Synthesis of isoprostanyl phosphatidylcholine and isoprostanyl phosphatidylethanolamine

Article abstract of DOI:10.1021/jo0518553

The syntheses of two isoprostanyl phospholipids are described. A newly established route to 15-F_2t-isoprostane and ent-15-epi-F _2t-isoprostane has allowed for the selective preparation of 15-F _2t-isoprostanyl phosphatidylethanolamine and ent-15-epi-F _2t-isoprostanyl phosphatidylcholine. The nature of the head-groups dictates the coupling strategy used to attach the appropriately protected isoprostanes to the corresponding lysophospholipids. Preliminary ¹H NMR and ³¹P NMR studies indicate that these isoprostanyl phospholipids aggregate in apolar solvents.

Full text of DOI:10.1021/jo0518553

Synthesis of Isoprostanyl Phosphatidylcholine and Isoprostanyl
Phosphatidylethanolamine
Manami Shizuka, Thomas O. Schrader, and Marc L. Snapper*
Department of Chemistry, Eugene F. Merkert Chemistry Center, Boston College, 2609 Beacon Street,
Chestnut Hill, Massachusetts 02467
marc.snapper@bc.edu
ReceiVed September 2, 2005
The syntheses of two isoprostanyl phospholipids are described. A newly established route to 15-F2t-
isoprostane and ent-15-epi-F2t-isoprostane has allowed for the selective preparation of 15-F2t-isoprostanyl
phosphatidylethanolamine and ent-15-epi-F2t-isoprostanyl phosphatidylcholine. The nature of the head-
groups dictates the coupling strategy used to attach the appropriately protected isoprostanes to the
corresponding lysophospholipids. Preliminary 1H NMR and 31P NMR studies indicate that these
isoprostanyl phospholipids aggregate in apolar solvents.
Isoprostanyl esters are generated through oxidation of phos-
phatidyl arachidonates (Figure 1).1 Phospholipase-mediated
hydrolysis of the resulting isoprostanyl phospholipids then
releases the isoprostanes from their lipid surroundings.2 The
biological activities of these lipid metabolites typically involve
inflammatory responses, as well as activities related to smooth
muscle growth and platelet aggregation factors.3 The isopros-
tanes are also recognized as important indicators of oxidative
stress,4 particularly in human maladies such as Alzheimer’s
disease,5 diabetes,6 and cancer.7 Although progress has been
(1) For free radical peroxidation of arachidonic acid, see: (a) Morrow,
J. D.; Hill, K. E.; Burk, R. F.; Nammour, T. M.; Badr, K. F.; Roberts, L.
J., II. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 9383. (b) Morrow, J. D.;
Harris, T. M.; Roberts, L. J., II. Anal. Biochem. 1990, 184, 1. (c) Morrow,
J. D.; Awad, J. A.; Boss, H. J.; Blair, I. A.; Roberts, L. J., II. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 10721. (d) Morrow, J. D.; Minton, T. A.;
Mukundan, C. R.; Campbell, M. D.; Zackert, W. E.; Daniel, V. C.; Badr,
K. F.; Blair, I. A.; Roberts, L. J., II. J. Biol. Chem. 1994, 269, 4317. (e)
Harrison, K. A.; Murphy, R. C. J. Biol. Chem. 1995, 270, 17273. (f) Morrow,
J. D.; Awad, J. A.; Wu, A.; Zackert, W. E.; Daniel, V. C.; Roberts, L. J.,
II. J. Biol. Chem. 1996, 271, 23185. (g) Basu, S. Prostaglandins,
Leukotrienes Essent. Fatty Acids 1998, 58, 319. (h) Roberts, L. J.; Fessel,
J. P. Chem. Phys. Lipids 2004, 128, 173. (i) Wang, D.; DuBois, R N. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 415. For review, see: (j) Fam, S. S.;
Morrow, J. D. Curr. Med. Chem. 2003, 10, 1723.
FIGURE 1. Formation of isoprostanes and isoprostanyl phospholipids
from arachidonic esters.
made in understanding the generation and distribution of the
isoprostanes, the biological activities and interactions of their
isoprostanyl phospholipid precursors are far less clear.
To begin addressing questions regarding the function of the
lipid-bound oxidative metabolites, we sought access to several
(2) Lawson, J. A.; Rokach, J.; FitzGerald, G. A. J. Biol. Chem. 1999,
274, 24441.
(3) (a) Natarajan, R.; Lanting, L.; Gonzales, N.; Nadler, J. Am. J. Physiol.
1996, 271, E159. (b) Kunapuli, P.; Lawson, J. A.; Rokach, J. A.; Meinkoth,
J. L.; FitzGerald, G. A. J. Biol. Chem. 1998, 273, 22442.
(4) Morrow, J. D.; Zackert, W. E.; Van Der Ende, D. S.; Reich, E. E.;
Terry, E. S.; Cox, B.; Sanchez, S. C.; Montine, T. J.; Roberts, L. J. Oxid.
Stress Dis. 2002, 8, 57.
(5) Quinn, J. F.; Montine, K. S.; Moore, M.; Morrow, J. D.; Kaye, J.
A.; Montine, T. J. J. Alzheimer’s Dis. 2004, 6, 93.
10.1021/jo0518553 CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/17/2006
1330
J. Org. Chem. 2006, 71, 1330-1334

Synthesis of Two Isoprostanyl Phospholipids
isoprostanyl phospholipids, as well as structural variants.8 These
compounds should allow for the identification of relevant
interactions of the oxidized phospholipids within the membrane
bilayer environment. The isoprostanyl phospholipids should also
provide valuable tools for studying phospholipase processing
of the oxidized phospholipids. As a preliminary step toward
these objectiVes, we report the synthesis of 15-F2t-isoprostanyl
phosphatidylethanolamine 1 and ent-15-epi-F2t-isoprostanyl
phosphatidylcholine 2.
SCHEME 1. Synthesis of 15-F2t-Isoprostanyl
Phosphatidylethanolamine 1a
a (a) DCC, DMAP, CH2Cl2; 74%. (b) NaI, 2-butanone, 80 °C; 93%. (c)
1 M HCl, THF/H2O; mixture of 6 and 1. (d) TFA, CH2Cl2; 32% from 5.
Martin.11 Coupling of tris-TBS-protected isoprostane 3, an
intermediate generated in our stereodivergent route to all the
isoprostane isomers,12 to the protected lysophosphatidyletha-
nolamine fragment 4 was accomplished using DCC and DMAP
to give the protected phospholipid 5 in 74% yield (Scheme 1).
The synthesis of 1 was completed by selective hydrolysis of
the phosphate methyl ester using Finkelstein conditions to give
the phosphate in 93% yield. An acid-mediated deprotection of
the silyl ethers and the Boc-protected amine gave the desired
15-F2t-isoprostanyl phosphatidylethanolamine 1 in 26% yield
along with fractions containing Boc-protected compound 6 after
silica gel chromatography. The mixture containing 6 was treated
with TFA in CH2Cl2 to deliver an additional 5% yield of the
desired phospholipid 1.
15-F2t-Isoprostanyl Phosphatidylethanolamine (1). On the
basis of 15-F2t-isoprostane’s well-established formation under
oxidative conditions,9 15-F2t-isoprostanyl phosphatidylethanol-
amine 1 was selected as an initial target. As illustrated in eq 1,
a convergent approach was envisioned for the preparation of
the 15-F2t-isoprostanyl phospholipid. Phospholipid 1 could be
Preliminary studies indicate that 15-F2t-isoprostanyl phos-
phatidylethanolamine 1 can serve as a substrate for secretory
phospholipase A2 (bee venom s-PLA2).13 Specifically, HPLC
analysis indicated that 15-F2t-isoprostane is liberated when 1 is
treated with s-PLA2. Although an interesting observation,
additional studies with more relevant phospholipases may be
necessary before any conclusions of biological significance can
be made.
ent-15-epi-F2t-Isoprostanyl Phosphatidylcholine (2). A
preliminary screen of the 15-F2-isoprostanes indicated that ent-
15-epi-F2t-isoprostane is more active in a whole blood platelet
aggregation assay than the known 15-F2t-isoprostane.14 This new
isoprostanyl activity inspired us to examine ent-15-epi-F2t-
isoprostanyl phosphatidylcholine 2, a likely cellular precursor
to ent-15-epi-F2t-isoprostane. With access to all the isoprostanes
in hand,12 a convergent esterification of the protected isoprostane
accessed readily by coupling the protected lysophosphatidyl-
ethanolamine derivative 4 with a tris-protected isoprostane 3.
Subsequent steps would then involve removal of the protecting
groups.
The known lysophosphatidylethanolamine derivative 4 was
(10) Delfino, J. M.; Schreiber, S. L.; Richards, F. M. Tetrahedron Lett.
1987, 28, 2327.
prepared using procedures reported by Schreiber/Richards10 and
(11) Martin, S. F.; Josey, J. A.; Wong, Y.-L.; Dean, D. W. J. Org. Chem.
1994, 59, 4805.
(12) (a) Schrader, T. O.; Snapper, M. L. J. Am. Chem. Soc. 2002, 124,
10998. (b) Schrader, T. O.; Snapper, M. L. Tetrahedron Lett. 2000, 41,
9685.
(13) s-PLA2 Assay Kit from Cayman Chemical, Ann Arbor, MI (Catalog
No. 765001). For related work from our laboratory, see: (a) Cheung, A.
K.; Murelli, R.; Snapper, M. L. J. Org. Chem. 2004, 69, 5712. (b) Cheung,
A. K.; Snapper, M. L. J. Am. Chem. Soc. 2002, 124, 11584.
(14) Schrader, T. O. Ph.D. Thesis, Boston College, Chestnut Hill, MA,
2002.
(6) Giovanni, D.; Falco, A.; Patrono, C. Chem. Phys. Lipids 2004, 128,
149.
(7) Camphausen, K.; Menard, C.; Sproull, M.; Goley, E.; Basu, S.;
Coleman, C. N. Int. J. Radiat. Oncol., Biol., Phys. 2004, 58, 1536.
(8) (a) Acharya, H. P.; Kobayashi, Y. Angew. Chem., Int. Ed. 2005, 44,
3481. (b) Jung, M. E.; Kers, A.; Subbanagounder, G.; Berliner, J. A. Chem.
Commun. 2003, 2, 196. (c) Jung, M. E.; Berliner, J. A.; Angst, D.; Yue,
D.; Koroniak, L.; Watson, A. D.; Li, R. Org. Lett. 2005, 7, 3933.
(9) Montuschi, P.; Barnes, P. J.; Roberts, L. J., II. FASEB J. 2004, 18,
1791.
J. Org. Chem, Vol. 71, No. 4, 2006 1331

Shizuka et al.
SCHEME 2. Synthesis of Lipid Fragment 9a
acid 7 with commercially available phosphatidylcholine alcohol
8 (eq 2) was envisioned as our preferred route to this likely
lipid metabolite. Unfortunately, no desired product was obtained
a (a) PMBCl, NaH, DMF, 0 °C to room temperature; 93%. (b) AcOH,
MeOH; 99%. (c) palmitic acid, DCC, DMAP, CH2Cl2; 78%. (d)
BOMCl, i-Pr2NEt, CH2Cl2; 90%. (e) DDQ, H2O/CH2Cl2; 90%. (f) (1)
Cl2(Od)PO(CH2)2Cl, Et3N, CH2Cl2, room temperature; (2) MeOH, Et3N,
room temperature; 63%. (g) Pd/C, H2, THF/H2O; 99%.
SCHEME 3. Completion of ent-15-epi-F2t-Isoprostanyl
Phosphatidylcholine 2a
using a variety of coupling protocols,15 and only starting material
was usually observed. The problem was thought to arise from
the zwitterionic nature of the lipid fragment 8. We decided,
therefore, to use the neutral lipid precursor 9 in coupling with
a protected form of 7, which can then be converted to
phospholipid 2 with the desired choline headgroup.
The initial steps toward 9 were performed under modified
Martin conditions from an optically pure, protected glycerol
(Scheme 2). PMB protection of the primary alcohol and
deprotection of the acetonide gave 10. DCC coupling of 10 with
palmitic acid, BOM protection of the secondary alcohol, and
oxidative removal of the PMB group with DDQ in CH2Cl2/
H2O yielded alcohol 11. The subsequent phosphorylation was
problematic with Martin’s procedure; instead, treating 11 with
2-chloroethyl phosphorodichloridate and Et3N in CH2Cl2, fol-
lowed by addition of methanol, and removal of the BOM group
by hydrogenolysis yielded the desired lipid fragment 9.
Esterification of the protected isoprostane 12 with 9 was now
successful using EDC/DMAP to obtain 13 in 55% yield (Scheme
3). Global deprotection of the TBS groups with 1 M HCl yielded
triol 14, which was then treated with excess anhydrous tri-
methylamine to give the desired ent-15-epi-F2t-isoprostanyl
phosphatidylcholine 2 in 80% yield.
a (a) EDC, DMAP, CH2Cl2; 55%. (b) 1 M HCl, THF/H2O; 92%. (c)
Me3N (excess), CHCl3/EtOH, 62 °C, 60 h; 80%.
structures are generated in apolar solvents. In contrast, isopros-
tanyl phospholipids (1 and 2) showed line broadening of all
1
signals in the H NMR spectra, suggesting that aggregates are
forming in CDCl3, whereas in more polar CD3OD, the molecule
appears to be monomeric.
In summary, the total synthesis of 15-F2t-isoprostanyl phos-
phatidylethanolamine 1 and ent-15-epi-F2t-isoprostanyl phos-
phatidylcholine 2 was accomplished via a convergent strategy
by an esterification of a protected isoprostane and a phosphatidyl
lipid fragment. 1H NMR spectroscopy indicated that aggregates
are forming in apolar solvents, whereas in more polar solvents,
1 and 2 appear to be monomeric. The synthetic phospholipids
will aid us to further understand mechanisms associated with
oxidative stress. Furthermore, our synthesis will allow us to
prepare photoaffinity probe reagents to assess the lipid interac-
tions of these cellular metabolites.
NMR analyses of phospholipids 1 and 2 revealed interesting
solvation effects. Although the 1H NMR spectra of both lipids
1
taken in CD3OD showed relatively sharp resonances, the H
NMR spectra taken in CDCl3 displayed broadened peaks.16
A
similar phenomenon was observed in the 31P NMR spectra.
Single resonances were observed when the 31P NMR spectra
were taken in CD3OD. However, no phosphorus resonance was
observed in CDCl3, even after several thousand scans. Kai et
al. also observed similar solvent-dependent behavior of phos-
pholipid bolaamphiphiles:17 Increased peak sharpening was
observed for the polar headgroup (R-NMe3+) with increasing
polarity of the solvent (9:1, 3:1, 1:1, 1:3, CDCl3/CD3OD), and
the reverse trend was observed for the terminal methyl groups
on the fatty acid chains. Their data suggested that micelle-like
structures are formed in polar solvents, whereas reverse micelle
Experimental Section
Tris-TBS-Protected Isoprostane (3). To a solution of bis-TBS
isoprostane 3b (41 mg, 0.070 mmol, 1 equiv) and Et3N (100 µL,
0.72 mmol, 10 equiv) in CH2Cl2 (700 µL, 0.10 M) was added
TBSCl (48 mg, 0.32 mmol, 4.6 equiv) followed by DMAP (8 mg,
0.07 mmol, 1 equiv). After stirring at 23 °C for 12 h, Et2O (15
mL) was added and the reaction mixture was washed sequentially
with HCl (1 M, 15 mL) and brine (15 mL). The organic layer was
dried over MgSO4, filtered, and concentrated. The residue was then
filtered through a 1 cm plug of silica gel (15:1, pentane/Et2O as
(15) The following coupling conditions were attempted: DCC/DMAP,
EDC/DMAP, CDI, and Yamaguchi esterification.
(16) The full 1H NMR spectral data are shown in the Supporting
Information.
(17) (a) Kai, T.; Sun, X.-L.; Faucher, K. M.; Apkarian, R. P.; Chaikof,
E. L. J. Org. Chem. 2005, 70, 2606. (b) Winnik, F. M.; Miyazawa, K.
Macromolecules 2002, 35, 2440.
1332 J. Org. Chem., Vol. 71, No. 4, 2006

Synthesis of Two Isoprostanyl Phospholipids
elutant) and concentrated. The resulting residue was dissolved in
THF (1 mL) and H2O (1 mL), and LiOH (10 mg, 0.41 mmol) was
added. The reaction was stirred at 23 °C for 2 h (judged complete
by TLC) at which time the reaction mixture was diluted with Et2O
and (15 mL) and HCl (0.5 M, 15 mL). The organic layer was
separated and washed with brine (15 mL). The organics were dried
over MgSO4, filtered, and concentrated to a crude oil that was
purified by silica gel chromatography (pentane/Et2O/AcOH; gradient
6:1:0.1 to 1:1:0.1; Rf ) 0.25, pentane/Et2O/AcOH, 6:1:0.1) to deliver
3 as a colorless oil (18 mg, 0.026 mmol, 42% yield based on
recovered 3b), followed by recovered 3b (Rf ) 0.0, pentane/Et2O/
AcOH, 6:1:0.1; 5 mg, 0.009 mmol, 12% recovery). 1H NMR
(CDCl3, 400 MHz): δ 5.46 (1H, dd, J ) 15.2, 6.0 Hz), 5.42 (1H,
m), 5.32 (1H, m), 5.29 (1H, dd, J ) 15.2, 10.4 Hz), 4.05 (1H, q,
J ) 6.4 Hz), 3.92 (1H, m), 3.77 (1H, q, J ) 6.8 Hz), 2.56 (1H, m),
2.34 (3H, m), 2.08 (4H, m), 1.82 (1H, m), 1.69 (2H, m), 1.56-
1.39 (3H, m), 1.36-1.21 (6H, m), 0.90-0.85 (30H, m), 0.04 (3H,
s), 0.03 (9H, s), 0.01 (6H, s). 13C NMR (CDCl3, 100 MHz): δ
178.9, 135.8, 129.9, 128.7, 127.6, 76.4, 76.2, 73.5, 52.8, 50.2, 44.6,
38.9, 33.6, 32.2, 27.0, 26.6, 26.4, 26.2, 25.3, 24.9, 23.0, 18.6, 18.4,
18.3, 14.4, -3.9, -4.0, -4.1, -4.20, -4.25, -4.33. IR (thin film,
NaCl): 2961 (s), 2936 (m), 2860 (m), 1721 (s), 1482 (m), 1262
(m), 1086 (m), 834 (w) cm-1. HRMS (ESI+): calcd for C38H76O5-
Si3Na (M + Na) 719.4898, found 719.4904.
were evaporated, and the material was purified by silica gel
chromatography (i-PrOH/H2O; 25:1, Rf ) 0.15) to give impure
fractions containing the Boc-protected compound 6 (multiple spots
by TLC, 3.5 mg) followed by pure 1 (1.1 mg, 1.4 µmol, 26% yield).
The material containing 6 was dissolved in TFA (250 µL) and
CH2Cl2 (250 µL) and stirred at 23 °C for 1 h. The volatiles were
evaporated (25 °C, 0.1 mmHg), and the material was purified by
silica gel chromatography (i-PrOH/H2O; 25:1) to give additional 1
1
(0.2 mg, 0.3 µmol, 32% total yield from 6). H NMR (CD3OD,
400 MHz): the proton NMR spectra for compound 1 was difficult
to interpret, so selected peaks are shown; δ 5.49 (1H, m), 5.48-
5.26 (2H, m), 5.22 (1H, m), 4.41 (1H, m), 4.21 (1H, dd, J ) 7.6,
4.0 Hz), 4.17 (1H, m), 4.12-3.83 (6H, m), 3.70-3.40 (2H, m),
3.61 (br s, impurity from silica gel), 3.14 (2H, m), 2.60 (4H, m),
2.42 (4H, t, J ) 7.6 Hz), 1.80-1.50 (6H, m), 1.48-1.22 (34H,
m), 0.90 (6H, t, J ) 6.8 Hz). 1H NMR (CDCl3, 400 MHz): δ 8.15
(2H, br s), 5.60-5.10 (4H, m), 4.40 (1H, m), 4.45-3.80 (7H, m),
3.80-3.30 (2H, m), 3.62 (br s, impurity from silica gel), 3.20 (2H,
br s), 2.40-2.20 (4H, m), 2.10-1.90 (4H, m), 1.80-1.48 (14H,
m), 1.38-1.18 (26H, m), 0.87 (6H, t, J ) 6.8 Hz). 31P NMR (CD3-
OD, 162 MHz): δ 4.30. IR (thin film, NaCl): 3415 (b), 2936 (s),
2867 (m), 1741 (m), 1470 (m), 1369 (w), 1224 (w), 1086 (m) cm-1
.
MS (ESI-): 788.42 (M - H), 770.42 (M - H3O). HRMS (ESI+):
calcd for C41H76NO11PNa (M + Na) 812.5054, found 812.5020.
HRMS (ESI-): calcd for C38H75NO11P (M - H) 788.5078, found
788.5080.
Tris-TBS-Isoprostanyl Phospholipid (5). To a solution of acid
3 (18 mg, 0.026 mmol, 1 equiv), alcohol 4 (15 mg, 0.026 mmol, 1
equiv), and DMAP (1.4 mg, 0.011 mmol, 0.4 equiv) in CH2Cl2
(370 µL, 0.07 M) was added a solution of DCC (8 mg, 0.04 mmol,
1.5 equiv) in CH2Cl2 (150 µL) over 30 min at 23 °C under N2.
After stirring for a total of 5.5 h (judged complete by TLC), the
reaction was filtered through a 1 cm layer of Celite and concen-
trated. The resulting material was dissolved in pentane (1 mL) and
filtered through cotton to remove insoluble impurities. The solution
was concentrated and purified by silica gel chromatography
(pentane/EtOAc; gradient 4:1, 1:1) to give recovered 3 (Rf ) 0.4,
pentane/EtOAc, 4:1; 3.5 mg, 0.0050 mmol, 19% recovery), followed
by the protected lipid 5 (Rf ) 0.15, pentane/EtOAc, 4:1; 19.3 mg,
0.0155 mmol, 74% yield based on recovered 3) isolated as a
BOM-Protected Phospholipid (11b). In a flame-dried 10 mL
Schlenk flask, alcohol 11 (265 mg, 0.59 mmol, 1 equiv) and CH2-
Cl2 (2.7 mL, 0.2 M) were added under N2. Et3N (164 µL, 1.18
mmol, 2 equiv) was added to the mixture, and the mixture was
stirred at 23 °C for 5 min. Reagent Cl2(Od)PO(CH2)2Cl (75 µL,
0.59 mmol, 1 equiv) was added dropwise, and the reaction was
stirred at 23 °C under N2 for 12 h. Et3N (82 µL, 0.59 mmol, 1
equiv) and methanol (36 µL, 0.88 mmol, 1.5 equiv) were added to
the reaction mixture, and the mixture was stirred for another 2 h at
23 °C. The reaction was opened to air, and the solution was filtered
through a cotton-plugged pipet, flushed with CH2Cl2 (2.0 mL), and
concentrated. The residue was purified by silica gel chromatography
(hexanes/EtOAc; gradient 2:1, 1:1, 1:10; Rf ) 0.3, hexane/EtOAc,
1:1) to give intermediate 11b as a pale yellow oil (225 mg, 0.37
mmol, 63% yield). 1H NMR (CDCl3, 400 MHz): δ 7.38-7.27 (5H,
m), 4.86 (2H, s), 4.66 (2H, s), 4.30-4.24 (3H, m), 4.21-4.14 (3H,
m), 4.10 (1H, q, J ) 5.1 Hz), 3.79 (3H, d, JH-P1 ) 11.3 Hz, d,
JH-P2 ) 11.3 Hz), 3.70-3.67 (2H, m), 2.30 (2H, t, J ) 7.4 Hz),
1.64-1.56 (2H, m), 1.28-1.24 (24H, m), 0.88 (3H, t, J ) 6.6 Hz).
13C NMR (CDCl3, 100 MHz): δ 173.4, 137.5, 128.5, 127.9, 94.1,
77.5, 77.2, 76.9, 73.5, 73.4, 69.9, 67.4, 67.3, 67.0, 66.9, 62.9, 42.7,
34.4, 32.2, 29.9, 29.8, 29.6, 29.5, 29.4, 25.2, 23.0, 14.5. 31P NMR
(CDCl3, 162 MHz): δ 0.93. IR (neat, NaCl): 2891 (s), 2859 (s),
1739 (s), 1463 (m), 1281 (s), 1174 (w), 1114 (m), 1040 (s), 1023
(s), 866 (w), 821 (w), 738 (w). HRMS (ESI+): calcd for C30H53-
ClO8P (M + H) 607.3161, found 607.3167.
Alcohol Phospholipid (9). Pd/C (10% by wt., dry, 13 mg) was
added to a stirred solution of BOM-phosphate 11b (100 mg, 0.16
mmol, 1 equiv) in THF (129 µL) and H2O (14.0 µL). Hydrogen
gas was bubbled into the reaction mixture for 5 min. The reaction
was stirred under H2 (1 atm) at 23 °C for 3 h (judged complete by
TLC). The solution was filtered through a pad of Celite and was
washed with EtOAc (5 mL). The mixture was dried over MgSO4,
filtered, and concentrated. The residue was purified by silica gel
chromatography (hexanes/EtOAc; 1:1; Rf ) 0.15) to give 9 as a
colorless oil (78 mg, 0.16 mmol, quantitative yield). 1H NMR
(CDCl3, 400 MHz): δ 4.27 (1H, td, J ) 5.5, 1.0 Hz), 4.25 (1H, td,
J ) 5.8, 1.0 Hz), 4.22-4.16 (3H, m), 4.16-4.12 (1H, m), 4.12-
4.08 (1H, m), 3.83 (3H, d, JH-P1 ) 11.3 Hz, d, JH-P2 ) 11.2 Hz),
3.72 (2H, t, J ) 5.6 Hz), 2.34 (2H, t, J ) 7.4 Hz), 1.66-1.59 (2H,
m), 1.29-1.25 (24H, m), 0.88 (3H, t, J ) 7.0 Hz). 13C NMR
(CDCl3, 100 MHz): δ 173.8, 69.3, 69.2, 69.1, 69.0, 67.6, 64.5,
55.1, 42.8, 42.7, 34.4, 32.3, 30.0, 29.91, 29.89, 29.8, 29.7, 29.6,
1
colorless oil. H NMR (CDCl3, 400 MHz): δ 5.45 (1H, dd, J )
15.6, 6.0 Hz), 5.42 (1H, m), 5.34-5.27 (2H, m), 5.24 (1H, m),
5.08 (1H, br s), 4.32 (1H, dd, J ) 11.6, 4.4 Hz), 4.22-4.04 (6H,
m), 3.92 (1H, m), 3.77 (3H, overlapping d, J ) 4.0 Hz), 3.76 (1H,
m), 3.41 (2H, m), 2.54 (1H, m), 2.32 (5H, m), 2.18-2.00 (4H, m),
1.82 (1H, m), 1.72-1.56 (3H, m), 1.55-1.34 (3H, m), 1.43 (9H,
s), 1.32-1.21 (31H, m), 0.90-0.84 (33H, m), 0.04 (3H, s), 0.02
(9H, s), 0.01 (6H, s). 13C NMR (CDCl3, 100 MHz): δ 173.2, 172.6,
172.5, 155.8, 135.9, 129.8, 128.6, 127.6, 79.8, 76.4, 76.2, 73.5,
69.6, 67.5, 65.7, 61.8, 54.9, 52.8, 50.2, 44.6, 41.2, 38.9, 34.3, 34.0,
32.3, 32.2, 30.0, 29.9, 29.8, 29.7, 29.6, 29.5, 28.7, 27.0, 26.6, 26.24,
26.20, 25.3, 25.2, 23.1, 23.0, 18.6, 18.4, 18.3, 14.5, 14.4, -3.88,
-3.94, -4.1, -4.20, -4.24, -4.3. 31P NMR (CDCl3, 162 MHz):
δ 1.23, 1.19. IR (thin film, NaCl): 3345 (b), 2967 (s), 2923 (s),
2849 (s), 1747 (s), 1734 (s), 1514 (m), 1469 (m), 1375 (m), 1262
(s), 1174 (s), 840 (s) cm-1. HRMS (ESI+): calcd for C65H128NO13-
PSi3Na (M + Na) 1268.8303, found 1268.8329.
15-F2t-Isoprostanyl Phosphatidylethanolamine (1). To a stirred
solution of 5 (11.7 mg, 9.38 µmol, 1 equiv) in 2-butanone (freshly
distilled from P2O5, 1.9 mL, 0.005 M) was added NaI (7.0 mg,
0.047 mmol, 5 equiv). The solution was refluxed (80 °C) for 2 h
(judged complete by TLC). After evaporation of the solvent, the
residue was suspended in CHCl3 (15 mL) and the solution was
washed with 5% aqueous HCl (2 × 15 mL) followed by H2O (15
mL). The aqueous layer was back-extracted with CHCl3 (25 mL),
and the combined organics were dried over Na2SO4, filtered, and
concentrated to give 6 as a pale yellow oil (10.8 mg, 8.76 µmol,
93% yield).
The protected lipid 6 (6.60 mg, 5.36 µmol, 1 equiv) was stirred
in a solution of HCl (1 M, 750 µL, 140 equiv) and THF (750 µL,
0.007 M) for 75 h at 23 °C (monitored by TLC). The volatiles
J. Org. Chem, Vol. 71, No. 4, 2006 1333

Shizuka et al.
29.5, 25.2, 23.0, 14.5. 31P NMR (CDCl3, 162 MHz): δ 1.89. IR
(neat, NaCl): 3388 (b), 2957 (s), 2891 (s), 2860 (s), 1739 (s), 1466
(s), 1377 (m), 1254 (s), 1176 (s), 1038 (s), 877 (m), 822 (m), 739
(m), 699 (m), 670 (m). HRMS (ESI+): calcd for C22H45ClO7P (M
+ H) 487.2591, found 487.2591.
complete by TLC). The reaction mixture was concentrated, and
the residue was purified by silica gel chromatography (CH2Cl2/
MeOH; 9:1; Rf ) 0.4, 0.38) to obtain the triol isoprostanyl lipid
13b as a colorless oil (12.0 mg, 0.014 mmol, 92% yield).
In a heavy-walled sealed tube, 13b (2.0 mg, 2.4 µmol) and
CHCl3/EtOH (100 µL/100 µL) were added and a rubber septum
was placed on top. Anhydrous trimethylamine (approximately 200
µL) was condensed to the solution at -78 °C until the volume
approximately doubled. The reaction mixture was allowed to warm
to 23 °C, and the rubber septum was replaced with a Teflon screw
cap. The reaction mixture was heated to 62 °C and was stirred at
this temperature for 60 h. The solution was allowed to cool to 23
°C and was concentrated. The crude residue was purified by silica
gel chromatography (CH3OH/H2O; gradient 100:0, 50:1, 10:1; Rf
) 0.25, CH3OH/H2O, 10:1) to obtain 2 as an off-white solid (1.3
mg, 1.6 µmol, 65% yield). 1H NMR (CD3OD, 400 MHz): δ 5.52-
5.49 (2H, m), 5.44 (1H, dd, J ) 11.8, 3.3 Hz), 5.39-5.33 (1H, m),
5.24-5.19 (1H, m), 4.40 (1H, dd, J ) 12.0, 3.3 Hz), 4.30-4.23
(3H, m), 4.17 (2H, m), 4.01-3.92 (3H, m), 3.84 (1H, dt, J ) 7.6,
5.4 Hz), 3.63-3.61 (2H, m), 2.66 (1H, m), 2.46 (1H, p, J ) 7.4
Hz), 2.32 (4H, m), 2.13-1.99 (5H, m), 1.65 (2H, m), 1.61-1.39
(12H, m), 1.27 (34H, m), 0.92 (3H, t, J ) 7.5 Hz), 0.89 (3H, t, J
) 7.1 Hz). 31P NMR (CD3OD, 162 MHz): δ 0.47. IR (thin film,
NaCl): 3355 (b), 3059 (m), 3027 (m), 2954 (s), 2892 (m), 2856
(s), 1737 (s), 1661 (w), 1465 (w), 1234 (m), 1085 (s), 971 (m).
HRMS (ESI+): calcd for C44H83NO11P (M + H) 832.5701, found
Tris-TBS-Isoprostanyl Phospholipid (13). Acid 12 (22.0 mg,
0.032 mmol, 1 equiv) was dissolved in CH2Cl2 (800 µL, 0.04 M).
EDC (11.0 mg, 0.057 mmol, 1.8 equiv) and DMAP (2.0 mg, 0.016
mmol, 0.5 equiv) were added to the solution followed by alcohol
9 (23.0 mg, 0.047 mmol, 1.5 equiv). The reaction mixture was
stirred for 12 h at 23 °C. The solution was diluted with CH2Cl2
(3.0 mL) and was washed with saturated NH4Cl (aq) (1 × 3 mL)
and brine (1 × 3 mL). The organic layer was dried over MgSO4,
filtered, and concentrated. The crude residue was purified by silica
gel chromatography (hexanes/EtOAc 5:1; Rf ) 0.15, 0.1) to obtain
13 as a colorless oil (20.0 mg, 0.017 mmol, 54% yield). 1H NMR
(CDCl3, 400 MHz): δ 5.46 (1H, dd, J ) 15.2, 6.0 Hz), 5.44 (1H,
m), 5.37-5.31 (2H, m), 5.27-5.21 (1H, m), 4.35-4.09 (6H, m),
4.04 (1H, q, J ) 6.2 Hz), 3.89 (1H, dt, J ) 6.8, 3.9 Hz), 3.80 (3H,
d, JH-P1 ) 11.3 Hz, d, JH-P2 ) 11.3 Hz), 3.70 (2H, t, J ) 5.7 Hz),
2.62-2.57 (1H, m), 2.36-2.29 (4H, m), 2.12-2.01 (4H, m), 1.88
(1H, q, J ) 7.1 Hz), 1.72-1.59 (3H, m), 1.54-1.40 (2H, m), 1.25
(34H, m), 0.88-0.86 (33H, m), 0.04 (3H, s), 0.03-0.01 (15H, m).
13C NMR (CDCl3, 100 MHz): δ 173.2, 172.5, 136.0, 129.9, 128.7,
128.1, 76.4, 76.3, 73.7, 69.7, 69.6, 67.51, 67.46, 65.9, 61.8, 55.0,
54.9, 52.6, 50.3, 44.6, 42.7, 42.6, 38.8, 34.3, 34.0, 32.27, 32.2,
30.04, 30.00, 29.97, 29.8, 29.7, 29.6, 29.5, 27.0, 26.6, 26.3, 26.22,
26.20, 25.3, 25.2, 23.1, 23.0, 18.6, 18.42, 18.37, 14.5, 14.4, -3.87,
-3.94, -4.2, -4.3. 31P NMR (CDCl3, 162 MHz): 0.77, 0.74. IR
(thin film, NaCl): 3059 (m), 3026 (s), 2950 (s), 2924 (s), 2859
(s), 1745 (s), 1462 (m), 1254 (s), 1144 (m), 1045 (s), 972 (w), 836
(s), 227 (m). HRMS (ESI+): calcd for C60H118ClO11Si3Na (M +
832.5704. [R]20 +46.3° (c ) 0.05, CHCl3).
D
Acknowledgment. We thank the National Institutes of
Health (R01-CA66617) for financial support and Dr. John L.
LaMattina for a Graduate Student Fellowship (M.S.). In addition,
we thank Prof. Mary F. Roberts for helpful discussions.
Na) 1187.7302, found 1187.7306. [R]20 -14° (c ) 1.0, CHCl3).
D
Supporting Information Available: Experimental details and
NMR spectra are provided. This material is available free of charge
via the Internet at http://pubs.acs.org.
ent-15-epi-F2t-Isoprostanyl Phosphatidylcholine (2). Aqueous
HCl (163 µL, 0.16 mmol, 10 equiv, 1 M) was added to a stirred
solution of 13 (19.0 mg, 0.016 mmol, 1 equiv) in THF (500 µL,
0.3 M), and the mixture was stirred at 23 °C for 10 h (judged
JO0518553
1334 J. Org. Chem., Vol. 71, No. 4, 2006