R. S.; Bhuyan, D. K. Mech. Aging DeV. 1986, 34, 289-96. (c)
Bhuyan, D. K.; Master, R. W.; Bhuyan, K. C. Biochim. Biophys. Acta 1996,
1285, 21-8. (d) Guichardant, M.; Taibi-Tronche, P.; Fay, L. B.; Lagarde,
M. Free Radical Biol. Med. 1998, 25, 1049-56. (e) Guichardant, M.;
Bernoud-Hubac, N.; Chantegrel, B.; Deshayes, C.; Lagarde, M. Prostag-
landins Leukot. Essent. Fatty Acids 2002, 67, 147-9. (f) James, P. F.;
Zoeller, R. A. J. Biol. Chem. 1997, 272, 23532-9.
(11) Wang, G.; Hollingsworth, R. I. J. Org. Chem. 1999, 64, 4140-
4147.
(12) Carpino, L. A.; Tsao, J.-H.; Ringsdorf, H.; Fell, E.; Hettrich, G. J.
Chem. Soc., Chem. Commun. 1978, 358-359.
(13) Shute, R. E.; Rich, D. H. Synthesis 1987, 346-349.
(14) Rosowsky, A.; Wright, J. E. J. Org. Chem. 1983, 48, 1539-1541.
The acids 15a-c could be obtained from the t-BOC
protected alkenoylphospholipids 13a-c by ozonolysis to give
(15) Pappas, J. J.; Keaveney, W. P.; Gancher, E.; Berger, M. Tetrahedron
Lett. 1966, 36, 4273-4278.
(16) Carpino, L. A.; Sau, A. C. J. Chem. Soc., Chem. Commun. 1979,
514.
(17) Ellison, R. A.; Lukenbach, E. R.; Chiu, C.-W. Tetrahedron Lett.
1975, 499.
2798
Org. Lett., Vol. 5, No. 16, 2003

Scheme 2a
Scheme 3a
a Reagents and conditions: (a) HO-PEB, DCC, DMAP, CHCl3,
72 h. (b) NBS, 1 h, -20 °C, then Py, 6 h, rt. (c) NaClO2/NaH2PO4,
2-methyl-2-butene, t-BuOH-H2O (5:1), 2 h, rt. (d) TFA 50%-
CHCl3 (1:2), 4 °C, 24 h.
a Reagents and conditions: (a) CH2dCH(CH2)nCO2H, DCC,
DMAP, CHCl3, 72 h. (b) O3, MeOH, -60 °C, then Me2S, MeOH,
-10 °C to room temperature. (c) NaClO2/NaH2PO4, 2-methyl-2-
butene, t-BuOH-H2O (5:1), 2 h, rt. (d) (OC-(CH2)n-2-CO)O, DCC,
DMAP, CHCl3, 72 h. (e) TFA, 4 °C, 30 min.
of isolated SAPE. These oxPEs exhibited bioactivities similar
to those of the corresponding oxPCs.2 Other recent studies
suggest that some oxPEs may have biological activities not
shared with oxPCs. Thus, the ability of oxidized LDL to
promote platelet prothrombinase activity appears to be
mediated primarily by the oxPE component of the lipo-
protein.19
Ongoing studies, to be reported elsewhere, confirm that
the oxidatively truncated ethanolamine phospholipids, identi-
cal with those prepared by the total syntheses detailed herein,
are formed by the oxidation of docosahexaenoic, arachidonic,
and linoleic acid esters of 2-lysoPE, and that 4b-c and 5b-c
induce monocyte binding to endothelial cells. As anticipated,
the aldehydic PE 4b is chemically unstable. Preliminary
results showed that 50% of 4b is lost within 1 h upon
standing in aqueous solution at room temperature in pH 7.4
phosphate buffered saline containing 5% methanol. The
ketoacids 6 are expected to be strong ligands for CD36 owing
to their structural similarity with the corresponding biologi-
cally active oxPCs.3 The affinity of the CD36 receptor for
structurally defined pure oxPL as well as their ability to cause
platelet activation are under investigation, and a study of
the chemistry of these oxPEs is in progress.
aldehydes 14a-c followed by NaClO2 oxidation (see steps
a to c in Scheme 2).9 However, a more practical route to
15a-c is available (Scheme 2, step d). The succinic, glutaric,
and azelaic acid monoesters of 2-lysoPE are best obtained
from the appropriate anhydride. Succinic (n ) 4) and glutaric
(n ) 5) anhydrides are commercially available while azelaic
anhydride (n ) 9) is readily available from azelaic acid.18
Coupling with t-BOC N-protected 2-lysoPE (HO-PEB) in
the presence of DMAP in anhydrous chloroform produces
the succinic, glutaric, and azelaic acid monoesters 15a-c
of t-BOC N-protected 2-lysoPE. Deprotection of 15a-c with
TFA at 0 °C delivers the corresponding monoesters of
2-lysoPE, 5a-c.
The ketoacids 6a-c, which are expected to be strong
ligands for CD36 due to their structural similarity with the
corresponding active phosphatidylcholines,3 were readily
available from the furyl acids 16a-c. Furylpropionic acid
16a is commercially available while 16b and 16c were
prepared according to Sun et al.8 Coupling of HO-PEB with
16a-c to give the furylalkanoyl monoesters of t-BOC
N-protected 2-lysoPE (17a-c), followed by a two-step
oxidation and deprotection delivers 6a-c (Scheme 3).
The compounds and methods reported in this letter will
be used to confirm the identities of biologically active oxPEs
present in oxidized LDL. Recent mass spectroscopic studies
indicate that oxPEs derived from 1-stearoyl-2-arachidonoyl-
sn-glycero-3-phosphorylethanolamine (SAPE) are present in
bovine LDL, and that 1-stearoyl-2-(5-oxovaleroyl)-PE and
1-stearoyl-2-glutaroyl-PE are produced upon autoxidation1
Acknowledgment. This work was supported in part by
NIH grant GM21249.
Supporting Information Available: Complete experi-
1
1
mental details including H and 13C (or H, HMQC, and
HMBC) NMR spectra for compounds 5a-c to 11a-c and
1
13a-c to 18a-c; for the unstable aldehydes 4a-c, H, but
not 13C, NMR spectra were recorded. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL034729Z
(19) Zieseniss, S.; Zahler, S.; Muller, I.; Hermetter, A.; Engelmann, B.
J. Biol. Chem. 2001, 276, 19828-35.
(18) Anderlini, F. Gazz. Chim. Ital. 1894, 24I, 476-480.
Org. Lett., Vol. 5, No. 16, 2003
2799