Synthesis of sn-1 functionalized phospholipids as substrates for secretory phospholipase A2

Article abstract of DOI:10.1016/j.chemphyslip.2006.12.006

Secretory phospholipase A₂ (sPLA₂) represents a family of small water-soluble enzymes that catalyze the hydrolysis of phospholipids in the sn-2 position liberating free fatty acids and lysophospholipids. Herein we report the synthesi

Full text of DOI:10.1016/j.chemphyslip.2006.12.006

Chemistry and Physics of Lipids 146 (2007) 54–66
Synthesis of sn-1 functionalized phospholipids as
substrates for secretory phospholipase A2
a
Lars Linderoth , Gunther H. Petersa,b, Kent Jørgensenc,
¨
Robert Madsena,d, Thomas L. Andresenc,∗
Department of Chemistry, Technical University of Denmark, Denmark
a
b
MEMPHYS-Center for Biomembrane Physics, Denmark
c
LiPlasome Pharma A/S, Denmark
d
Center for Sustainable and Green Chemistry, Technical University of Denmark, Denmark
Received 23 July 2006; received in revised form 4 December 2006; accepted 22 December 2006
Available online 7 January 2007
Abstract
Secretory phospholipase A2 (sPLA2) represents a family of small water-soluble enzymes that catalyze the hydrolysis of phospho-
lipids in the sn-2 position liberating free fatty acids and lysophospholipids. Herein we report the synthesis of two new phospholipids
(1 and 2) with bulky allyl-substituents attached to the sn-1 position of the glycerol backbone. The synthesis of phospholipids 1 and
2 is based upon the construction of a key aldehyde intermediate 3 which locks the stereochemistry in the sn-2 position of the final
phospholipids. The aldehyde functionality serves as the site for insertion of the allyl-substituents by a zinc mediated allylation.
Small unilamellar liposomes composed of phospholipids 1 and 2 were subjected to sPLA2 activity measurements. Our results show
that only phospholipid 1 is hydrolyzed by the enzyme. Molecular dynamics simulations revealed that the lack of hydrolysis of
phospholipid 2 is due to steric hindrance caused by the bulky side chain of the substrate allowing only limited access of water
molecules to the active site.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: sPLA2 activity; Biomarkers; Molecular dynamics simulations; Phospholipid; Substrate specifity; Synthesis
1. Introduction
has been studied extensively, the substrate specificity
with relation to the active site architecture of the enzyme
still needs to be addressed and understood, since the
enzyme seems to be able to catalyze the hydrolysis of
lipids with very diverse chemical structures (Andresen
and Jørgensen, 2005a; Bonsen et al., 1972a,b; Slotboom
et al., 1976).
Secretory phospholipase A2 (sPLA2) constitutes a
family of interfacially active mammalian enzymes. To
date, 10 members (IB, IIA, IIC, IID, IIE, IIF, III, V, X
and XIIA) of this family have been reported (Murakami
and Kudo, 2004). Even though this group of enzymes
This family of enzymes catalyzes the hydrolysis of
phospholipids in the sn-2 position generating free fatty
acids and lysophospholipids (Six and Dennis, 2000).
Moreover, the group of enzymes shares several char-
acteristics as they have a low molecular weight ranging
from 14 to 19 kDa, require Ca2+ for enzymatic activa-
∗
Corresponding author at: Risø National Laboratory, Technical Uni-
versity of Denmark, DK-4000 Roskilde, Denmark.
Tel.: +45 46775480; fax: +45 46774791.
E-mail address: thomas.andresen@risoe.dk (T.L. Andresen).
0009-3084/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2006.12.006

L. Linderoth et al. / Chemistry and Physics of Lipids 146 (2007) 54–66
55
tion and are secreted from cells (Laye and Gill, 2003).
sPLA2 catalyzesthehydrolysisofphospholipidsthrough
a highly conserved catalytic site, where His48, Asp99
and Ca2+ are of particular importance (Dennis, 1994).
Hydrolysis proceeds through activation and specific ori-
entation of a water molecule in the catalytic site (Scott
et al., 1990). The hydrolysis is achieved by hydrogen
bonding of the water molecule to His48 assisted by
Asp99 where Ca2+ stabilizes the intermediate of the
hydrolysis through coordination to the sn-2 carbonyl of
the phospholipid substrate (Dennis, 1994; Scott et al.,
1990).
An interesting aspect present in the sPLA2 family of
enzymes is the fact that thedifferentsubtypesof enzymes
show different specificity towards lipid substrates. This
is reflected in the activity profile of, e.g., the sPLA2-IIA
subtype, which is mainly active towards anionic phos-
pholipids, whereas the sPLA2 V and X subtypes catalyze
the hydrolysis of both anionic and zwitterionic phospho-
lipids (Murakami and Kudo, 2004). Substrate specificity
has been proposed to be dependent on the ability of the
enzymes to bind to the lipid-membrane interface, which
is achieved by the electrostatic interactions between the
enzyme and the surface of the lipid substrate (Leidy et
al., 2006).
Inthispaper, weinvestigatethecatalyticsiteofsPLA2
by the synthesis of two new phospholipids 1 and 2
(Fig. 1), which contain allyl-substituents in the sn-1
position. Testing these phospholipids towards hydroly-
sis catalyzed by sPLA2 can provide valuable insight into
the structural determinants that govern substrate speci-
ficity, and reveal whether sPLA2 can tolerate a bulky
substituent close to the glycerol backbone. Additionally,
the two new phospholipids can function as biomarker
precursors, since the allyl group can serve as a functional
handle for further chemical manipulation by insertion of,
e.g., fluorescent molecular probes such as naphthalene.
Biomarkers positioned in close proximity to the glyc-
erol backbone are important with respect to obtaining
information on the microstructure and interfacial proper-
ties of lipid membranes (Bagatolli, 2003; Bagatolli and
Gratton, 2001). Most of the biomarkers known today
report changes in membranes occurring along the fatty
acid chains or at the end of the phosphate headgroup.
It can therefore be difficult to obtain information at
the membrane–water interface of phospholipid bilayers.
However, incorporating a fluorescent probe in the sn-1
position of the glycerol backbone offers unique opportu-
nities to investigate changes in the vicinity of the glycerol
backbone of phospholipids.
sPLA2-IIA has proven to be especially interest-
ing in therapeutic intervention, since elevated levels
of sPLA2-IIA is present in the microenvironment sur-
rounding tumors, e.g., high levels of sPLA2-IIA have
been observed in human colorectal adenomas and in
neoplastic prostatic tissue (Laye and Gill, 2003). More-
over, sPLA2 plays a role in the release of arachidonic
acid that is metabolized downstream by cyclooxygenase
(COX) and lipoxygenase (LOX) enzymes to give, e.g.,
prostaglandinsandleukotrienes(Andresenetal., 2005b).
The elevated levels of sPLA2 in tumor tissue have pro-
vided the basis of a new tumor activated drug delivery
concept, which relies on the administration of sPLA2
degradable liposomes (Andresen et al., 2005c; Andresen
et al., 2004a). This leads to a site-specific release of
the therapeutics at the tumor target site. The liposomes
can entrap and transport conventional chemotherapeu-
tics such as cisplatin and doxorubicin and release the
therapeutics by using sPLA2 as a tumor-specific trigger
(Andresen et al., 2005d).
The primary challenge in this work has been to design
a suitable synthetic strategy that incorporates an allyl-
substituent in the sn-1 position of the final phospholipid.
As a consequence, the conventional ways of generat-
ing phospholipids (Bhatia and Hajdu, 1988; Martin et
al., 1994; Paltauf and Hermetter, 1994; Roodsari et al.,
1999) cannot be used, and a new and more complex syn-
theticroutehasthereforebeeninvestigated. Wereportthe
synthesis of phospholipids 1 and 2 by the use of a key
aldehyde intermediate 3, which locks the stereochem-
istry in the sn-2 position of the final phospholipid. The
aldehyde functionality furthermore serves as the site for
incorporating the allyl-substituents in the sn-1 position
of the final phospholipids. This aldehyde intermediate
is used for the first time in phospholipid synthesis, and
as our results indicate, will be useful for future synthetic
procedures towards derivatives of natural phospholipids,
where new functional groups are introduced. sPLA2
activity measurements in combination with molecular
dynamics (MD) simulations of the two new phospho-
Fig. 1. Structure of the two target phospholipids.

56
L. Linderoth et al. / Chemistry and Physics of Lipids 146 (2007) 54–66
lipids have provided useful insight on a molecular level
that can explain the specificity of sPLA2 towards the two
phospholipids.
solution, which had turned white overnight, was filtered
and the filtrate was extracted with CH2Cl2 (3 × 200 mL).
The combined organic extracts were dried over MgSO4,
filtered and concentrated in vacuo to give white crystals
(33.0 g). The mixture was used without further purifi-
cation in the next step. Rf 0.77 (10% CH3OH/CH2Cl2).
Crude 5 was dissolved in isopropanol (350 mL) under
nitrogen, and NaBH4 (7.20 g, 190 mmol) was added in
small portions over 6 min, which gave rise to a small
increase in the temperature. The reaction was heated to
70 ◦C and stirred for 30 min. The solution was cooled
to room temperature, and sat. aq. NH4Cl (50 mL) was
slowly added after which the solution was concentrated
in vacuo to a volume of 40 mL. H2O (200 mL) and
EtOAc (100 mL) were added and the phases were sep-
arated. The aqueous phase was extracted with EtOAc
(8 × 100 mL) and the combined organic extracts were
dried over Na2SO4, filtered and concentrated in vacuo
to afford a colorless oil (32.0 g). The oil was used with-
out further purification in the next step. Rf 0.08 (50%
EtOAc/heptane). Sat. NaHCO3 (50 mL) was added to a
solution of crude 6 in CH2Cl2 (350 mL). NaIO4 (40.1 g,
187 mmol) was added in small portions, and the reac-
tion stirred overnight at room temperature. The mixture,
which now contained white crystals, was dried over
MgSO4 and filtered. Na2S2O3 (20.0 g, 130 mmol) was
added to the filtrate and the mixture stirred at room
temperature for 25 min. The mixture was filtered and
concentrated in vacuo to give a yellow syrup, which
was distilled at 80 ◦C, 3.5 mmHg using a Vigreux dis-
tillation apparatus furnishing 18.6 g (64%) of 3 as a
colorless syrup. Rf 0.29 (50% EtOAc/heptane). [α]D
2. Experimental procedures
2.1. Organic synthesis
Dioxane, THFandtolueneweredistilledfromsodium
benzophenone, while CH2Cl2 was distilled from CaH2.
Thin layer chromatography was performed on aluminum
plates coated with silica (Merck, Kieselgel 60 F254).
Visualization of the compounds were done by heating
after dipping the plates in a solution of Ce(SO4)2 (2.5 g)
and (NH4)6Mo7O24 (6.25 g) in 10% aqueous H2SO4
(250 mL). Flash chromatography was performed with
silica gel 60. NMR spectra were recorded on a Varian
Mercury 300 spectrometer with (CH3)4Si (δH = 0.0 ppm)
1
as an internal reference for H NMR and with CDCl3
(δC = 77.0 ppm) as an internal reference for 13C NMR.
Optical rotations were measured on a Perkin-Elmer 241
polarimeter and microanalyses were obtained at the
¨
Mikroanalytisches Laboratorium, Universitat Wien.
2.1.1. (2S,5S,6S)-5,6-Dimethoxy-5,6-dimethyl-
[1,4]dioxane-2-carbaldehyde (3)
Trimethyl orthoformate (62.0 mL, 567 mmol) and
2,3-butanedione (14.0 mL, 160 mmol) were added to
a solution of l-ascorbic acid (25.0 g, 142 mmol) in
dry CH3OH (250 mL) under nitrogen after which the
solution turned yellow. BF3·OEt2 (1.80 mL, 14.2 mmol)
was added, and the solution was stirred for 42 h at
room temperature. The reaction mixture was neutral-
ized to pH 7 with Et3N and concentrated in vacuo to
give a reddish foam. This was dissolved in CH2Cl2
(250 mL) and washed with brine (400 mL). The aque-
ous phase was extracted with CH2Cl2 (3 × 100 mL) and
EtOAc (2 × 100 mL), and the combined organic extracts
were dried over MgSO4, filtered and concentrated in
vacuo to afford a reddish foam (55.5 g). Rf 0.40 (10%
CH3OH/CH2Cl2). Crude 4 was dissolved in a solution of
K2CO3 (39.4 g, 285 mmol) in H2O (400 mL) and cooled
to0 ◦C. 35%(v/v)H2O2 (28.0 mL, 288 mmol)wasadded
dropwise over 30 min to the reddish solution, and the
reaction was stirred for 20 min at 0 ◦C. The solution
was allowed to warm to room temperature and stirred
overnight after which NaHCO3 (53.6 g, 638 mmol) was
added and the reaction was stirred for 1 h. (CH3)2SO4
(60.0 mL, 630 mmol) was added dropwise over a period
of 1 h, and the solution was heated to reflux. The mixture
was refluxed for 4 h after which the yellow solution was
cooled to room temperature and stirred overnight. The
1
+102.8 (2.06 g/100 mL, CHCl3). H NMR (300 MHz,
CDCl3): δ 9.59 (s, 1H), 4.30 (dd, J 5.6, 9.5 Hz, 1H),
3.70–3.61 (m, 2H), 3.26 (s, 3H), 3.20 (s, 3H), 1.32 (s,
3H), 1.24 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 200.3,
99.7, 98.3, 72.3, 58.3, 48.4, 48.2, 17.6, 17.5.
2.1.2. (2S,5S,6S)-2-(1ꢀ-(Hydroxy)but-3ꢀ-enyl)-5,6-
dimethoxy-5,6-dimethyl-[1,4]dioxane (7)
Zinc (12.0 g) was activated by stirring with 1 M HCl
(60 mL) for 15 min. The solution was filtered, and the fil-
ter cake was quickly washed with H2O (2 × 25 mL) and
Et2O (2 × 20 mL), after which the zinc was transferred to
a flame dried flask. The zinc was dried with a heatgun for
5 min under reduced pressure (2 mmHg) and allowed to
cool under reduced pressure (2 mmHg) for at least 1 h.
Aldehyde 3 (10.0 g, 49.1 mmol) was dissolved in dis-
tilled THF (100 mL) under nitrogen. The solution was
cooled to 0 ◦C, and activated zinc (6.42 g, 98.2 mmol)
and allyl bromide (8.50 mL, 98.2 mmol) were added. Sat.
aq. NH4Cl (50 mL) was added dropwise over 15 min and

L. Linderoth et al. / Chemistry and Physics of Lipids 146 (2007) 54–66
57
the reaction was stirred for 10 min at 0 ◦C. The mix-
ture was filtered and the filter cake rinsed with CH2Cl2
(100 mL). H2O (100 mL) was added to the filtrate and the
phases were separated. 0.5 M HCl (14 mL) was added to
the aqueous phase, which turned the aqueous phase into a
clear solution, and the aqueous phase was extracted with
CH2Cl2 (2 × 100 mL). The combined organic extracts
were washed with sat. NaHCO3 (200 mL) and H2O
(200 mL), dried over MgSO4, filtered and concentrated
in vacuo to afford a colorless syrup. Purification by
flash chromatography (25% EtOAc/CH2Cl2) gave 9.03 g
(74%) of 7 as a 5:1 mixture of diastereomers (colorless
oil). Rf 0.48 (50% EtOAc/heptane). Microanalysis cal-
culated (C12H22O5): C 58.52; H 9.00. Analysis result:
The combined organic extracts were washed with H2O
(2 × 70 mL), dried over MgSO4 and concentrated in
vacuo to give a yellow oil. The crude product was puri-
fied by dry column flash chromatography (1:0 → 3:1,
heptane/EtOAc) and then by flash chromatography (5%
Et2O/heptane) to afford 8.10 g (60%) of 9 as a colorless
oil and 2.38 g (18%) of its diastereomer as a colorless oil.
Rf 0.12(5%Et2O/heptane). [α]D +82.30(2.00 g/100 mL,
CHCl3). Microanalysis calculated (C28H54O5): C 71.44;
1
H 11.56. Analysis result: C 71.09; H 11.57. H NMR
(300 MHz, CDCl3): δ 5.88–5.74 (m, 1H), 5.08–4.97 (m,
2H), 3.75 (ddd, J 3.9, 7.9, 10.7 Hz, 1H), 3.66–3.54 (m,
2H), 3.45 (dt, J 6.3, 8.7 Hz, 1H), 3.32–3.23 (m, 2H), 3.21
(s, 3H), 3.19 (s, 3H), 2.49–2.40 (m, 1H), 2.23 (dt, J 6.9,
14.4 Hz, 1H), 1.44–1.40 (m, 2H), 1.22 (s, 3H), 1.21 (s,
3H), 1.18 (br. s, 26H), 0.81 (t, J 7.0 Hz, 3H). 13C NMR
(CDCl3, 75 MHz): δ 134.5, 117.1, 99.0, 97.9, 79.2, 70.5,
68.2, 61.7, 48.1, 48.0, 35.0, 31.9, 30.0, 29.7, 29.4, 29.3,
26.1, 22.7, 17.8, 17.6, 14.1.
1
C 57.87; H 8.96. Both isomers: H NMR (300 MHz,
CDCl3): δ 5.85–5.68 (m, 1H), 5.13–5.03 (m, 2H),
3.76–3.47 (m, 4H), 3.20 (s, 6H), 2.42–2.33 (m, 1H),
2.23–2.06 (m, 1H), 1.94 (br. s, OH), 1.23 (s, 3H), 1.22
(s, 3H). Major isomer: 13C NMR (CDCl3, 75 MHz): δ
133.9, 118.6, 99.2, 97.7, 70.7, 69.2, 60.0, 48.0, 37.2,
17.8, 17.5. Minor isomer: 13C NMR (CDCl3, 75 MHz):
δ 133.9, 117.8, 99.2, 97.9, 70.1, 69.5, 60.3, 48.0, 37.4,
17.8, 17.5.
2.1.5. (1Rꢀ,2S,5S,6S)-2-(1ꢀ-(Hexadecyloxy)2ꢀ,2ꢀ-
dimethyl-but-3ꢀ-enyl)-5,6-dimethoxy-5,6-dimethyl-
[1,4]dioxane (10)
Cetyl triflate was synthesized in the following
way: trifluoromethanesulfonic anhydride (0.90 mL,
5.36 mmol) was added to a solution of dry pyridine
(0.44 mL, 5.36 mmol) in dry CH2Cl2 (10 mL) at 0 ◦C
under nitrogen. The ice bath was removed, and a solu-
tion of cetyl alcohol (1.0 g, 4.12 mmol) in dry CH2Cl2
(4 mL) was added dropwise over a period of 10 min.
The reaction was stirred for 2 h at room temperature
and was then quenched with H2O (10 mL). CH2Cl2
(50 mL) was added, and the solution was washed with
H2O (2 × 50 mL). The combined aqueous phases were
extracted with CH2Cl2 (50 mL), and the combined
organic extracts were washed with brine (50 mL). The
organic phase was dried over MgSO4, filtered and con-
centrated in vacuo. The residue was dissolved in hexane
(10 mL) and filtered through a plug of silica gel with
hexane and EtOAc. The solvent was removed by concen-
tration in vacuo giving 1.41 g (92%) of cetyl triflate. Rf
0.50 (7% heptane/Et2O). 1H NMR (300 MHz, CDCl3): δ
4.46 (t, J 6.5 Hz, 2H), 1.80–1.71 (m, 2H), 1.39–1.18 (m,
26H), 0.80(t, J6.7 Hz, 3H). 13CNMR(CDCl3, 75 MHz):
δ 120.7, 77.8, 31.9, 29.7, 29.6, 29.5, 29.3, 29.2, 28.8,
25.0, 22.7, 14.1. 1,8-Bis(dimethylamino)naphthalene
(proton sponge, 281 mg, 1.31 mmol) was added to a
solution of 8 (200 mg, 0.729 mmol) and cetyl triflate
(492 mg, 1.31 mmol) in dry CH2Cl2 (7 mL) under nitro-
gen. The yellow solution was heated to reflux and
stirred for 6 days. CH2Cl2 (50 mL) was added, and the
solution was washed with brine (200 mL). The aque-
2.1.3. (2S,5S,6S)-2-(1ꢀ-(Hydroxy)2ꢀ,2ꢀ-dimethyl-
but-3ꢀ-enyl)-5,6-dimethoxy-5,6-dimethyl-
[1,4]dioxane (8)
Performed as described for 7 giving 170 mg (70%)
of 8 (4:1) as a clear oil. Microanalysis calculated
(C14H26O5): C 61.29; H 9.55. Analysis result: C 60.99;
1
H 9.59. Both isomers: H NMR (300 MHz, CDCl3): δ
5.98–5.82 (m, 1H), 5.07–5.00 (m, 2H), 3.98 (dt, J 3.5,
11.0 Hz, 1H), 3.81 (t, J 11.3 Hz, 1H), 3.58 (dd, J 3.3,
11.5 Hz, 1H), 3.48 (d, J 3.9 Hz, 1H), 3.28 (s, 3H), 3.26
(s, 3H), 2.08 (br. s, OH), 1.28 (s, 3H), 1.27 (s, 3H), 1.10
(s, 3H), 1.09 (s, 3H). Major isomer: 13C NMR (CDCl3,
75 MHz): δ 144.4, 112.9, 99.4, 97.6, 79.0, 67.9, 59.9,
48.3, 48.0 40.7, 24.8, 22.8, 17.8, 17.5.
2.1.4. (1Rꢀ,2S,5S,6S)-2-(1ꢀ-(Hexadecyloxy)but-3ꢀ-
enyl)-5,6-dimethoxy-5,6-dimethyl-[1,4]dioxane (9)
Alcohol 7 (7.10 g, 28.8 mmol) was dissolved in DMF
(30 mL) under nitrogen. The solution was cooled to 0 ◦C,
and cetyl iodide (36.2 mL, 115 mmol) and 50% (w/w)
NaHdispersioninoil(4.15 g, 86 mmol)wereadded, after
which gas evolved from the solution. After 10 min, the
solution was heated to 50 ◦C and stirred for 20 h. The
mixture was allowed to cool to room temperature, and
CH3OH (10 mL) was added. The solution was stirred
for 10 min and Et2O (50 mL) was added, after which
the solution was washed with brine (50 mL), and the
aqueous phase was extracted with Et2O (4 × 50 mL).

58
L. Linderoth et al. / Chemistry and Physics of Lipids 146 (2007) 54–66
ous phase was extracted with CH2Cl2 (3 × 50 mL), the
combined organic extracts were dried over MgSO4, fil-
tered and concentrated in vacuo. Purification by flash
chromatography gave 142 mg (39%) of 10 and 63 mg
(18%) of its diastereomer as colorless oils. Rf 0.10 (5%
Et2O/heptane). Microanalysis calculated (C30H58O5): C
2.1.8. Octadecanoic acid ((2S,3R)-1-(tert-
butyldiphenylsilyloxy)-3-(hexadecyloxy)hex-5-en-2-
yl)ester (13)
4-(Dimethylamino)pyridine (226 mg, 1.85 mmol)
and
tert-butyl-diphenylsilylchloride
(0.518 mL,
1.99 mmol) were added to a solution of 11 (0.455 g,
1.28 mmol) in dry CH2Cl2 (30 mL) under nitrogen,
and the solution was stirred at room temperature.
After 26 h, octadecanoic acid (726 mg, 2.55 mmol)
and 4-(dimethylamino)pyridine (10.0 mg, 0.08 mmol)
were added and the solution was cooled to 0 ◦C. N,Nꢀ-
Dicyclohexylcarbodiimide (1 M) in CH2Cl2 (2.55 mL,
2.55 mmol) was added dropwise over a period of 4 min.
The solution was allowed to warm to room temperature
and stirred for 24 h. The mixture was filtered through
a GF/A filter, and the filtrate was concentrated in
vacuo. The residue was dissolved in Et2O (50 mL) and
washed with H2O (2 × 50 mL) and brine (50 mL). The
combined organic extracts were dried over Na2SO4,
filtered and concentrated in vacuo. Purification by flash
chromatography (100:1 → 100:20 heptane/Et2O) gave
918 mg (84%) of 13 as a colorless oil. Rf 0.15 (2.5%
heptane/Et2O). [α]D +7.51 (1.97 g/100 mL, CHCl3).
Microanalysis calculated (C56H96O4Si): C 78.08; H
11.23. Analysis result: C 77.93; H 11.26. 1H NMR
(300 MHz, CDCl3): δ 7.62–7.57 (m, 4H), 7.36–7.27
(m, 6H), 5.81–5.67 (m, 1H), 4.99–4.94 (m, 3H), 3.80
(dd, J 5.9, 11.1 Hz, 1H), 3.70 (dd, J 3.8, 11.1 Hz, 1H),
3.62–3.56 (m, 1H), 3.42–3.34 (m, 2H), 2.23–2.13 (m,
4H), 1.63–1.50 (m, 4H), 1.18 (br. s, 54H), 0.97 (s, 9H),
0.81 (t, J 6.7 Hz, 6H). 13C NMR (CDCl3, 75 MHz): δ
173.0, 135.6, 135.5, 134.6, 133.4, 133.3, 130.0, 129.7,
127.7, 117.0, 77.5, 74.6, 70.6, 62.2, 36.2, 35.2, 31.9,
30.0, 29.7, 29.6, 29.3, 26.7, 26.1, 25.1, 22.7, 19.1,
14.1.
1
72.24; H 11.72. Analysis result: C 72.47; H 11.96. H
NMR (300 MHz, CDCl3): δ 5.90 (dd, J 10.8, 17.5 Hz,
1H), 5.02–4.95 (m, 2H), 3.97 (dt, J 2.9, 10.9 Hz, 1H),
3.84–3.72 (m, 2H), 3.52 (dd, J 3.2, 11.5 Hz, 1H),
3.47–3.42 (m, 1H), 3.25 (s, 3H), 3.23 (s, 3H), 3.06 (d, J
2.6 Hz, 1H), 1.56–1.52 (m, 2H), 1.26 (br. s, 32H), 1.06
(s, 3H), 1.02 (s, 3H), 0.88 (t, J 6.7 Hz, 3H). 13C NMR
(CDCl3, 75 MHz): δ 145.1, 111.9, 99.2, 97.6, 88.0, 73.4,
68.4, 60.6, 48.2, 47.8, 41.2, 31.9, 30.2, 29.7, 29.6, 29.4,
26.3, 24.5, 23.3, 22.7, 17.9, 17.6, 14.1.
2.1.6. (2S,3R)-3-Hexadecyloxy-hex-5-ene-1,2-diol
(11)
94% Trifluoroacetic acid in H2O (5 mL) was added
to a solution of 9 (332 mg, 0.705 mmol) in CH2Cl2
(5 mL), and the mixture was stirred at room tempera-
ture for 25 min. The solution was diluted with CH2Cl2
(20 mL), washed with H2O (20 mL) and the aqueous
phase was extracted with CH2Cl2 (10 mL). The com-
bined organic extracts were dried over MgSO4, filtered
and concentrated in vacuo. Purification by flash chro-
matography (30% EtOAc/heptane) gave 190 mg (76%)
of 11 as white crystals. Rf 0.2 (30% EtOAc/heptane).
[α]D − 15.28 (2.03 g/100 mL, CHCl3). mp 38–39 ◦C
(Et2O). Microanalysis calculated (C22H44O3): C 74.10;
1
H 12.44. Analysis result: C 73.96; H 12.55. H NMR
(300 MHz, CDCl3): δ 5.84–5.70 (m, 1H), 5.09–5.00 (m,
2H), 3.65–3.54 (m, 3H), 3.51 (dt, J 6.7, 9.1 Hz, 1H),
3.40–3.22 (m, 2H), 2.38–2.14 (m, 2H), 1.50–1.45 (m,
2H), 1.19 (br. s, 26H), 0.81 (t, J 6.5 Hz, 3H). 13C NMR
(CDCl3, 75 MHz): δ 134.2, 117.4, 81.1, 72.1, 70.7, 63.4,
35.0, 31.9, 30.0, 29.7, 29.6, 29.4, 29.3, 26.1, 22.6, 14.1.
2.1.9. Octadecanoic acid ((2S,3R)-1-(tert-
butyldiphenylsilyloxy)-3-(hexadecyloxy)-4,4-
dimethyl-hex-5-en-2-yl)ester (14)
The synthesis of 14 was carried out as described for
the synthesis of 13 to afford 1.18 g (84%) of 14. 1H NMR
(300 MHz, CDCl3): δ 7.69–7.64 (m, 4H), 7.43–7.35 (m,
6H), 5.80 (dd, J 10.5, 17.9 Hz, 1H), 5.15–5.10 (m, 1H),
4.87–4.81 (m, 2H), 3.84–3.83 (m, 2H), 3.59 (dt, J 6.4,
8.8 Hz, 1H), 3.35 (dt, J 6.6 Hz, 8.9 Hz, 1H), 3.12 (d,
J 3.4 Hz, 1H), 2.26–2.21 (m, 2H), 1.70–1.57 (m, 2H),
1.43–1.39 (m, 2H), 1.25 (br. s, 54H), 1.10 (s, 6H), 1.02 (s,
9H), 0.88 (t, J 6.7 Hz, 6H). 13C NMR (CDCl3, 75 MHz):
δ 172.9, 144.8, 135.7, 135.6, 129.6, 129.5, 127.7, 127.6,
111.3, 87.1, 75.8, 73.6, 63.1, 41.2, 36.2, 34.7, 31.9, 30.1,
29.7, 29.5, 29.4, 29.3, 29.2, 26.9, 26.8, 26.1, 25.1, 24.8,
24.5, 23.5, 22.7, 19.2, 14.1.
2.1.7. (2S,3R)-3-Hexadecyloxy-4,4-dimethyl-hex-5-
ene-1,2-diol (12)
Performed as described for 11 giving 666 mg (76%)
of 12 as a clear oil. [α]D − 173.8 (1.0 g/100 mL, CHCl3).
Microanalysis calculated (C24H48O3): C 74.94; H 12.58.
Analysis result: C 75.15; H 12.75. 1H NMR (300 MHz,
CDCl3): δ 6.00 (dd, J 10.5, 17.2 Hz, 1H), 5.07–5.01 (m,
2H), 3.72–3.64 (m, 4H), 3.57–3.50 (m, 1H), 3.16–3.15
(m, 1H), 1.58–1.53 (m, 2H), 1.26 (br. s, 26H), 1.08 (s,
6H), 0.88 (t, J 6.6 Hz, 3H). 13C NMR (CDCl3, 75 MHz):
δ 145.5, 112.0, 88.1, 74.6, 72.4, 64.0, 41.5, 31.9, 30.4
29.7, 29.5, 29.3, 26.1, 25.0, 22.7 22.4, 14.1.

L. Linderoth et al. / Chemistry and Physics of Lipids 146 (2007) 54–66
59
2.1.10. (2ꢀS,3ꢀR,4S)-2,2-Dimethyl-[1,3]dioxolan-
4-ylmethyl 2ꢀ-octadecanoyloxy-3ꢀ-hexadecyloxy-
hex-5ꢀ-enyl methyl phosphate (17)
29.6, 29.4, 29.3, 29.1, 26.7, 26.1, 25.2, 24.8, 22.7,
14.1.
2.1.11. (2ꢀS,3ꢀR,4S)-2,2-Dimethyl-[1,3]dioxolan-
4-ylmethyl 2ꢀ-octadecanoyloxy-3ꢀ-hexadecyloxy-
4ꢀ,4ꢀ-dimethyl-hex-5ꢀ-enyl methyl
Imidazole (81.0 mg, 1.19 mmol) was added to a
solution of 13 (292 mg, 0.339 mmol) in dry THF
(25 mL) under nitrogen, and the solution was cooled
to −15 ◦C. 0.1 M tetrabutylammonium fluoride in
THF (10.2 mL, 1.02 mmol) was added, and the mix-
ture was stirred for 2 h. The solution was diluted
with CH2Cl2 (30 mL), washed with sat. NaHCO3
(3 × 100 mL), dried over Na2SO4, filtered and concen-
trated in vacuo. The crude product 15 was dissolved
in dry CH2Cl2 (7 mL) followed by addition of
2,2,6,6-tetramethylpiperidine (0.12 mL, 0.711 mmol)
phosphate (18)
Performedasdescribedfor17giving332 mg(58%)of
18 as a clear oil. Microanalysis calculated (C49H95O9P):
C 68.49; H 11.14. Analysis result: C 68.54; H 11.12. 1H
NMR (300 MHz, CDCl3): δ 5.92 (dd, J 10.5, 17.9 Hz,
1H), 5.21–5.17 (m, 1H), 5.04–4.99 (m, 2H), 4.38–4.35
(m, 2H), 4.20–3.94 (m, 4H), 3.86–3.80 (m, 1H), 3.77 (d,
J 3.5 Hz, 3H), 3.69–3.64 (m, 1H), 3.46–3.38 (m, 1H),
3.13 (d, J 2.8 Hz, 1H), 2.34–2.28 (m, 2H), 1.63–1.52 (m,
4H), 1.42 (s, 3H), 1.36 (s, 3H), 1.25 (br. s, 54H), 1.09
(s, 3H), 1.06 (s, 3H), 0.88 (t, J 6.7 Hz, 6H). 13C NMR
(CDCl3, 75 MHz): δ 172.7, 144.2, 112.1, 109.8, 87.1,
74.0, 73.9, 73.8, 67.5, 67.3, 66.1, 54.4, 41.3, 34.4, 31.9,
30.1, 29.7, 29.5, 29.3, 29.2, 29.1, 26.7, 26.2, 25.2, 24.7,
24.3, 23.6, 22.6, 14.1.
and
methyl-N,N-diisopropylchlorophosphoramidite
(0.13 mL, 0.678 mmol) under nitrogen at room temper-
ature, after which the reaction was stirred for 45 min.
The solution was diluted with CH2Cl2 (15 mL), washed
with sat. NaHCO3 (3 × 20 mL), dried over Na2SO4,
filtered and concentrated in vacuo. The residue was
co-concentrated with toluene, kept under reduced pres-
sure overnight and used directly in the next reaction. Rf
0.68 (20% EtOAc/heptane). Activated molecular sieves
2.1.12. (2S,2ꢀS,3ꢀR)-2,3-Dihydroxypropyl
˚
2ꢀ-octadecanoyloxy-3ꢀ-hexadecyloxy-hex-5ꢀ-enyl
phosphate (1)
(1 g, 4 A) and 97% 2,3-O-isopropylidene-sn-glycerol
(0.09 mL, 0.678 mmol) were added to a solution of
the crude compound in dry CH2Cl2 (7 mL) under
nitrogen, and the solution was stirred at room tem-
perature for 40 min. The reaction was cooled to 0 ◦C,
and 5-phenyl-1H-tetrazole (104 mg, 0.712 mmol) was
added after which the mixture was allowed to warm to
room temperature and stirred for 90 min. The solution
was then cooled to 0 ◦C. 5.5 M t-BuOOH in decane
(0.129 mL, 0.712 mmol) was added and the solution was
stirred for 1 h. 1 M Na2SO3 (10 mL) and sat. NaHCO3
(10 mL) were added, and the mixture was allowed to
warm to room temperature and stirred for 20 min. The
solution was extracted with CH2Cl2 (4 × 20 mL), and
the combined organic extracts were washed with brine
(15 mL), dried over Na2SO4, filtered and concentrated
in vacuo to give a white slurry. Purification by flash
chromatography (30% EtOAc/heptane) gave 145 mg
(51%) of 17 as a mixture of diastereomers (white
slurry). Rf 0.2 (30% EtOAc/heptane). Microanalysis
calculated (C47H91O9P): C 67.91; H 11.04. Analysis
result: C 68.28; H 11.09. 1H NMR (300 MHz, CDCl3):
δ 5.82–5.60 (m, 1H), 5.05–4.96 (m, 3H), 4.26–4.11
(m, 3H), 4.02–3.88 (m, 3H), 3.78–3.67 (m, 4H),
3.48–3.37 (m, 3H), 2.68–2.13 (m, 4H), 1.57–1.42
(m, 4H), 1.35 (s, 3H), 1.29 (s, 3H), 1.18 (br. s, 54H),
0.80 (t, J 6.9 Hz, 6H). 13C NMR (CDCl3, 75 MHz):
δ 172.8, 133.6, 117.7, 109.8, 77.0, 74.0, 72.7, 70.7,
67.5, 66.0, 65.9, 54.4, 35.2, 34.3, 31.9, 29.9, 29.7,
Compound 17 (210 mg, 0.244 mmol) was dissolved
in a solution of CH2Cl2 (6 mL), CH3CN (6 mL), iso-
propanol (8 mL) and 40% (CH3)3N (7 mL), which was
stirred at room temperature. After stirring overnight,
EtOH (20 mL) and toluene (20 mL) were added, and the
mixture was concentrated in vacuo. The residue was dis-
solved in a 7:1 CH2Cl2/CH3OH (17.5 mL) solution and
extracted with 1 M HCl (30 mL). 1 M HCl (1 mL) and
additional CH3OH (4 mL) were added to the organic
extract, which gave a 65:25:4 CH2Cl2/CH3OH/1 M HCl
solution. The solution was stirred at room temperature
overnight, and NaHCO3 (286 mg, 3.4 mmol) was added
followed by stirring for an additional 24 h. The neutral
(pH 7) solution was dried over Na2SO4, filtered and con-
centrated in vacuo. Purification by flash chromatography
(15% CH3OH/CH2Cl2) gave 122 mg (60%) of 1 as a
colorless oil. Rf 0.12 (15% CH3OH/CH2Cl2). 1H NMR
(300 MHz, 30% CD3OD/CDCl3): δ 5.92–5.79 (m, 1H),
5.18–5.04 (m, 3H), 4.07–3.87 (m, 4H), 3.80–3.76 (m,
1H), 3.62–3.53 (m, 5H), 2.38–2.29 (m, 4H), 1.65–1.48
(m, 4H), 1.27 (br. s, 54H), 0.89 (t, J 6.7 Hz, 6H). 13C
NMR (30% CD3OD/CDCl3, 75 MHz): δ 173.5, 133.9,
116.8, 77.7, 73.3, 70.6, 70.4, 66.1, 63.5, 62.0, 34.7,
34.0, 31.5, 29.6, 29.3, 29.2, 29.1, 29.0, 28.7, 25.7, 24.5,
22.3, 13.5. 31P NMR (162 MHz, 30% CD3OD/CDCl3):
δ 3.55. ESI HRMS calcd. for C43H85O9P [M−H]− m/z
775.5847, found: m/z 775.5849.

60
L. Linderoth et al. / Chemistry and Physics of Lipids 146 (2007) 54–66
2.1.13. (2S,2ꢀS,3ꢀR)-2,3-Dihydroxypropyl
(CD3)2SO): δ 4.97 (dd, J 2.5, 5.8 Hz, 1H), 4.95 (d, J
4.9 Hz, 1H), 4.59 (dd, J 5.5 Hz, 1H), 4.08 (ddd, J 5.4 Hz,
1H), 3.71–3.65 (m, 1H), 3.33–3.40 (m, 2H), 3.19 (s,
3H), 1.97 (ddd, J 2.6, 6.4, 13.1 Hz, 1H), 1.87 (dt, J 5.7,
13.2 Hz, 1H). 13C NMR (CD3OD, 75 MHz): δ 105.4,
2ꢀ-octadecanoyloxy-3ꢀ-hexadecyloxy-4ꢀ,4ꢀ-dimethyl-
hex-5ꢀ-enyl phosphate (2)
Performed as described for 1 giving 61 mg (79%)
of 2 as a colorless oil. 1H NMR (300 MHz, 30%
CD3OD/CDCl3): δ 5.98–5.88 (m, 1H), 5.22–5.20 (m,
1H), 5.04–4.99 (m, 2H), 4.26–3.96 (m, 4H), 3.91–3.85
(m, 1H), 3.81–3.57 (m, 5H), 2.31–2.29 (m, 2H),
1.61–1.53 (m, 4H), 1.26 (br. s, 54H), 1.09 (s, 6H), 0.83
(br. s, 6H). 13C NMR (30% CD3OD/CDCl3, 75 MHz):
δ 173.4, 144.3, 111.7, 87.3, 74.8, 73.6, 70.7, 66.1,
65.3, 62.0, 44.5, 41.1, 34.3, 31.7, 29.9, 29.4, 29.3,
29.2, 29.0, 25.9, 24.6, 24.2, 23.1, 22.4, 13.8. 31P NMR
(162 MHz, 30% CD3OD/CDCl3): δ 3.83. ESI HRMS
calcd. for C45H89O9P [M−H]− m/z 803.6160, found:
m/z 803.6162.
1
87.0, 71.6, 63.7, 54.2, 41.1. 21 (␤-anomer): H NMR
(500 MHz, (CD3)2SO): δ 4.89 (dd, J 2.4, 5.8 Hz, 1H),
4.81 (d, J 5.5 Hz, 1H), 4.59 (dd, J 5.7 Hz, 1H), 3.91 (ddd,
J 4.9, 5.3, 8.1 Hz, 1H), 3.71–3.65 (m, 1H), 3.49 (ddd, J
3.3, 5.2, 11.7 Hz, 1H), 3.33–3.40 (m, 1H), 3.22 (s, 3H),
2.25 (ddd, J 5.8, 8.1, 13.9 Hz, 1H), 1.63 (ddd, J 2.3, 4.8,
13.8 Hz, 1H). 13C NMR (CD3OD, 75 MHz): δ 105.1,
85.6, 71.1, 62.0, 54.0, 41.1. 22 (␣-anomer): 13C NMR
(CDCl3, 75 MHz): δ 98.1, 85.4, 70.9, 62.7, 41.0. 22 (␤-
anomer): 13C NMR (CDCl3, 75 MHz): δ 98.1, 85.8, 71.2,
61.5, 41.2. 23 (␣-anomer): 13C NMR (CDCl3, 75 MHz):
δ 93.9, 67.3, 66.5, 66.1, 33.7. 23 (␤-anomer): 13C NMR
(CDCl3, 75 MHz): δ 91.7, 67.5, 64.6, 62.8, 35.1.
2.1.14. Stereochemistry of 7R
Alcohol 7 (410 mg, 1.7 mmol) was dissolved in
dry CH2Cl2 (15 mL) and 4-(dimethylamino)pyridine
(19 mg, 0.16 mmol), followed by addition of acetic
anhydride (0.32 mL, 3.4 mmol) and Et3N (0.43 mL,
3.4 mmol) under nitrogen. The solution was stirred for
45 min and then concentrated in vacuo. The residue was
purified by flash chromatography (9% EtOAc/heptane)
to give 350 mg (72%) of 19 and 60 mg (12%) of its
diastereomer as colorless oils. To the solution of 19
(0.31 g, 1.1 mmol) in CH2Cl2 (5 mL) was added 95%
trifluoroacetic acid in H2O (5 mL) and the yellow solu-
tion was stirred at room temperature for 45 min. The
mixture was concentrated in vacuo, and Na (15.5 mg,
0.67 mmol) dissolved in dry CH3OH (5 mL) was added.
The flask was fitted with a CaCl2-tube and after stirring
for 30 min additional Na (15.3 mg, 0.67 mmol) in dry
CH3OH (5 mL) was added. The reaction was stirred for
45 min and subsequently quenched with ion exchange
resin (1.4 mL, Amberlite IR-120 H+, 1.9 meq/mL). The
mixture was stirred for 35 min, filtered and concen-
trated in vacuo. Purification by flash chromatography
gave 112 mg (79%) of 20 as white crystals. [α]D
+10.25 (2.00 g/100 mL, CHCl3). Microanalysis calcu-
lated (C14H26O6): C 54.53; H 9.15. Analysis result: C
54.62; H 9.19. Triol 20 (0.105 g, 0.80 mmol) was dis-
solved in CH3OH (50 mL). The solution was cooled
to −78 ◦C and treated with ozone for 30 min (CH3)2S
(0.4 mL, 5.4 mmol) was added, and the solution was
stirred under nitrogen for 18 h. The mixture was con-
centrated in vacuo, filtered through reverse phase silica
with CH3OH and concentrated in vacuo. Purification
by flash chromatography (12% CH3OH/CH2Cl2) gave
55 mg (52%) of 22 and 23 and 20 mg (17%) of 21
2.1.15. Stereochemistry of 8R
NaH 50% (w/w) dispersion in oil (93 mg, 1.94 mmol)
was added to a solution of 8 (177 mg, 0.65 mmol) in
dry DMF (7 mL), and the solution was stirred at room
temperature under nitrogen for 30 min. The mixture was
cooled to 0 ◦C, and BnBr (0.25 mL, 2.06 mmol) and
Bu4NI (24 mg, 0.065 mmol) were added, and the mix-
ture stirred at room temperature overnight. The reaction
was quenched with H2O (1 mL), toluene (20 mL) was
added, and the mixture was concentrated in vacuo. Et2O
(100 mL) and H2O (10 mL) were added, and the mix-
ture was washed with 1 M HCl (2 × 50 mL) and H2O
(2 × 50 mL), dried over Na2SO4, filtered and concen-
trated in vacuo. Purification by flash chromatography
(6% Et2O/heptane) gave 162 mg (68%) of 24 (and 50 mg
of the diastereoisomer, 21%) as a colorless oil. Rf
0.34 (10% EtOAc/heptane). Benzyl ether 24 (162 mg,
0.44 mmol) was dissolved in CH2Cl2 (5 mL), and 90%
trifluoroacetic acid in H2O (5 mL) was added. The yel-
low solution was stirred at room temperature for 20 min,
where after CH2Cl2 (20 mL) was added and the solu-
tion was washed with H2O (20 mL). The aqueous phase
was extracted with CH2Cl2 (2 × 30 mL), and the com-
bined organic extracts were dried over MgSO4, filtered
and concentrated in vacuo. Purification by flash chro-
matography (20% EtOAc/heptane) gave 60 mg (54%)
of 25 as a colorless oil. Rf 0.46 (25% EtOAc/heptane).
Diol 25 (60 mg, 0.24 mmol) was dissolved in t-BuOH
(3 mL) and hydrogenated overnight at 1 atm with 10%
Pd/C (6 mg). The mixture was filtered through a GF/A
filter, and the filter cake was rinsed with CH2Cl2 (10 mL)
andEtOAc(10 mL), whereafterthesolventwasremoved
by concentration in vacuo. Crude 26 (38 mg, 0.23 mmol)
1
as colorless oils. 21 (␣-anomer): H NMR (500 MHz,

L. Linderoth et al. / Chemistry and Physics of Lipids 146 (2007) 54–66
61
was dissolved in dry CH2Cl2 (5 mL) under nitrogen,
and p-toluenesulfonic acid (3.5 mg, 0.02 mmol) and ben-
zaldehyde dimethylacetal (0.04 mL, 0.23 mmol) were
added to the solution. The mixture was stirred at room
temperature overnight, where after the solution was
washed with sat. NaHCO3 (10 mL), and the aqueous
phase was extracted with CH2Cl2 (2 × 10 mL). The com-
bined organic extracts were washed with brine (10 mL),
dried over MgSO4, filtered and concentrated in vacuo.
The crude compound (27) was dissolved in dry CH2Cl2
(1 mL) under nitrogen, and 4-(dimethylamino)pyridine
(337 mg, 2.76 mmol) and benzoyl chloride (0.28 mL,
2.3 mmol) were added to the solution. The mixture was
stirred at room temperature overnight, where after H2O
(20 mL) was added and the mixture was extracted with
CH2Cl2 (3 × 10 mL). The combined organic extracts
were dried over MgSO4, filtered and concentrated
in vacuo. Purification by flash chromatography (6%
Et2O/heptane) gave 63 mg (77%) of 28 as a white slurry.
Rf 0.2 (6% Et2O/heptane). [α]D +35.9 (1.0 g/100 mL,
CHCl3). Microanalysis calculated (C22H26O4): C 74.55;
H 7.39. Analysis result: C 74.30; H 7.14. 1H NMR
(300 MHz, CDCl3): δ 8.18–8.15 (m, 2H), 7.60–7.35 (m,
8H), 5.56 (s, 1H), 5.38 (td, J 5.3, 9.4 Hz, 1H), 4.44 (dd,
J 5.3, 10.7 Hz, 1H), 3.82 (d, J 9.5 Hz, 1H), 3.68 (dd, J
9.4, 10.7 Hz, 1H), 1.64–1.52 (m, 1H), 1.38–1.26 (m, 1H),
1.02(s, 3H), 0.99(s, 3H), 0.89(t, J7.6 Hz, 3H). 13CNMR
(CDCl3, 75 MHz): δ 165.1, 137.9, 133.4, 130.5, 129.7,
128.8, 128.5, 128.2, 126.0, 100.8, 83.6, 67.9, 64.8, 37.0,
31.6, 23.5, 8.1.
has been substracted from the thermograms to afford the
melting enthalpies of the liposomes.
2.4. Activity measurements
The conditions used to perform the sPLA2 activity
measurements were as follows: 0.15 mM phospholipid
as SUVs, 150 nM sPLA2, 0.15 M KCl, 30 ␮M CaCl2,
10 ␮M EDTA, 10 ␮M HEPES (pH 7.5). The catalytic
reaction was initiated by addition of 8.9 ␮L of a 42 ␮M
snake (Agkistrodon piscivorus piscivorus) venom sPLA2
stock solution to a 2.5 mL liposome suspension ther-
mostated prior to the addition of the enzyme. The
time-dependent action of sPLA2 was monitored from the
changes in the 90◦ static light scattering giving informa-
tion of changes in the lipid morphology as non-bilayer
forming lysophopholipids and fatty acids are gener-
ated. High-performance liquid chromatography (HPLC)
quantification of the products from the enzymatic reac-
tion was performed with a 5 ␮m diol column. Two
different mobile phases (hereafter referred to as mobile
phase A and mobile phase B) were used. Mobile phase
A was a chloroform/methanol/25% ammonium hydrox-
ide (800:195:5) solution, while mobile phase B was a
chloroform/methanol/water/25% ammonium hydroxide
(600:340:50:5) solution. The following gradients were
used at a flow rate of 1 mL/min 0–14 min: 100% A to
100% B using a linear gradient; 14–25 min: 100% B;
25–30 min: 100% B to 100% A using a linear gradient;
30–45 min: 100% A to regenerate. An evaporative light
scattering detector was used for detection. The turnover
of the hydrolysis was followed by HPLC. Samples
were collected at different time intervals by collecting
100 ␮L of the reaction mixture and rapidly mixing with
a 0.5 mL chloroform/methanol/acetic acid (2:4:1) solu-
tion to quench the reaction. The solution was washed
with 0.5 mL of water, and 50 ␮L of the organic phase
was used for HPLC. Fluorescence measurements were
performed using an SLM DMX 1100 spectrofluorome-
ter. Purified snake venom PLA2 was a generous gift from
Dr. R.L. Biltonen (University of Virginia, VA, USA).
2.2. Preparation of liposomes
Phospholipids 1 and 2 were hydrated in an aque-
ous buffer (150 mM KCl, 10 mM HEPES, 30 ␮M
CaCl2, 10 ␮M EDTA, pH 7.5). MLVs formed sponta-
neously. To ensure complete hydration, the lipids were
hydrated for 1 h at 60 ◦C. During the hydration, the
lipids were vortexed every 15 min. SUVs were pre-
pared by sonication of MLVs for 1 h, 5 ◦C above the
lipid main phase transition temperature regime. The
total concentration of lipids in the buffer solutions was
2 mM.
2.5. Molecular dynamics simulations
The crystal structures of bee-venom (Apis Mellifera)
phospholipase A2 complexed with the transition-state
analogue, l-1-O-octyl-2-heptylphosphonyl-sn-glycero-
3-phosphoethanolamine (diC8(2Ph)PE), resolved to
2.3. Differential scanning calorimetry (DSC)
Differential scanning calorimetry of 2 mM multil-
amellar liposomes was performed by using a Microcal
MC-2 (Northhampton, MA, USA) ultrasensitive power
compensating scanning calorimeter equipped with a
nanovoltmeter. The scans were performed in the upscan
mode at a scan rate of 10 ◦C/h. An appropriate baseline
˚
2.0 A (White et al., 1990) and human phospholipase
A2 IIA complexed with 6-phenyl-4(R)-(7-phenyl-
˚
heptanoylamino)-hexanoic acid resolved to 2.1 A
(Hansford et al., 2003) were obtained from the Protein

62
L. Linderoth et al. / Chemistry and Physics of Lipids 146 (2007) 54–66
Data Bank ((Bernstein et al., 1977); entry codes: 1 poc
and 1 kqu, respectively).
The simulations were carried out for 10 ns in the NPT
ensemble. A time step of 1 fs was used throughout all
simulations. An isotropic constant ambient pressure
of 1 atm was imposed using the Langevin piston
method (Feller et al., 1995) with a damping coefficient
of 5 ps−1, a piston period of 200 fs, and a decay of
500 fs. The Particle Mesh Ewald method was used for
computation of the electrostatic forces (Essmann et al.,
1995; Darden et al., 1993). The grid spacing applied
The initial modeling step involved placing
diC8(2Ph)PE into the binding cleft of human phospholi-
pase A2 IIA, which was done by (i) deleting the inhibitor
in 1 kqu (keeping calcium ions and water molecules
in the structure), (ii) deleting calcium ions and water
molecules in 1 poc, (iii) aligning 1 poc with 1 kqu,
and (iv) finally deleting the bee-venom phospholipase
A2 structure. The structures for the phospholipids 1
and 2 were built from diC8(2Ph)PE using SPARTAN
Version 1.0.2 (Wavefunction Inc., Irvine, California,
USA). Missing distance, angle and torsion parameters
for the allyl side chains (located at the sn-1 position
in the phospholipids 1 and 2) were obtained from
the CHARMM27 parameter set describing similar
atom types. The structures complexed with either
phospholipid 1 or 2 were solvated using the program
˚
was approximately 1.0 A, and a fourth order spline was
used for the interpolation. The long range part of the
electrostatic forces was evaluated every fourth fs. Van
˚
der Waals interactions were cut off at 12 A using a
˚
switching function starting at 10 A. Periodic boundary
conditions were applied in x, y and z-directions. The
analyses of the trajectories were performed using VMD
(Humphrey et al., 1996).
¨
SOLVATE (by Grubmuller). Eighteen water molecules
3. Results and discussion
were randomly replaced with chloride ions to neutralize
the systems. The final systems contained 4888 water
molecules, and the simulation cell dimensions were
52.7 A × 51.7 A × 67.3 A. For all simulations, NAMD
(Kale et al., 1999) was used with the Charmm27 all
hydrogens parameter set and with the TIP3 water model
(Jorgensen et al., 1983). Initially, the energy of the
systems was minimized for 5000 steps. This was fol-
lowed by 100 ps of heating of the systems to T = 300 K.
3.1. Synthesis of aldehyde 3
˚
˚
˚
The synthesis of the key aldehyde 3 is based on a
procedure originally reported by Michel and Ley (2002,
2003). However, we raised the overall yield from 35
to 64% by modifying the workup of the intermediates
(Scheme 1). l-Ascorbic acid was butanedione protected
by reaction with 2,3-butanedione, boron trifluoride ethyl
Scheme 1. Reagents and conditions: (a) 2,3-butanedione, BF3·OEt2, CH(OCH3)3, CH3OH; (b) H2O2, K2CO3, H2O then (CH3)2SO4, NaHCO3,
reflux; (c) NaBH4, isopropanol, 70 ◦C; (d) NaIO4, NaHCO3, H2O, CH2Cl2; (e) Zn, allyl bromide or 3,3-dimethylallyl bromide, sat. NH4Cl,
THF, 0 ◦C; (f) cetyl iodide, NaH, DMF, 50 ◦C or cetyl triflate, 1,8-bis(dimethylamino)naphthalene, CH2Cl2, reflux (g) trifluoroacetic acid,
CH2Cl2; (h) (i) tert-butyldiphenylsilyl chloride, 4-(dimethylamino)pyridine, CH2Cl2; (ii) octadecanoic acid, N,Nꢀ-dicyclohexylcarbodiimide, 4-
(dimethylamino)pyridine, CH2Cl2;(i)tetrabutylammoniumfluoride, imidazole, THF, −15 ◦C;(j)(i)methyl-N,N-diisopropylchlorophosphoramidite,
2,2,6,6-tetramethylpiperidine, CH2Cl2; (ii) 2,3-O-isopropylidene-sn-glycerol, 5-phenyl-1H-tetrazole, CH2Cl2 then tert-butylhydroperoxide, 0 ◦C;
(k) (i) (CH3)3N/CH2Cl2/CH3CN/isopropanol; (ii) CH2Cl2/CH3OH/HCl; (iii) NaHCO3.

L. Linderoth et al. / Chemistry and Physics of Lipids 146 (2007) 54–66
63
etherate and trimethyl orthoformate in methanol. The
generated compound 4 was used without purification in
the next reaction, where it was treated with hydrogen per-
oxide followed by dimethyl sulfate yielding methylester
5. Crude 5 was reduced to diol 6 with sodium borohy-
dride, and subsequently diol 6 was oxidatively cleaved
with sodium periodate to afford aldehyde 3. The only
purification during the synthesis involved a simple dis-
tillation of the final aldehyde 3.
to the low yield, the reaction conditions were changed
to the procedure by Heyes et al. (2002) using cetyl
triflate and 1,8-bis(dimethylamino)naphthalene (proton
sponge), which improved the yield to 57%. Again, it
was not possible to run the reaction to completion, and
25% of the starting material was recovered after 6 days.
In contrast to the reaction with NaH and cetyl iodide,
the recovered starting material consisted of 8 as a 4:1
mixture, which shows that in this case there is no differ-
enceinreactivitybetweenthetwodiastereomers. Further
reaction of 9 and 10 with TFA removed the butane-
dione protecting group in 76% yield in both cases. In
order to verify the stereochemistry of 7R, the reactions
in Scheme 2a were carried out. The stereochemistry of
7R was then confirmed by comparing the NMR data of
compound 21, 22 and 23 with literature data (Birk et al.,
1997; Dyatkina and Azhayev, 1984). The stereochem-
istry of 8R was verified by the generation of compound
28 and analyzing the 1H NMR data of the six-membered
ring (Scheme 2b).
3.2. Synthesis of diol 11 and 12
The synthesis of diol 11 and 12 required the intro-
duction of a new stereogenic center. This was done
by a zinc mediated allylation of aldehyde 3 with allyl
bromide giving the alcohol 7 as a 5:1 diastereomeric
mixture in 74% yield (Scheme 1). Allylation with 3,3-
dimethylallyl bromide using the same conditions, gave
8 as a 4:1 diastereomeric mixture in 70% yield. It was
not possible to separate the diastereomeric mixtures by
flash chromatography. However, it was found that the
diastereomers could be separated after the next reac-
tion. Reaction of the diastereomeric mixture of alcohol 7
with NaH and cetyl iodide gave 9R and its diastereoiso-
mer in a total yield of 78%. Under the same reaction
conditions the diastereomeric mixture of 8 only gave
48% yield of 10 as a 3:2 mixture with the wrong iso-
mer being the major compound. It was found that the
reaction had not gone to completion, and 44% of the
starting material could be recovered. Interestingly, NMR
of the recovered starting material only showed the pres-
ence of the desired diastereomer 8R which indicates that
the wrong diastereomer 8S is more reactive than 8R. Due
3.3. Synthesis of phospholipid 1 and 2
The synthesis of phospholipids 1 and 2 relied on
the introduction of a tert-butyldiphenylsilyl protect-
ing group on the primary alcohol of diol 11 and 12
followed by insertion of the fatty acid chain in the
secondary position with N,Nꢀ-dicyclohexylcarbodiimide
and 4-(dimethylamino)pyridine (Scheme 1). This was
carried out in 84% yield for both diol 11 and 12. The
tert-butyldiphenylsilyl group was then removed from 13
and 14 with tetrabutylammonium fluoride at −15 ◦C.
As the fatty acid chain easily migrates to the primary
Scheme 2. (A) Reagents and conditions: (a) acetic anhydride, 4-(dimethylamino)pyridine, Et3N, CH2Cl2; (b) (i) trifluoroacetic acid, H2O, CH2Cl2;
(ii) Na, CH3OH; (c) O3, (CH3)2S, CH3OH. (B) Reagents and conditions: (a) BnBr, NaH, Bu4NI, DMF; (b) 90% aq. trifluoroacetic acid in CH2Cl2;
(c) H2, t-BuOH, Pd/C; (d) PhCH(OCH3)2, p-toluenesulfonic acid, CH2Cl2; (e) PhC(O)Cl, 4-(dimethylamino)pyridine, CH2Cl2.

64
L. Linderoth et al. / Chemistry and Physics of Lipids 146 (2007) 54–66
position, the deprotection can be troublesome. Acyl
migration was prevented by adding imidazole to the
reaction mixture as described by Qin et al. (1999).
After removal of the tert-butyldiphenylsilyl protecting
group, the phosphate headgroup was inserted into the
primary position by use of the phosphoramidite method
(Beaucage and Iyer, 1993). Crude alcohols 15 and
16 were reacted with commercially available methyl-
N,N-diisopropylchlorophosphoramidite in the presence
of 2,2,6,6-tetramethylpiperidine to afford the corre-
sponding amidites, which were not purified but reacted
with 2,3-O-isopropylidine-sn-glycerol in combination
with 5-phenyl-1H-tetrazole (Andresen et al., 2004b).
The generated phosphites were oxidized to the cor-
responding phosphates 17 and 18 by treatment with
tert-butylhydroperoxide in an overall yield of 51% from
13 and 58% from 14. Treatment of 17 and 18 with
(CH3)3N followed by reaction of the crude compound
with a CH2Cl2/CH3OH/HCl solvent system afforded the
desired phospholipids 1 and 2 in 60% and 79% yield,
respectively.
In the light of these findings, MD simulations were
carried out in order to further understand the selectivity
of sPLA2 towards phospholipids 1 and 2. Simulations
in general can provide insight at a molecular level and
might reveal the structural requirements that govern sub-
strate specificity (Peters, 2004a,b). Figs. 3A and B show
snapshots taken from the MD simulations of phospho-
lipids 1 and 2, respectively. The images are close-up of
the active site of sPLA2. The secondary protein structure
is shown in ribbon mode. His48 and Asp99 involved in
the hydrolysis, the substrate and a water molecule are
displayed in liquorice mode. Major differences between
the snapshots are observed in the orientation of His48
and the location of the water molecule. For phospholipid
1 (Fig. 3A), Asp99-His48-water are perfectly aligned
for hydrolysis. In this configuration, His48 is able to
abstract a proton from the water molecule that is per-
fectly positioned to perform a nucleophilic attack on the
sn-2 carbonyl. Clearly, the allyl-substituent in phospho-
lipid 1 does not seem to interfere with the incoming water
molecule participating in the hydrolysis. In contrast,
Fig. 3B shows that one of the two methyl substituents in
phospholipid 2 prevents a water molecule from getting in
place to take part in the hydrolysis. Furthermore, His48
is not optimal aligned to participate in the hydrolysis.
Hence, the sn-1 side chain of phospholipid 2 is too bulky
for allowing free access of a water molecule and perfect
alignment of His48. A detailed analysis of the simula-
tions will be provided elsewhere (unpublished material).
In summary, we have described the preparation of
phospholipids 1 and 2 from l-ascorbic acid. In the
synthesis, aldehyde 3 served as a key intermediate for
introduction of the allyl-substituents and for control-
ling the stereochemistry at the sn-2 position of the final
phospholipids. Difficulties in incorporating the ether
3.4. sPLA2 activity measurements and molecular
dynamics simulations
Differential scanning calorimetry (DSC) results of the
two new phospholipids 1 and 2 revealed the thermody-
namic properties of the liposomes. DSC of phospholipid
1 shows the main phase transition Tm at 47 ◦C
(T1/2 = 1.7 ◦C, ꢀH = 10.5 kcal/mol) as a single peak, and
DSC of phospholipid 2 shows the main phase tran-
sition Tm at 44 ◦C (T1/2 = 0.48 ◦C, ꢀH = 9.2 kcal/mol)
as a single peak. Small unilamellar liposomes com-
posed of phospholipids 1 and 2 were subjected to
hydrolysis with sPLA2. The enzymatic reaction was
carried out at Tm and monitored by light scattering
and HPLC. Increased intensity changes in the 90◦ light
scattering were measured for liposomes composed of
phospholipid 1 indicating morphology changes caused
by the production of nonbilayer forming fatty acids and
lysophospholipids and hence hydrolytic activity. This
was further supported by HPLC measurements showing
that there was a decrease in the substrate concentration
upon addition of the enzyme (Fig. 2). Light scattering
measurements of sPLA2 action towards phospholipid
2 composed liposomes did not reveal any morphol-
ogy changes, suggesting that sPLA2 is not able to
catalyze the hydrolysis of this particular phospholipid.
The inability of sPLA2 to hydrolyze phospholipid 2
was verified by HPLC, which showed no changes in
the concentration of the phospholipid upon addition of
the enzyme.
Fig. 2. sPLA2-catalyzed hydrolysis at 47 ◦C of liposomes composed
of phospholipid 1. The HPLC chromatograms show the amount of
phospholipid 1 before sPLA2 was added (0 s), and the amount of
unhydrolyzed lipids 1000 and 6000 s after addition of sPLA2.

L. Linderoth et al. / Chemistry and Physics of Lipids 146 (2007) 54–66
65
Fig. 3. Snapshots of the active site of sPLA2. Images are taken from simulations of phospholipids 1 (A) and 2 (B). The secondary protein structure
is shown in the ribbon mode and is colored green. His48, Asp99, the substrate and a water molecule are shown in the liquorice mode and are colored
according to the atom type (H: white, N: blue, O: red, P: gold). The Ca2+ essential for sPLA2 activity is shown in van der Waals mode and is colored
gold. Lipid structures to the right of the images define the region shown in the images. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article.)
moiety in the sn-1 position arose in the case of alco-
hol 8 and consequently, the reaction conditions were
changed from sodium hydride and cetyl iodide to cetyl
triflate and 1,8-bis(dimethylamino)naphthalene, which
improved the yield of the reaction. Additionally, sPLA2
activity measurements of the phospholipids in combina-
tion with molecular dynamics simulations revealed that
the limited space available in the binding cleft prohibits
hydrolysis of phospholipid 2, whereas phospholipid 1
seems to be a good substrate for sPLA2.
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Acknowledgements
Financial support from the Technical University
of Denmark and the Lundbeck Foundation are grate-
fully acknowledged. Center for Biomembrane Physics
(MEMPHYS) and Center for Sustainable and Green
Chemistry are supported by the Danish National
Research Foundation.
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