The total synthesis of 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine-1,3,1′-13C3 and -2,1′-13C2 by a novel chemoenzymatic method

Article abstract of DOI:10.1016/j.chemphyslip.2009.08.001

2-O-Arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine was synthesized with carbon-13 enrichment of the three glycerol carbons and the carbonyl of the stearoyl group. Phospholipase A₂ was utilized to give optically pure lyso-PC, and only 3% acyl migration occurred during reacylation with arachidonic acid anhydride. This phospholipid is an important biosynthetic precursor of arachidonic acid metabolites as well as the endocannabinoid 2-arachidonoylglycerol (2-AG), and is now available for NMR studies.

Full text of DOI:10.1016/j.chemphyslip.2009.08.001

Chemistry and Physics of Lipids 163 (2010) 102–109
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
The total synthesis of
2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine-1,3,1ꢀ-13C3
and -2,1ꢀ-13C2 by a novel chemoenzymatic method
Richard I. Duclos Jr.∗
Center for Drug Discovery, Northeastern University, 116 Mugar Life Sciences Building, 360 Huntington Avenue, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 June 2009
Received in revised form 25 July 2009
Accepted 6 August 2009
Available online 13 August 2009
2-O-Arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine was synthesized with carbon-13 enrich-
ment of the three glycerol carbons and the carbonyl of the stearoyl group. Phospholipase A2 was utilized
to give optically pure lyso-PC, and only 3% acyl migration occurred during reacylation with arachidonic
acid anhydride. This phospholipid is an important biosynthetic precursor of arachidonic acid metabolites
as well as the endocannabinoid 2-arachidonoylglycerol (2-AG), and is now available for NMR studies.
© 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords:
2-O-Arachidonoyl-1-O-stearoyl-sn-glycero-
3-phosphocholine
2-O-Arachidonoyl-1-O-stearoyl-sn-glycerol
13C-labeled
PLA2
PLC
1. Introduction
extremely useful in the determination of the depth of insertion, pre-
ferred orientation, and conformation of drug molecules and integral
Probing model lipid membranes at the molecular level with
NMR has utilized 1,2-diacyl-sn-glycero-3-phosphocholines (PCs)
isotopically labeled in the acyl groups (Stockton et al., 1974;
Schmidt et al., 1977; Seelig, 1977; Davis, 1979; Wittebort et al.,
1981; Blume et al., 1982; Wittebort et al., 1982; Perly et al., 1984;
Ruocco et al., 1985; Makriyannis et al., 1986, 1990; Makriyannis
and Mavromoustakos, 1989; Yang et al., 1992; Huang et al., 1993;
Rajamoorthi et al., 2005) and the phosphocholine head group
(Stockton et al., 1974; Seelig, 1977; London et al., 1979; Roux et
al., 1983; Ruocco et al., 1985; Yang et al., 1992; Lin et al., 1997). In
addition to the phosphorus nuclei (Yeagle, 1978; Gorenstein, 1984;
Burt, 1987; Deihl, 2002; Seydel and Wiese, 2002), the stable isotopi-
cally labeled nuclei of these lipids have been utilized to provide
the molecular details of structural and dynamic properties, such
as lipid phase transitions of model membranes and the perturba-
tion effects of drug molecules. Labeled membrane components are
membrane proteins present within the lipid matrix of such model
membranes using solid state NMR (Makriyannis et al., 1986, 1989,
1990; Yang et al., 1992; Holzgrabe et al., 1999; Toke et al., 2004;
Tian et al., 2005; Tang et al., 2007; Su et al., 2009).
Stable isotopic labeling of the glycerol backbone of PCs
would be particularly useful for studying the critical inter-
facial region of membranes. Such novel glycerol-labeled PCs
have the potential to reveal information by probing the region
between the polar membrane surfaces and the central hydropho-
bic environment of model membrane bilayers. Introducing stable
isotopic labels in the glycerol backbone represents the next chal-
lenge for lipid synthetic chemistry. The only previous synthetic
approach to glycerol-labeled lipids utilized glycerol kinase in
a chemoenzymatic synthesis of a perdeuterated 1,2-diacyl-sn-
glycero-3-phosphocholine (Kingsley and Feigenson, 1979). Also,
Pasquaré utilized tissue incubations in the related preparations
of [sn-2-3H]glycerophospholipids (Pasquaré de Garcia and Giusto,
1986; Pasquaré and Giusto, 1993; Pasquaré et al., 2006).
This reportdetails an alternativeapproach developed to produce
optically pure diacyl-sn-glycero-3-phosphocholines on a 300 mg
scale that are labeled in the glycerol portion of the molecule. Of
particular interest are 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-
3-phosphocholine (Hindenes et al., 2000) and the corresponding
diglyceride 2-O-arachidonoyl-1-O-stearoyl-sn-glycerol that is the
Abbreviations:
DG, diacylglycerol; DSPC, 1,2-di-O-stearoyl-sn-glycero-3-
phosphocholine; PC, 1,2-diacyl-sn-glycero-3-phosphocholine; PLA2, phospholipase
A2; PLC, phospholipase C; SAPC, 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-
phosphocholine.
∗
Tel.: +1 617 373 3163; fax: +1 617 373 7493.
E-mail address: duclos@neu.edu.
0009-3084/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2009.08.001

R.I. Duclos Jr. / Chemistry and Physics of Lipids 163 (2010) 102–109
103
Scheme 1. Synthesis of 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine-1,3,1ꢀ-13C3 and -2,1ꢀ-13C2 (11, SAPC-13C4).
predominant diacylglycerol (DG) component in brain (Jung et al.,
2007) and neural tissue (Eichberg and Zhu, 1992). Thus, the phos-
phocholine group is also utilized as a protecting group (Duclos et
al., 2009) for synthetic 1,2-diacyl-sn-glycerols that are isotopically
labeled in the glycerol backbone. As a protecting group, the phos-
phocholine group is easily removed enzymatically (Zwaal et al.,
1971; Mavis et al., 1972; Majerus and Prescott, 1982; Dennis, 1983;
Duclos et al., 2009), and it is particularly suited to arachidonoyl
glycerides where reductive or acidic deprotection conditions and
extensive chromatographic purifications cannot be used.
diacylglycerol byproduct can be seen by 1D and 2D TLC (Thomas
et al., 1965; Pollack et al., 1971; Kodali et al., 1990) and by the
attenuation of the ı 5.08 and 3.72 resonances in the 1H NMR
with the appearance of the rearrangement product resonances
between ı 4.05 and 4.21 ppm. Conversion to the corresponding
racemic phosphatidylcholine 7 (rac-DSPC-13C5) was performed in
63% by the mild method that we have previously used for phos-
phocholines (Duclos and Makriyannis, 1992; Duclos, 1993; Duclos
et al., 1994; Lin et al., 2001). The absolute stereospecificity of
the PLA2 enzyme (Gupta et al., 1977; Dennis, 1994) was utilized
to perform the hydrolysis of the sn-2 acyl chain to give only
the desired enantiomer of the corresponding lyso-PC-13C4 8. The
lyso-PC-13C4 8 was separated from the unhydrolyzed enantiomer
ent-DSPC-13C5 9 and from free stearic-1-13C acid by a variation
of a reported method using DEAE-cellulose column chromatogra-
phy (Barlow et al., 1988). The 13C NMR spectrum demonstrated
the presence of 10% of the isomeric lyso-PC, labeled 2-O-stearoyl-
sn-glycero-3-phosphocholine, however. A small amount of acyl
rearrangement (Kodali et al., 1990), along with the possibil-
ity of some phosphoester rearrangement (Lammers et al., 1978;
Plückthun and Dennis, 1982), occurred during the lengthy purifica-
tion of lyso-PC-13C4 8. This necessitated the facile re-esterification
to give pure 1,2-di-O-stearoyl-sn-glycero-3-phosphocholine (10,
DSPC-13C5) in 98% yield. The PLA2 enzymatic hydrolysis (100%
yield) followed immediately by reacylation (Mason et al.,
1981) with arachidonic anhydride (84% yield) gave the opti-
cally pure mixed-chain phosphatidylcholine product, labeled
2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine (11,
SAPC-13C4). The C4 and C16 protons of the arachidonoyl chain
(see Fig. 1) were easily assigned based on the reported TOCSY
of arachidonic acid (Singer et al., 1996). The carbon resonances
(see Fig. 2) were assigned based on the work of Gundstone
(http://www.lipidlibrary.co.uk). The three glycerol carbon reso-
nances were readily distinguished by their chemical shifts and
couplings. The carbonyl region (see Fig. 3) shows the enriched
2. Results and discussion
Consideration of the optimal signal-to-noise intensities of the
carbon-13 NMR signals from the three glycerol resonances required
a strategy such that there were no adjacent labeled carbons where
13C–13C couplings (1JCC) would occur. This coupling would be par-
ticularly problematic for the sn-2 carbon resonance if it were to
3
have two adjacent carbon-13 labels in addition to the strong JCP
coupling of glycero-3-phospholipids. The intensities of the glycerol
carbon resonances were optimized by starting with a 50:50 mix-
ture of glycerol-1,3-13C2 (1) and glycerol-2-13C (2) (see Scheme 1)
which was first converted to the corresponding trityl derivative
3. This mono-protected glycerol 3 was prepared from the rela-
tively insoluble labeled glycerols 1 and 2 with one recycle of ditrityl
byproduct to improve the conversion yield to rac-3-tritylglycerol
3. The racemic trityl-protected 1,2-diacylglycerol 4 was prepared
by diesterification of diol 3 with stearic-1-13C acid. Deprotection
by the method of Kodali and Duclos (1992) gave the racemic
1,2-diacylglycerol 5 in 90% yield. The rac-1,2-diacylglycerol 5 had
100% 13C enrichment in the two carbonyls, and as for all subse-
quent compounds, labeling enrichment of 50% in each of the three
glycerol backbone positions. The 1,2-diacylglycerol 5 was used
promptly due to the possibility of rearrangement (Mattson and
Volpenhein, 1962; Kodali et al., 1990; Stella et al., 1997). Any 1,3-

104
R.I. Duclos Jr. / Chemistry and Physics of Lipids 163 (2010) 102–109
The 13C enrichment of four carbons was accomplished in
this synthesis of a 1:1 mixture of the title compounds 2-O-
arachidonoyl-1-O-stearoyl-sn-glycerol-1,3,1ꢀ-13C3 and -2,1ꢀ-13C2.
This labeled SAPC-13C4 11 will be useful for NMR and mass spectral
studies of hydrolysis by phospholipases A2, C, D, and arachidonic
acid utilization (Nicolaou and Kokotos, 2004). There is consider-
able recent interest in the phospholipase C hydrolysis to give the
corresponding 1,2-diglyceride that is the biosynthetic precursor of
the endocannabinoid 2-AG (Sugiura et al., 2006; Ahn et al., 2008;
Duclos et al., 2009). Preliminary experiments with unlabeled 2-
O-arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine run on
a 10 mg scale gave a quantitative conversion to unlabeled 2-O-
arachidonoyl-1-O-stearoyl-sn-glycerol.
Fig. 1. 1H NMR spectrum of 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-
phosphocholine-1,3,1ꢀ-13C3 and -2,1ꢀ-13C2 (11, SAPC-13C4).
3. Experimental
3.1. General
The CH2Cl2, CHCl3 (amylene stabilized), and CCl4 used for reac-
tion solvents were distilled twice from P2O5 immediately prior to
use. Diisopropylethylamine and pyridine were distilled from CaH2.
Et2O was freshly distilled from benzophenone ketyl. Glycerol-1,3-
13C2 (1), glycerol-2-13C (2), and stearic-1-13C acid were obtained
from Cambridge Isotope Laboratories (Andover, MA). The stearic-1-
13C acid contained less than 1% of palmitic acid as the only impurity
by GC (5% DEGS-PS on 100/120 supelcoport, 160 ◦C, FID) following
methylation. All other reagents and solvents were purchased from
Sigma–Aldrich (Milwaukee, WI) and were used without further
purification. All reactions were performed with magnetic stirring.
Organic phases were dried over Na2SO4, solvents removed by
rotary evaporation under reduced pressure, and flash column chro-
matography employed silica gel 60 (230–400 mesh, E. Merck). All
compounds were demonstrated to be homogeneous by analyti-
cal thin-layer chromatography on aluminum pre-coated silica gel
TLC plates (UV254, layer thickness 250 ␮m, E. Merck), and chro-
matograms were visualized under ultraviolet light or by charring
(Bitman and Wood, 1982). Phosphocholines were readily identi-
fied on duplicate plates with molybdic acid reagent (Dittmer and
Lester, 1964; Ryu and MacCoss, 1979). All solvent ratios are by vol-
ume. Melting points were determined on a microapparatus and
are uncorrected. 1H and 13C NMR spectra were recorded on Bruker
AMX-500 spectrometer. Unless otherwise stated, all NMR spectra
were recorded using CDCl3 as solvent which shows a residual pro-
ton resonance at ı 7.26 and a carbon triplet centered at ı 77.02.
However, in 2:1 CDCl3/CD3OD the chloroform residual proton res-
onance is observed at ı 7.49 and the carbon triplet is centered at ı
77.71. Chemical shifts are reported in ppm relative to tetramethyl-
silane as an internal standard with multiplicities indicated as br
(broadened), s (singlet), d (doublet), t (triplet), q (quartet), quintet
(quintet), and m (multiplet).
Fig. 2. 13C NMR spectrum of 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-
phosphocholine-1,3,1ꢀ-13C3 and -2,1ꢀ-13C2 (11, SAPC-13C4).
carbonyl of the sn-1 stearoyl group at ı 174.27 with its satellite
peaks at ı 174.51 and 174.06 (each approximately 0.55%) result-
ing from coupling (1JCC = 55 Hz) to the 1.1% natural abundance of
13C in the C2ꢀ-methylene carbon alpha to the carbonyl. The 1ꢀꢀ-
carbonyl of the sn-2 arachidonoyl group appeared at ı 173.63,
identical to unlabeled SAPC (Avanti Polar Lipids, Inc.). The carbonyl
resonance at ı 173.91 indicated the presence of approximately
3% of the isomeric 1-O-arachidonoyl-2-O-stearoyl-sn-glycero-3-
phosphocholine-1,3,1ꢀꢀ-13C3 and -2,1ꢀꢀ-13C2, an isomeric PC that
typically results during the acylation of lyso-PCs.
3.2. rac-3-O-Tritylglycerol-1,3-13C2 and -2-13C (3)
A 1:1 mixture of labeled glycerols (0.954 g, 10.2 mmol) from
0.5 g of glycerol-1,3-13C2 (1) and 0.5 g of glycerol-2-13C (2) with
2.66 mL (1.97 g, 15.2 mmol, 150 mol %) of Hunig’s base in 20 mL
of CH2Cl2 was stirred vigorously during the addition of 2.84 g
(10.2 mmol, 100 mol %) of trityl chloride in 10 mL of CH2Cl2 over
30 min. Some fuming was observed, and the reaction became
homogeneous, clear, and yellow during an additional 2 h of stir-
ring. Chromatography (95:4.5:0.5 CH2Cl2/MeOH/Et3N) separated
ditrityl byproduct (Rf 0.80) from the product 3 (Rf 0.27) by TLC
(95:4.5:0.5 CH2Cl2/MeOH/Et3N). The byproduct was de-tritylated
with 0.86 mL (0.99 g) of BF3-etherate in 15 mL of 2:1 CH2Cl2/MeOH,
the recovered labeled glycerol was triturated with benzene, and
Fig. 3. The carbonyl region 13C NMR of 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-
3-phosphocholine-1,3,1ꢀ-13C3 and -2,1ꢀ-13C2 (11, SAPC-13C4) demonstrates the
presence of 3% of the 1-O-arachidonoyl-2-O-stearoyl-PC-13C4 isomer formed during
reacylation of lyso-PC-13C4.

R.I. Duclos Jr. / Chemistry and Physics of Lipids 163 (2010) 102–109
105
recycled one additional time to improve the monotritylation yield.
3.5. rac-1,2-Di-O-stearoylglycero-3-phospho-ˇ-bromoethanol-
The product 3 (1.40 g, 4.17 mmol, 41%) was a white crystalline solid:
mp 97–98 ◦C; 1H NMR ı 6.90–7.60 (m, 15H, aromatics), 3.60–4.30
(m, 1H, 2), 3.30–3.70 (m, 2H, 1), 3.10–3.40 (m, 2H, 3); 13C NMR
ı 143.72, 128.67, 127.96, and 127.22 (aromatics), 87.02 (C-Ph3),
71.18, 65.07, and 64.32 (glycerol).
1,3,1ꢀ,1ꢀꢀ-13C4 and -2,1ꢀ,1ꢀꢀ-13C3
(6)
Pyridine (3.64 mL, 3.56 g, 45 mmol) in 5 mL of Et2O was added
dropwise to a stirred solution of 2.54 g (10.5 mmol, 350 mol %) of
␤-bromoethyldichlorophosphate (Duclos and Makriyannis, 1992;
Duclos, 1993; Duclos et al., 1994; Lin et al., 2001) in 10 mL of Et2O
under argon over 5 min. After stirring for 15 min, a solution of 1.89 g
(3.01 mmol, 100 mol %) of rac-DG-13C5 5 in 40 mL of Et2O was added
over 15 min. The reaction mixture was then refluxed gently for
4 h using an oil bath at 55 ◦C. The reaction mixture was cooled
to 0 ◦C, and 12 mL of H2O was added dropwise over 2 min. After
5 min, 20 mL of Et2O was added and the reaction was allowed to
stir overnight. The reaction mixture was partitioned between 60 mL
of 2 N HCl and 90 mL of 10:90 MeOH/CH2Cl2. The organic phase
was washed with 100 mL of water, dried, and the solvent removed
to give a white solid foam. Chromatography on 225 g of silica gel
eluting with a gradient of 8–49% methanol in CH2Cl2 gave 1.69 g
(2.08 mmol, 69%) of ␤-bromoethylphosphodiester 6 as a white
solid (20:80 MeOH/CH2Cl2, Rf 0.23; 60:30:4 CHCl3/MeOH/H2O, Rf
0.57): mp 72–4 ◦C; 1H NMR (2:1, CDCl3/CD3OD) ı 5.24 (m; and,
3.3. rac-1,2-Di-O-stearoyl-3-O-tritylglycerol-1,3,1ꢀ,1ꢀꢀ-13C4 and
-2,1ꢀ,1ꢀꢀ-13C3 (4)
To a stirred solution of 1.40 g (4.17 mmol, 100 mol %) of diol
3, 2.50 g (8.76 mmol, 210 mol %) of stearic-1-13C acid, and 1.15 g
(9.41 mmol, 225 mol %) of 4-dimethylaminopyridine (DMAP) in
50 mL of CCl4 was added a solution of 2.15 g (10.4 mmol, 250 mol
%) of dicyclohexylcarbodiimide (DCC) in 10 mL of CCl4 dropwise
over 10 min. The reaction became cloudy and was allowed to stir
overnight, during which time a precipitate of dicyclohexylurea
had formed. The reaction was warmed to 30–35 ◦C, the byprod-
uct was removed by filtration through a 10–20 ␮m fritted glass
funnel, and the CCl4 filtrate and washings were combined and
concentrated. Chromatography on a warmed column (wrapped
with heating tape from Ace Glass, Cat. No. 12063) eluting with
30–35 ◦C 49.5:50:0.5 CH2Cl2/hexanes/Et3N gave 3.04 g (3.49 mmol,
84%) of diester 4 as a white solid which was homogeneous by TLC
(49.5:50:0.5 CH2Cl2/hexanes/Et3N, Rf 0.19): mp 33.5 ◦C (soften),
48.0–48.5 ◦C (melt): 1H NMR ı 7.10–7.70 (m, 15H, aromatic), 5.25
1
1
br d, JCH = 144 Hz, 1H, 2), 4.40 (m; and, br d, JCH = 150 Hz, 2H,
1
1), 4.15 (m, 2H, P–O–CH2), 4.02 (br s; and, br d, JCH = 144 Hz,
2H, 3), 3.54 (t, 2H, J = 6.2 Hz, CH2–Br), 2.33 (apparent quintet,
4H, 2ꢀ and 2ꢀꢀ), 1.50–1.70 (m, 4H, 3ꢀ and 3ꢀꢀ), 1.15–1.50 (m, 56H,
4–17ꢀ and 4–17ꢀꢀ), 0.89 (t, 6H, J = 6.9 Hz, 18ꢀ and 18ꢀꢀ); 13C NMR
(2:1, CDCl3/CD3OD) ı 174.25 and 173.91 (1ꢀ and 1ꢀꢀ carbonyls),
1
1
(m; and, d, JCH = 150 Hz, 1H, 2), 4.31 (m; and, d, JCH = 150 Hz,
1
3
2
2H, 1), 3.59 (br s; and, br d, JCH = 144 Hz, 2H, 3), 2.33 and 2.22
70.85 (d, JCP = 6.9 Hz, 2), 65.85 (d, JCP = 4.9 Hz, P–O–CH2), 64.11
(d, 2JCP = 5.5 Hz, 3), 62.98 (1), 34.59 and 34.42 (two d, 1JCC = 57.3 Hz,
2ꢀ and 2ꢀꢀ), 32.24 (16ꢀ and 16ꢀꢀ), 31.19 (m, CH2–Br), 29.48-30.02 (4ꢀ-
15ꢀ and 4ꢀꢀ-15ꢀꢀ), 25.21 (3ꢀ and 3ꢀꢀ), 22.97 (17ꢀ and 17ꢀꢀ), 14.21 (18ꢀ
and 18ꢀꢀ).
(two dt, 4H, J2,3 = 7.3, JCH = 7.3 Hz, 2ꢀ and 2ꢀꢀ), 1.40–1.70 (m, 4H,
2
3ꢀ and 3ꢀꢀ), 0.90–1.40 (m, 56H, 4–17ꢀ and 4–17ꢀꢀ), 0.88 (t, 6H,
J = 6.3 Hz, 18ꢀ and 18ꢀꢀ); 13C NMR ı 173.35 and 172.99 (1ꢀ and 1ꢀꢀ
carbonyls), 143.66, 128.67, 127.87, and 127.14 (aromatics), 86.73
(C-Ph3), 70.47, 62.85, and 62.29 (glycerol), 34.42 and 34.14 (two d,
1JCC = 57.5 Hz, 2ꢀ and 2ꢀꢀ), 31.96 (16ꢀ and 16ꢀꢀ), 29.11-29.73 (4–15ꢀ
and 4–15ꢀꢀ), 24.99 and 24.89 (3ꢀ and 3ꢀꢀ), 22.71 (17ꢀ and 17ꢀꢀ), 14.12
(18ꢀ and 18ꢀꢀ).
3.6.
rac-1,2-Di-O-stearoylglycero-3-phosphocholine-1,3,1ꢀ,1ꢀꢀ-13C4
and -2,1ꢀ,1ꢀꢀ-13C3 (7, rac-DSPC-13C5)
3.4. rac-1,2-Di-O-stearoylglycerol-1,3,1ꢀ,1ꢀꢀ-13C4 and
Excess trimethylamine was added to a solution of bromide
6 (1.68 g, 2.06 mmol) in 25 mL of 2:2:1 CHCl3/i-propanol/N,N-
-2,1ꢀ,1ꢀꢀ-13C3 (5, rac-DG-13C5)
dimethylformamide in
a thick walled pressure tube with a
A stirred solution of 2.92 g (3.35 mmol, 100 mol %) of trityl
compound 4 in 40 mL of dry CH2Cl2 under argon was cooled to
−20 ◦C (N2/CCl4). A solution of 1.21 g (10.0 mmol, 300 mol %) of
bromodimethylborane (caution: Me2BBr is spontaneously flammable
in air) in 4.3 mL (2.5 M) CH2Cl2 was added dropwise over 15 min.
After 3 h, the reaction was quenched by the dropwise addition of
27 mL of cold saturated aqueous NaHCO3. Then 200 mL of Et2O
was added followed by shaking, and two washes with 75 mL H2O.
The Et2O phase was dried and evaporated. Column chromatogra-
phy on 250 g of boric acid treated silica gel (Kodali and Duclos,
1992; Paltauf and Hermetter, 1994) prepared according to Buchnea
(1974) and Bergelson (1980) gave 1.90 g (3.02 mmol, 90%) of 1,2-
diglyceride 5 as a white solid that was nearly homogeneous by
TLC (5:95 acetone/CH2Cl2, Rf 0.40 with E. Merck silica gel, Rf 0.37
with Analtech silica gel G treated with boric acid). Two dimensional
TLC demonstrated that the 1,3-diglyceride rearrangement byprod-
uct (5:95 acetone/CH2Cl2, Rf 0.52 with E. Merck silica gel, Rf 0.52
with Analtech silica gel G treated with boric acid) forms to a very
PTFE-lined screw-top. The reaction mixture was stirred magnet-
ically and heated with a 50 ◦C oil bath for 1 day. The solvents
were removed under reduced pressure to give a white solid. Chro-
matography (warmed column) eluting with a 30–35 ◦C gradient
of 60:30:0 to 60:30:4 CHCl3/MeOH/H2O gave a white solid which
was re-dissolved in CHCl3 and filtered through a 0.5 ␮m pore PTFE
membrane. Solvent removal and lyophilization from t-butanol gave
1.53 g (1.88 mmol, 91%) of racemic phosphocholine 7 (rac-DSPC-
13C5) as a fluffy white solid that was homogeneous by TLC (60:30:5
CHCl3/MeOH/H2O, Rf 0.24; 25:15:4:2 CHCl3/MeOH/HOAc/H2O, Rf
0.28; 60:30:5 CHCl3/MeOH/NH4OHaq, Rf 0.20): mp 80–82 ◦C; 1H
NMR (2:1, CDCl3/CD3OD) ı 5.24 (m; and, br d, 1JCH = 144 Hz, 1H, 2),
1
4.40 (m; and, br d, JCH = 150 Hz, 1H, 1a), 4.26 (m, 2H, P–O–CH2),
1
4.16 (m; and, br d, JCH = 150 Hz, 1H, 1b), 4.00 (m; and, br d,
1JCH = 144 Hz, 2H, 3), 3.62 (m, 2H, CH2–N), 3.23 (m, 9H, N–(CH3)3),
2.32 (apparent quintet, 4H, 2ꢀ and 2ꢀꢀ), 1.55–1.65 (m, 4H, 3ꢀ and
3ꢀꢀ), 1.20–1.45 (m, 56H, 4–17ꢀ and 4–17ꢀꢀ), 0.89 (t, 6H, J = 6.9 Hz, 18ꢀ
and 18ꢀꢀ); 13C NMR (2:1, CDCl3/CD3OD) ı 174.36 and 174.01 (1ꢀ
and 1ꢀꢀ carbonyls), 70.92 (br s, 2), 66.93 (br s, CH2–N), 64.08 (br s,
small extent under the analytical TLC conditions: mp 70–71 ◦C; 1
H
1
2
NMR ı 5.08 (m; and, br d, JCH = 144 Hz, 1H, 2), 4.27 (m; and, br
3), 63.13 (1), 59.57 (d, JCP = 4.2 Hz, P–O–CH2), 54.49 (N–(CH3)3),
1
1
1
d, JCH = 150 Hz, 2H, 1), 3.72 (br s; and, br d, JCH = 144 Hz, 2H, 3),
2.34 (apparent quintet, 4H, 2ꢀ and 2ꢀꢀ), 1.50–1.70 (m, 4H, 3ꢀ and 3ꢀꢀ),
1.15–1.40 (m, 56H, 4–17ꢀ and 4–17ꢀꢀ), 0.88 (t, 6H, J = 6.6 Hz, 18ꢀ and
18ꢀꢀ).
34.62 and 34.47 (two d, JCC = 57.4 Hz, 2ꢀ and 2ꢀꢀ), 32.31 (16ꢀ and
16ꢀꢀ), 29.54–30.07 (4–15ꢀ and 4–15ꢀꢀ), 25.33 and 25.28 (3ꢀ and 3ꢀꢀ),
23.02 (17ꢀ and 17ꢀꢀ), 14.20 (18ꢀ and 18ꢀꢀ); and was a broad doublet
resonance with unreferenced 31P NMR in 2:1 CDCl3/CD3OD.

106
R.I. Duclos Jr. / Chemistry and Physics of Lipids 163 (2010) 102–109
3.7. 1-O-Stearoyl-sn-glycero-3-phosphocholine-1,3,1ꢀ-13C3 and
-2,1ꢀ-13C2 (8, lyso-PC-13C4); and,
1-13C acid in 30 mL of CCl4 and 0.723 g (3.50 mmol, 100 mol %)
of dicyclohexylcarbodiimide in 10 mL of CCl4 were shaken in a
125 mL separatory funnel for 5 min, then the reaction was allowed
to proceed for 18 h. The mixture was filtered through What-
man #1 filter paper, and the white dicyclohexylurea precipitate
was washed with three 5 mL portions of CCl4 that were also fil-
tered and combined. The solvent was removed to give 1.99 g
(3.60 mmol, quantitative) of anhydride that was used without fur-
2,3-di-O-stearoyl-sn-glycero-1-phosphocholine-1,3,1ꢀꢀ,1ꢀꢀꢀ-13C4
and -2,1ꢀꢀ,1ꢀꢀꢀ-13C3 (9, ent-DSPC-13C5) via PLA2 hydrolysis
According to the reported method (Mason et al., 1981), 1.49 g
(1.84 mmol) rac-DSPC-13C5 7 was dispersed in 56 mL of buffer
(100 mM NaEDTA, 100 mM Tris, pH 8.1) and mixed by rotation
on a rotary evaporator for 1 h. The mixture was extracted with
150 mL of 30–35 ◦C 2:1 CHCl3/MeOH, followed by two additional
extractions of 50 mL each. The combined warm extracts were
dried and rotary evaporated. The white residue was given one
chase with CHCl3, and was lyophilized from t-butanol to give
1.44 g of a fluffy white solid suitable for dispersion in solution
and enzymatic hydrolysis. The phosphocholine 7 (1.43 g, 1.76 mol)
was dissolved in 120 mL of 1:5 MeOH/Et2O and stirred vigor-
ously magnetically with heating from a 29 ◦C bath. A solution of
185 mg of Crotalus adamanteus snake venom in 15 mL of buffer
(220 mM NaCl, 20 mM CaCl2, 1 mM Na2EDTA, 50 mM Tris, pH
7.4) was added in five 3 mL portions at 20 min intervals, then
the mixture was stirred for an additional 1 h 40 min (total of
3 h). The organic phase was separated and saved. The (15 mL)
aqueous phase was extracted with four 15 mL portions of 2:1
CHCl3/MeOH, and the combined organic phases were rotary evap-
orated without heating above 35 ◦C to give a white foam. The
foam was dissolved in CHCl3, some water was separated, then the
CHCl3 solution was dried, and rotary evaporated to give 1.42 g
of a mixture of lysophosphocholine 8, phosphocholine 9, and
stearic-1-13C acid. The chromatographic separation was performed
using the method of Sigler (Barlow et al., 1988). A 1500 cm3
column of microgranular preswollen DEAE-cellulose (DE-52, What-
man) in H2O was poured and washed with 4200 mL of HOAc,
8400 mL of MeOH, 4200 mL of 1:1 CHCl3/MeOH, followed by
5500 mL of CHCl3. A solution of the crude product mixture in
5 mL of CHCl3 was loaded and eluted with a stepwise gradient
of methanol in CHCl3. The ent-DSPC-13C5 9 byproduct (1.05 g)
eluted between 4 and 7% methanol and was identical by NMR
to rac-DSPC-13C5 7. The gradient was then steeply increased to
33% MeOH and then eluted isocratically to give 0.33 g (0.61 mmol,
69% theoretical yield) of lysophosphocholine 8 as a white solid
following lyophilization from cyclohexane. The product lyso-PC-
13C4 8 (Rf 0.21) was free of phosphatidylcholine (Rf 0.36) by TLC
(60:35:8 CHCl3/MeOH/H2O) and was used promptly: 1H NMR
(2:1, CDCl3/CD3OD) ı 4.34 (m, 2H, P–O–CH2), 4.14 (m; and, br d,
ther purification: 1H NMR ı 2.44 (dt, 4H, J2,3 = 7.4, JCH = 7.4 Hz,
2
2), 1.60–1.70 (m, 4H, 3), 1.20–1.40 (m, 56H, 4–17), 0.88 (t,
1
6H, J = 6.9 Hz, 18); 13C NMR ı 169.64 (1), 35.31 (d, JCC = 58 Hz,
2), 31.96 (16), 28.90–29.75 (4–15), 24.26 (3), 22.72 (17), 14.13
(18).
3.9.
1,2-Di-O-stearoyl-sn-glycero-3-phosphocholine-1,3,1ꢀ,1ꢀꢀ-13C4
and -2,1ꢀ,1ꢀꢀ-13C3 (10, DSPC-13C5)
To a cloudy solution of lyso-PC-13C4 8 (0.321 g, 0.590 mmol)
and 0.979 g (1.77 mmol, 300 mol %) of stearic-1,1ꢀ-13C2 anhydride
in 5 mL of CHCl3 was added a solution of 0.175 g (1.18 mmol,
200 mol %) of 4-pyrrolidinopyridine (4-PPY) in 2 mL of CHCl3.
The reaction was initially warmed for 10 min to 30–35 ◦C with a
water bath and became homogeneous after 3 h. TLC of samples
of the homogeneous reaction mixture showed that the reaction
was essentially complete after stirring overnight. The volume was
adjusted to 20 mL with CHCl3 and 10 mL of MeOH was then
added. The organic solution was washed with 15 mL of 0.25 N
HCl. The aqueous extract was backwashed twice with CHCl3 and
the backwashes were combined with the organic phase, dried,
and the solvent removed. Column chromatography was performed
with a wrapped warmed column and elution with 30–35 ◦C sol-
vents. Byproducts were first eluted with a gradient of 60:30:0 to
60:30:5 CHCl3/MeOH/H2O. The pure product DSPC-13C5 10 sub-
sequently eluted and the solvent was removed. The DSPC-13C5
10 was dissolved in 90:10 CHCl3/MeOH and filtered through a
0.5 ␮m pore PTFE filter, the solvent was removed, and lyophiliza-
tion from t-butanol gave 0.407 g (0.501 mmol, 98%) of a fluffy
white solid that was homogeneous by TLC (Rf 0.28, 25:15:4:2
CHCl3/MeOH/HOAc/H2O): mp 83–85 ◦C; 1H, 13C, and 31P NMR were
identical to the racemic material rac-DSPC-13C5 7 detailed above
and to ent-DSPC-13C5 9; [␣]D24 + 6.8◦ (c 4.59, 1:1 CHCl3/MeOH)
[rotations for unlabeled DSPC: Lit. (Patel et al., 1979) [␣]D + 6.4◦ (c
4.59, 1:1 CHCl3/MeOH); Lit. (Baer and Buchnea, 1959) [␣]29 + 6.2◦
1
1JCH = 150 Hz, 2H, 1), 3.98–4.04 (m; and, br d, JCH = 150 Hz, 2H, 2
D
1
(c 10, 1:1 CHCl3/MeOH)].
and 3a), 3.95 (m; and, br d, JCH = 144 Hz, 1H, 3b), 3.65 (m, 2H,
CH2–N), 3.23 (m, 9H, N–(CH3)3), 2.35 (dt, 4H, J2,3 = 7.4, 2JCH = 7.4 Hz,
2ꢀ), 1.55–1.64 (m, 2H, 3ꢀ), 1.20–1.40 (m, 28H, 4–17ꢀ), 0.88 (t, 3H,
J = 7.1 Hz, 18ꢀ); 13C NMR (2:1, CDCl3/CD3OD) ı 174.83 (1ꢀ carbonyl),
3.10. Arachidonic anhydride
3
68.88 (d, JCP = 6.9 Hz, 2), 67.03 (br s, 3 and CH2–N), 65.17 (1),
59.33 (d, 2JCP = 4.5 Hz, P–O–CH2), 54.23 (t, 1JCN = 4.0 Hz, N–(CH3)3),
34.18 (d, 1JCC = 57.0 Hz, 2ꢀ), 32.04 (16ꢀ), 29.29–29.81 (4–15ꢀ), 24.98
(3ꢀ), 22.79 (17ꢀ), 14.10 (18ꢀ). The presence of 10% isomeric 2-O-
stearoyl-sn-glycero-3-phosphocholine-1,3,1ꢀꢀ-13C3 and -2,1ꢀꢀ-13C2
was indicated by enriched nuclei resonance signals at ı 174.36 (1ꢀꢀ
carbonyl), 73.26 (d, 3JCP = 7.3 Hz, 2), 63.35 (d, 2JCP = 5.3 Hz, 3), 60.01
(1); and, a second small broad doublet resonance that was observed
upfield of the broad doublet resonance with unreferenced 31P NMR
in 2:1 CDCl3/CD3OD.
Anhydrous CCl4 was vacuum degassed, then further degassed by
argon bubbling and 20 mL was used to dissolve 1.00 g (3.28 mmol,
200 mol %) of arachidonic acid and 0.376 g (1.82 mmol, 100 mol %)
of dicyclohexylcarbodiimide. The reaction mixture was allowed to
stand overnight under an argon atmosphere. The white precipi-
tate that formed was removed by paper filtration and the filtrate
washed with additional CCl4. The solvent was removed to give a
quantitative yield of anhydride as a clear very faint yellow liquid
that was used without further purification: 1H NMR ı 5.30–5.45
(m, 16H, 5,6,8,9,11,12,14,15), 2.80–2.85 (m, 12H, 7,10,13), 2.46 (t,
4H, J = 7.4 Hz, 2), 2.15 (dt, 4H, J = 7.2, 7.2 Hz, 4), 2.05 (dt, 4H, J = 7.1,
7.1 Hz, 16), 1.71–1.77 (m, 4H, 3), 1.20–1.60 (m, 12H, 17,18,19), 0.89
(t, 6H, J = 6.8 Hz, 20); 13C NMR ı 169.31 (1), 130.52, 129.36, 128.64,
128.46, 128.37, 128.02, 127.83, 127.55 (olefinics), 34.59 (2), 31.55
(18), 29.35 (17), 27.25 (16), 26.25 (4), 25.66 (br s, 7,10,13), 24.05
(3), 22.60 (19), 14.08 (20).
3.8. Stearic-1,1ꢀ-13C2 anhydride
The labeled anhydride was prepared by the method used for the
unlabeled compound (Selinger and Lapidot, 1966). A slightly warm
(30–35 ◦C) solution of 2.02 g (7.08 mmol, 200 mol %) of stearic-

R.I. Duclos Jr. / Chemistry and Physics of Lipids 163 (2010) 102–109
107
1
3.11. 2-O-Arachidonoyl-1-O-stearoyl-sn-glycero-3-
phosphocholine-1,3,1ꢀ-13C3 and -2,1ꢀ-13C2 (11,
SAPC-13C4)
2H, P–O–CH2), 4.16 (m; and, br d, JCH = 150 Hz, 1H, 1b), 4.00 (m;
1
and, br d, JCH = 149 Hz, 2H, 3), 3.61 (m, 2H, CH2–N), 3.23 (m, 9H,
N–(CH3)3), 2.75–2.90 (m, 6H, 7ꢀꢀ, 10ꢀꢀ, 13ꢀꢀ), 2.36 (t, 2H, J2,3 = 7.4, 2ꢀꢀ),
2.31 (dt, 2H, J2,3 = 7.4, JCH = 7.4 Hz, 2ꢀ), 2.13 (dt, 4H, J = 7.0, 7.0 Hz,
2
4ꢀꢀ), 2.06 (dt, 4H, J = 7.1, 7.0 Hz, 16ꢀꢀ), 1.65–1.75 (m, 2H, 3ꢀꢀ), 1.55–1.65
(m, 2H, 3ꢀ), 1.20–1.45 (m, 34H, 4–17ꢀ and 17–19ꢀꢀ), 0.89 (apparent q,
6H, 18ꢀ and 20ꢀꢀ); 13C NMR (2:1, CDCl3/CD3OD) ı 174.27 (1ꢀ), 173.91
(1ꢀꢀ of the rearrangement byproduct), 173.63 (1ꢀꢀ), 130.74, 129.31,
129.04, 128.90, 128.61, 128.36, 128.11, 127.85 (olefinics), 70.87 (d,
3JCP = 7.6 Hz, 2), 66.86 (br s, CH2–N), 63.92 (d, 2JCP = 4.2 Hz, 3), 63.01
According to the reported method (Mason et al., 1981), 0.393 g
(0.484 mmol) of optically pure labeled DSPC-13C5 10 was dispersed
in 15 mL of buffer (100 mM NaEDTA, 100 mM Tris, pH 8.1) and
mixed by rotation on a rotary evaporator for 1 h. The mixture was
extracted with 30 mL of 30 ◦C 2:1 CHCl3/MeOH, followed by two
additional extractions of 15 mL each. The combined extracts were
dried and rotary evaporated. The white residue was given one chase
with CHCl3, and was lyophilized from t-butanol to give 0.388 g of
a fluffy white solid suitable for dispersion in solution and enzy-
matic hydrolysis. The phosphatidylcholine DSPC-13C5 10 (0.388 g,
0.478 mol) was dissolved in 36 mL of 1:5 MeOH/Et2O and stirred
vigorously magnetically with heating from a 29 ◦C bath. A solution
of 49.4 mg of C. adamanteus snake venom in 4 mL of buffer (220 mM
NaCl, 20 mM CaCl2, 1 mM Na2EDTA, 50 mM Tris, pH 7.4) was added
in five 0.8 mL portions at 20 min intervals, then the mixture was
stirred for an additional 1 h 40 min (total of 3 h). The organic phase
was separated and the solvent removed. The (4 mL) aqueous phase
was extracted with six 5 mL portions of Et2O. The Et2O extracts
were combined with the residue from the first organic phase, con-
centrated to 5 mL, and backwashed twice with 2 mL portions of
water. The three aqueous phases were combined (8 mL). (The com-
bined Et2O extracts contained 0.126 g of fatty acid and very little
lyso-PC.) The aqueous phase was extracted with three 10 mL por-
tions of 4:1 CHCl3/MeOH with phase separation performed rapidly
using a tabletop centrifuge. The combined organic phases were
dried, rotary evaporated without heating above 35 ◦C, given one
chase with CHCl3, lyophilized from t-butanol, re-lyophilized from
cyclohexane, and then dried at high vacuum overnight in the pres-
ence of P2O5 to give 0.262 g (quantitative yield) of a fluffy white
powder that was nearly homogeneous by TLC (Rf 0.07, 60:30:5
CHCl3/MeOH/H2O) containing only trace amounts of PC (Rf 0.27)
and fatty acid (Rf 0.86): mp 116–120 ◦C soften, 240–245 ◦C melt.
To a cloudy solution of the lysophosphocholine intermediate
(0.260 g, 0.478 mmol, 100 mol %) and 0.970 g (1.64 mmol, 343 mol
%) of arachidonic anhydride in 5 mL of CHCl3 was added a solu-
tion of 0.142 g (0.958 mmol, 200 mol %) of 4-pyrrolidinopyridine
(4-PPY) in 2 mL of CHCl3. The reaction mixture became homo-
geneous within a half hour. TLCs (60:30:5 CHCl3/MeOH/H2O and
25:15:4:2 CHCl3/MeOH/HOAc/H2O) of samples of the homoge-
neous reaction mixture showed that the starting material was
completely consumed after stirring overnight. The reaction mix-
2
(1), 59.34 (d, JCP = 5.1 Hz, P–O–CH2), 54.43 (N–(CH3)3), 34.36 (d,
1JCC = 57.4 Hz, 2ꢀ), 33.96 (2ꢀꢀ), 32.22 (16ꢀ), 31.82 (18ꢀꢀ), 29.43–29.98
(4–15ꢀ and 17ꢀꢀ), 27.50 (16ꢀꢀ), 26.80 (4ꢀꢀ), 25.91 (s, 7ꢀꢀ, 10ꢀꢀ, 13ꢀꢀ),
25.17 and 25.11 (3ꢀ and 3ꢀꢀ), 22.95 and 22.84 (17ꢀ and 19ꢀꢀ), 14.19
and 14.16 (18ꢀ and 20ꢀꢀ); and was a broad doublet resonance with
unreferenced 31P NMR in 2:1 CDCl3/CD3OD.
3.12. Unlabeled 2-O-arachidonoyl-1-O-stearoyl-sn-glycerol (DG)
via phospholipase C
To a 1.5 mL screw-top Eppendorf tube containing 10.1 mg
(1.22 × 10−5 mol) of 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-
phosphocholine (SAPC, Avanti 850469C) in 150 ␮L of buffer
(100 mM NaCl, 50 mM Tris, 10 mM CaCl2, 0.1 mM ZnCl2, pH 7.0)
was added 1.2 mL of hexane. A solution of phospholipase C (Sigma
P 9439 from Bacillus cereus, 50 U) in 250 ␮L of buffer was deliv-
ered by pipette to the bottom (aqueous) phase in 50 ␮L portions at
20 min intervals. With the screw cap in place and under an argon
atmosphere, the reaction was gently stirred magnetically. A sample
of the hexane phase was removed after 2 h and found to contain
>95% product 1,2-diacyl-sn-glycerol (DG) by TLC (Rf 0.50, 30:70
acetone/hexane) with very little unlabeled DSPC starting material
(Rf 0.00, 30:70 acetone/hexane). The bottom (aqueous) phase was
removed by pipette and was discarded. The top (hexane) phase
was centrifuged at 10K × g for 2 min. The top (hexane) phase was
transferred to a clean PTFE-lined screw-top vial and was dried
briefly over Na2SO4. The dry hexane solution was transferred to a
new vial, and to this solution (250 ␮L) of crude product was added
107 ␮L of acetone. A plug of silica gel (150 mg, 0.3 cm3) over a bed
of glass wool and sand in a disposable 1 mL syringe body fitted
with a 0.2 ␮m PTFE filtration membrane was thoroughly washed
with 30:70 acetone/hexane and used to remove origin material.
The solution of crude product in 30:70 acetone/hexane was rapidly
forced through the column and immediately washed with an addi-
tional 400 ␮L portion of 30:70 acetone/hexane. The column elution
and wash fractions were combined to give a quantitative yield of
2-O-arachidonoyl-1-O-stearoyl-sn-glycerol (DG) that was free of
starting phosphatidylcholine (SAPC). There was no evidence of the
rearrangement 1,3-diacyl-sn-glycerol byproduct that would have
had a slightly higher Rf than that for the 1,2-diacyl-sn-glycerol (Rf
0.50, 30:70 acetone/hexane) (Gaffney and Reese, 1997; Hindenes et
al., 2000), and the stability of the 2-O-arachidonoyl-1-O-stearoyl-
sn-glycerol was demonstrated by 2D TLC. The product was stored in
the acetone/hexane solution under an argon atmosphere at −20 ◦C
when not in use: 1H NMR (CD3OD) ı 5.25–5.40 (m, 8H, 5ꢀꢀ, 6ꢀꢀ, 8ꢀꢀ, 9ꢀꢀ,
11ꢀꢀ, 12ꢀꢀ, 14ꢀꢀ, 15ꢀꢀ), 5.00–5.10 (m, 1H, 2), 4.34 (dd, 1H, J = 12.0, 3.4 Hz,
1a), 4.10 (dd, 1H, J = 12.0, 6.7 Hz, 1b), 3.59–3.65 (m, 2H, 3), 2.78–2.85
(m, 6H, 7ꢀꢀ, 10ꢀꢀ, 13ꢀꢀ), 2.33 and 2.27 (two t, 4H, J = 7.4 Hz, 2ꢀ and
2ꢀꢀ), 2.11 (dt, 2H, J = 6.9, 6.9 Hz, 4ꢀꢀ), 2.04 (dt, 2H, J = 7.0, 7.0 Hz, 16ꢀꢀ),
1.55–1.86 (m, 4H, 3ꢀ and 3ꢀꢀ), 1.25–1.37 (m, 34H, 4–17ꢀ and 17–19ꢀꢀ),
0.87 and 0.86 (two t, 6H, J = 6.8 Hz, 18ꢀ and 20ꢀꢀ); 13C NMR ı 173.79
and 173.16 (1ꢀ and 1ꢀꢀ), 130.54, 129.06, 128.79, 128.65, 128.34,
128.12, 127.86, 127.56 (olefinics), 72.25, 61.98, 61.57 (glycerol),
34.12 and 33.66 (2ꢀ and 2ꢀꢀ), 31.95 (16ꢀ), 31.54 (18ꢀꢀ), 29.15–29.72
(4–15ꢀ and 17ꢀꢀ), 27.25 (16ꢀꢀ), 26.51 (4ꢀꢀ), 25.67, 25.65, and 25.64 (7ꢀꢀ,
ture was washed with 5.6 mL of 0.25
N HCl. The aqueous
extract was backwashed twice with CHCl3 and the backwashes
were combined with the organic phase, dried, and the solvent
removed. Column chromatography with argon-degassed solvent
mixtures first eluted byproducts with a gradient of 60:30:0
to 60:30:3 CHCl3/MeOH/H2O. The product SAPC-13C4 11 sub-
sequently eluted and the solvent was removed. TLC (25:15:4:2
CHCl3/MeOH/HOAc/H2O) showed 4-pyrrolidinopryidine (Rf 0.56)
to be the only impurity present with the product (Rf 0.31). The
SAPC-13C4 11 was dissolved in 30 mL of 90:10 CHCl3/MeOH,
washed with 5 mL of 0.25 M HCl, the phases separated using cen-
trifugation, the organic phase dried, and the solvent removed.
The product was re-chromatographed isocratically with argon-
degassed 60:30:3 CHCl3/MeOH/H2O, then dissolved in 90:10
CHCl3/MeOH, filtered through a 0.5 ␮m pore PTFE membrane, the
solvent was removed, and then lyophilization from t-butanol gave
0.334 g (0.402 mmol, 84%) of an off-white solid that was homo-
geneous by TLC (Rf 0.34, 25:15:4:2 CHCl3/MeOH/HOAc/H2O): mp
62–64 ◦C; 1H NMR (2:1, CDCl3/CD3OD) ı 5.30–5.45 (m, 8H, 5ꢀꢀ, 6ꢀꢀ,
8ꢀꢀ, 9ꢀꢀ, 11ꢀꢀ, 12ꢀꢀ, 14ꢀꢀ, 15ꢀꢀ), 5.24 (m; and, br d, JCH = 144 Hz, 1H, 2),
1
4.42 (br d, 2J1a,1b = 15 Hz; and, br d, 1JCH = 145 Hz, 1H, 1a), 4.25 (m,

108
R.I. Duclos Jr. / Chemistry and Physics of Lipids 163 (2010) 102–109
10ꢀꢀ, 13ꢀꢀ), 24.91 and 24.78 (3ꢀ and 3ꢀꢀ), 22.71 and 22.59 (17ꢀ and 19ꢀꢀ),
14.12 and 14.08 (18ꢀ and 20ꢀꢀ).
Kodali, D.R., Tercyak, A., Fahey, D.A., Small, D.M., 1990. Acyl migration in 1,2-
dipalmitoyl-sn-glycerol. Chem. Phys. Lipids 52, 163–170.
Kodali, D.R., Duclos Jr., R.I., 1992. Debenzylation and detritylation by bro-
modimethylborane: synthesis of mono-acid or mixed-acid 1,2- or 2,3-diacyl-
sn-glycerols. Chem. Phys. Lipids 61, 169–173.
Lammers, J.G., Liefkens, T.J., Bus, J., van der Meer, J., 1978. Syntheses and
spectroscopic properties of ␣- and ␤-phosphatidylcholines and phos-
phatidylethanolamines. Chem. Phys. Lipids 22, 293–305.
Lin, S., Duclos Jr., R.I., Makriyannis, A., 1997. Syntheses of 1,2-di-O-palmitoyl-sn-
glycero-3-phosphocholine (DPPC) and analogs with 13C- and 2H-labeled choline
head groups. Chem. Phys. Lipids 86, 171–181.
Acknowledgements
This work was supported by the National Institutes of Health
Research Grants HL-26335 (PD: D. Atkinson) and DA-24842 (PD:
R. Duclos). I am grateful to Donald M. Small, G. Graham Shipley,
Alexandros Makriyannis, Jianxin Guo, and Xiaoyu Tian for their
interest in this work and their helpful discussions. I also grate-
fully acknowledge Jan-Ove Hindenes for assistance with several
chromatographies, Mike Gigliotti for gas chromatographic analyses
of derivatized commercial fatty acids, and Jon Vural for assistance
with the NMR instrument.
Lin, S., Duclos Jr., R.I., Makriyannis, A., 2001. Preparation of optically pure 4-
(hydroxymethyl)-2-pentadecyl-1,3-dioxolanes and their corresponding phos-
phodiester ether lipid derivatives. Chem. Phys. Lipids 113, 111–122.
London, R.E., Walker, T.E., Wilson, D.M., Matwiyoff, N.A., 1979. Application of doubly
1
decoupled 13C { H, 14N}NMR spectroscopy to the studies of the conformation
and dynamics of the choline headgroup of phospholipids. Chem. Phys. Lipids 25,
7–14.
Majerus, P.W., Prescott, S.M., 1982. Characterization and assay of diacylglycerol
lipase from human platelets. Methods Enzymol. 86, 11–17.
Makriyannis, A., Siminovitch, D.J., Das Gupta, S.K., Griffin, R.G., 1986. Studies on the
interaction of anesthetic steroids with phosphatidylcholine using 2H and 13C
solid state NMR. Biochim. Biophys. Acta 859, 49–55.
References
Makriyannis, A., Banijamali, A., Jarrell, H.C., Yang, D.-P., 1989. The orientation of
(−)-ꢀ9-tetrahydrocannabinol in DPPC bilayers as determined from solid-state
2H-NMR. Biochem. Biophys. Acta 986, 141–145.
Makriyannis, A., Mavromoustakos, T., 1989. Studies on drug-membrane interac-
tions using solid state NMR, small angle X-ray diffraction and differential
scanning calorimetry. Rev. Clin. Pharmacol. Pharmacokinetics, Intl. Ed. 3, 95–
114.
Makriyannis, A., Yang, D.-P., Griffin, R.G., Das Gupta, S.K., 1990. The perturbation of
model membranes by (−)-ꢀ9-tetrahydrocannabinol. Studies using solid-state
2H- and 13C-NMR. Biochim. Biophys. Acta 1028, 31–42.
Mason, J.T., Broccoli, A.V., Huang, C., 1981. A method for the synthesis of isomeri-
cally pure saturated mixed-chain phosphatidylcholines. Anal. Biochem. 113, 96–
101.
Mattson, F.H., Volpenhein, R.A., 1962. Synthesis and properties of glycerides. J. Lipid
Res. 3, 281–296.
Mavis, R.D., Bell, R.M., Vagelos, P.R., 1972. Effect of phospholipase C hydroly-
sis of membrane phospholipids on membranous enzymes. J. Biol. Chem. 247,
2835–2841.
Nicolaou, A., Kokotos, G. (Eds.), 2004. Bioactive Lipids. The Oily Press, Bridgwater,
UK.
Paltauf, F., Hermetter, A., 1994. Strategies for the synthesis of glycerophospholipids.
Prog. Lipid Res. 33, 239–328.
Pasquaré de Garcia, S.J., Giusto, N.M., 1986. Phosphatidate phosphatase activity in
isolated rod outer segment from bovine retina. Biochim. Biophys. Acta 875,
195–202.
Pasquaré, S.J., Giusto, N.M., 1993. Differential properties of phosphatidate phospho-
hydrolase and diacylglyceride lipase activities in retinal subcellular fractions
and rod outer segments. Comp. Biochem. Physiol. B 104, 141–148.
Pasquaré, S.J., Salvador, G.A., Giusto, N.M., 2006. Age-associated changes of insulin
action on the hydrolysis of diacylglycerol generated from phosphatidic acid.
Comp. Biochem. Physiol. B 144, 311–318.
Patel, K.M., Morrisett, J.D., Sparrow, J.T., 1979. A convenient synthesis of phos-
phatidylcholines: acylation of sn-glycero-3-phosphocholine with fatty acid
anhydride and 4-pyrrolidinopyridine. J. Lipid Res. 20, 674–677.
Perly, B., Dufourc, E.J., Jarrell, H.C., 1984. Facile and high yielding syntheses of phos-
phatidylcholines and phosphatidylethanolamines containing 2H-labeled acyl
chains. J. Labelled Compd. Radiopharm. 21, 1–13.
Plückthun, A., Dennis, E.A., 1982. Acyl and phosphoryl migration in lysophospho-
lipids: importance in phospholipid synthesis and phospholipase specificity.
Biochemistry 21, 1743–1750.
Pollack, J.D., Clark, D.S., Somerson, N.L., 1971. Four-directional-development thin-
layer chromatography of lipids using trimethyl borate. J. Lipid Res. 12, 563–
569.
Rajamoorthi, K., Petrache, H.I., McIntosh, T.J., Brown, M.F., 2005. Packing and vis-
coelasticity of polyunsaturated ␻-3 and ␻-6 lipid bilayers as seen by 2H NMR
and X-ray diffraction. J. Am. Chem. Soc. 127, 1576–1588.
Roux, M., Huynh-Dinh, T., Igolen, J., Prigent, Y., 1983. Simple preparation of 1,2-
dipalmitoyl-sn-glycero-3-phosphoric acid and deuterated choline derivatives.
Chem. Phys. Lipids 33, 41–45.
Ruocco, M.J., Makriyannis, A., Siminovitch, D.J., Griffin, R.G., 1985. Deuterium NMR
investigation of ether- and ester-linked phosphatidylcholine bilayers. Biochem-
istry 24, 4844–4851.
Ahn, K., McKinney, M.K., Cravatt, B.F., 2008. Enzymatic pathways that regulate endo-
cannabinoid signaling in the nervous system. Chem. Rev. 108, 1687–1707.
Baer, E., Buchnea, D., 1959. Synthesis of saturated and unsaturated
l-␣-lecithins. Acylation of the cadmium chloride compound of l-␣-
glycerylphosphorylcholine. Can. J. Biochem. Physiol. 37, 953–959.
Barlow, P.N., Vidal, J.-C., Lister, M.D., Hancock, A.J., Sigler, P.B., 1988. Synthesis and
some properties of constrained short-chain phosphatidylcholine analogues:
(+)- and (−)-(1,3/2)-1-O-(phosphocholine)-2,3-O-dihexanoylcyclopentane-
1,2,3-triol. Chem. Phys. Lipids 46, 157–164.
Bergelson, L.D., 1980. Lipid Biochemical Preparations. Elsevier, New York.
Bitman, J., Wood, D.L., 1982. An improved copper reagent for quantitative densito-
metric thin-layer chromatography of lipids. J. Liquid Chromatogr. 5, 1155–1162.
Blume, A., Wittebort, R.J., Das Gupta, S.K., Griffin, R.G., 1982. Phase equi-
libria, molecular conformation, and dynamics in phosphatidylcholine/
phosphatidylethanolamine bilayers. Biochemistry 21, 6243–6253.
Buchnea, D., 1974. Detritylation by silicic acid boric acid column chromatography.
Lipids 9, 55–57.
Burt, C.T. (Ed.), 1987. Phosphorus NMR in Biology. CRC Press, Boca Raton.
Davis, J.H., 1979. Deuterium magnetic resonance study of the gel and liquid crys-
talline phases of dipalmitoyl phosphatidylcholine. Biophys. J. 27, 339–358.
Deihl, B.W.K., 2002. 31P-NMR in phospholipid analysis. Lipid Technol. 14, 62–65.
Dennis, E.A., 1983. Phospholipases. In: Boyer, P.D. (Ed.), The Enzymes. Academic
Press, New York, pp. 308–357.
Dennis, E.A., 1994. Diversity of group types, regulation, and function of phospholi-
pase A2. J. Biol. Chem. 269, 13057–13060.
Dittmer, J.C., Lester, R.L., 1964. A simple, specific spray for the detection of phospho-
lipids on thin-layer chromatograms. J. Lipid Res. 5, 126–127.
Duclos Jr., R.I., Makriyannis, A., 1992. Syntheses of all four stereoisomers which are
conformationally constrained 1,4-dioxanyl analogs of the antineoplastic ether
lipid ET-18-OCH3. J. Org. Chem. 57, 6156–6163.
Duclos Jr., R.I., 1993. Synthesis of 1-palmitoyl-2-hexadecyl-sn-glycero-3-
phosphocholine (PHPC). Chem. Phys. Lipids 66, 161–170.
Duclos Jr., R.I., Chia, H.H., Abdelmageed, O.H., Esber, H., Fournier, D.J., Makriyannis, A.,
1994. Syntheses of racemic and nearly optically pure ether lipids and evaluation
of in vitro antineoplastic activities. J. Med. Chem. 37, 4147–4154.
Duclos Jr., R.I., Gatley, S.J., Bhatt, S.R., Johnston, M., 2009. The preparation of 2-O-[1ꢀ-
14C]arachidonoyl-1-O-stearoyl-sn-glycerol. J. Labelled Compd. Radiopharm. 52,
324–326.
Eichberg, J., Zhu, X., 1992. Diacylglycerol composition and metabolism in peripheral
nerve. Adv. Exp. Med. Biol. 318, 413–425.
Gaffney, P.R.J., Reese, C.B., 1997. Preparation of 2-O-arachidonoyl-1-O-stearoyl-
sn-glycerol and other di-O-acyl glycerol derivatives. Tetrahedron Lett. 38,
2539–2542.
Gorenstein, D. (Ed.), 1984. Phosphorus-31 NMR. Academic Press, New York.
Gunstone, F.D., http://www.lipidlibrary.co.uk.
Gupta, C.M., Radhakrishnan, R., Khorana, H.G., 1977. Glycerophospholipid synthesis:
improved general method and new analogs containing photoactivable groups.
Proc. Natl. Acad. Sci. U.S.A. 74, 4315–4319.
Hindenes, J.-O., Nerdal, W., Guo, W., Di, L., Small, D.M., Holmsen, H., 2000.
Physical properties of the transmembrane signal molecule, sn-1-stearoyl 2-
arachidonoylglycerol. Acyl chain segregation and its biochemical implications.
J. Biol. Chem. 275, 6857–6867.
Ryu, E.K., MacCoss, M., 1979. Modification of the Dittmer–Lester reagent for the
detection of phospholipid derivatives on thin-layer chromatograms. J. Lipid Res.
20, 561–563.
Schmidt, C.F., Barenholz, Y., Huang, C., Thompson, T.E., 1977. Phosphatidyl-
choline 13C-labeled carbonyls as a probe of bilayer structure. Biochemistry 16,
3948–3954.
Seelig, J., 1977. Deuterium magnetic resonance: theory and application to lipid mem-
branes. Quart. Rev. Biophys. 10, 353–418.
Selinger, Z., Lapidot, Y., 1966. Synthesis of fatty acid anhydrides by reaction with
dicyclohexylcarbodiimide. J. Lipid Res. 7, 174–175.
Holzgrabe, U., Wawer, I., Diehl, B. (Eds.), 1999. NMR Spectroscopy in Drug Develop-
ment and Analysis. Wiley-VCH, New York.
Huang, T.-H., Lee, C.W.B., Das Gupta, S.K., Blume, A., Griffin, R.G., 1993. A 13C and 2
H
nuclear magnetic resonance study of phosphatidylcholine/cholesterol interac-
tions: characterization of liquid–gel phases. Biochemistry 32, 13277–13287.
Jung, K.-M., Astarita, G., Zhu, C., Wallace, M., Mackie, K., Piomelli, D., 2007. A key
role for diacylglycerol lipase-␣ in metabotropic glutamate receptor-dependent
endocannabinoid mobilization. Mol. Pharmacol. 72, 612–621.
Kingsley, P.B., Feigenson, G.W., 1979. The synthesis of a perdeuterated phospho-
lipid: 1,2-dimyristoyl-sn-glycero-3-phosphocholine-d72. Chem. Phys. Lipids 24,
135–147.
Seydel, J.K., Wiese, M., 2002. Drug–membrane Interactions. Wiley-VCH, Weinheim.

R.I. Duclos Jr. / Chemistry and Physics of Lipids 163 (2010) 102–109
109
Singer, S., Sivaraja, M., Souza, K., Millis, K., Corson, J.M., 1996. 1H-NMR detectable
fatty acyl chain unsaturation in excised leiomyosarcoma correlate with grade
and mitotic activity. J. Clin. Invest. 98, 244–250.
Tian, X., Guo, J., Yao, F., Yang, D.P., Makriyannis, A., 2005. The conformation, loca-
tion, and dynamic properties of the endocannabinoid ligand anandamide in a
membrane bilayer. J. Biol. Chem. 280, 29788–29795.
Stella, N., Schweitzer, P., Piomelli, D., 1997. A second endogenous cannabinoid that
modulates long-term potentiation. Nature 388, 773–778.
Stockton, G.W., Polnaszek, C.F., Leitch, L.C., Tulloch, A.P., Smith, I.C., 1974. A study
of mobility and order in model membranes using 2H NMR relaxation rates and
quadrupole splittings of specifically deuterated lipids. Biochem. Biophys. Res.
Commun. 60, 844–850.
Toke, O., Maloy, W.L., Kim, S.J., Blazyk, J., Schaefer, J., 2004. Secondary structure and
lipid contact of a peptide antibiotic in phospholipid bilayers by REDOR. Biophys.
J. 87, 662–674.
Wittebort, R.J., Schmidt, C.F., Griffin, R.G., 1981. Solid-state carbon-13 nuclear
magnetic resonance of the lecithin gel to liquid-crystalline phase transition.
Biochemistry 20, 4223–4228.
Su, Y., Doherty, T., Waring, A.J., Ruchala, P., Hong, M., 2009. Roles of arginine and
lysine residues in the translocation of a cell-penetrating peptide from 13C, 31P,
and 19F solid-state NMR. Biochemistry 48, 4587–4595.
Sugiura, T., Kishimoto, S., Oka, S., Gokoh, M., 2006. Biochemistry, pharmacology
and physiology of 2-arachidonoylglycerol, an endogenous cannabinoid receptor
ligand. Prog. Lipid Res. 45, 405–446.
Tang, M., Waring, A.J., Hong, M., 2007. Phosphate-mediated arginine insertion into
lipid membranes and pore formation by a cationic membrane peptide from
solid-state NMR. J. Am. Chem. Soc. 129, 11438–11446.
Wittebort, R.J., Blume, A., Huang, T.-H., Das Gupta, S.K., Griffin, R.G., 1982. Carbon-13
nuclear magnetic resonance investigations of phase transitions and phase equi-
libria in pure and mixed phospholipid bilayers. Biochemistry 21, 3487–3502.
Yang, D.-P., Mavromoustakos, T., Beshah, K., Makriyannis, A., 1992. Amphipathic
interactions of cannabinoids with membranes. A comparison between ꢀ8-THC
and its O-methyl analog using differential scanning calorimetry, X-ray diffrac-
tion and solid state 2H-NMR. Biochim. Biophys. Acta 1103, 25–36.
Yeagle, P.L., 1978. Phospholipid headgroup behavior in biological assemblies. Acc.
Chem. Res. 11, 321–327.
Thomas III, A.E., Scharoun, J.E., Ralston, H., 1965. Quantitative estimation of iso-
meric monoglycerides by thin-layer chromatography. J. Am. Oil Chem. Soc. 42,
789–792.
Zwaal, R.F., Roelofsen, B., Comfurius, P., van Deenen, L.L., 1971. Complete purification
and some properties of phospholipase C from Bacillus cereus. Biochim. Biophys.
Acta 233, 474–479.