Total synthesis and mass spectrometric analysis of a: Mycobacterium tuberculosis phosphatidylglycerol featuring a two-step synthesis of (R)-tuberculostearic acid

Article abstract of DOI:10.1039/c7ob01786c

We report the total synthesis of (R)-tuberculostearic acid-containing Mycobacterium tuberculosis phosphatidylglycerol (PG). The approach features a two-step synthesis of (R)-tuberculostearic acid, involving an (S)-citronellyl bromide linchpin, and the pho

Full text of DOI:10.1039/c7ob01786c

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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DOI: 10.1039/C7OB01786C
Organic and Biomolecular Chemistry
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Total synthesis and mass spectrometric analysis of a
Mycobacterium tuberculosis phosphatidylglycerol featuring a two-
step synthesis of (R)-tuberculostearic acid
Received 00th January 20xx,
Accepted 00th January 20xx
Satvika Burugupalli, Mark B. Richardson, Spencer J. Williams*
DOI: 10.1039/x0xx00000x
We report the total synthesis of (R)-tuberculostearic acid-containing Mycobacterium tuberculosis phosphatidylglycerol
(PG). The approach features a two-step synthesis of (R)-tuberculostearic acid, involving an (S)-citronellyl bromide linchpin,
and the phosphoramidite-assisted assembly of the full PG structure. Collision-induced dissociation mass spectrometry of
two chemically-synthesized PG acyl regioisomers revealed diagnostic product ions formed by preferential loss of
carboxylate at the secondary (sn-2) position.
www.rsc.org/
phosphatidylethanolamine,10
and
phosphatidylinositol
mannosides.11 Possibly because of its role as a biosynthetic
intermediate, Mtb PG occurs at low levels and the challenges of
its isolation4 from the natural source are compounded by the
pathogenicity and low growth rate of Mtb. Recent work has
shown that Mtb PG can be presented by the CD1d antigen
presenting molecule to natural killer T cells that display ‘type
2’ T cell receptors.4 Type 2 NKT cells are distinguished from
their better studied type I variants, by lacking reactivity to the
Introduction
Phosphatidylglycerols are phospholipids produced by a wide
range of organisms including bacteria, fungi, plants and
animals.1 Within animal cells PG is mainly found in the
mitochondrial membranes,2 and is believed to be released into
the cellular milieu only upon cellular damage or stress. PGs are
also produced by a wide range of bacteria, including important
human pathogens such as Listeria
Mycobacterium tuberculosis Mtb). PGs can be presented on
, Streptococcus, and
prototypical NKT ligand
α-galactosyl ceramide, and through
(
possessing a diverse array of αβ chains that comprise the T cell
receptor.12 While most studies have focused on type I NKT
cells, type II NKT cells are more abundant in humans, and are
functionally distinct from type I NKT cells.13 However, studies
of type 2 NKT cells have been limited by a lack of well-defined
lipid antigens.
several antigen presenting proteins of the cluster of
differentiation 1 (CD1) class, specifically CD1b3 and CD1d,4, 5
where they are recognized by natural killer T cells and lipid-
reactive T cells. Thus PGs appear to be a molecular signature
allowing immune recognition of bacterial infection or damaged
self.
In mycobacteria, PG is an intermediate in the biosynthesis
of lysinylated PG,6 phosphatidylinositol,7 and cardiolipin.7, 8 It
has been proposed that PG is synthesized in two steps. Step 1 is
Herein we report the total synthesis of the TBSA-containing
Mtb PG (
effort we have devised the shortest synthesis yet reported of
TBSA ( ) in just two steps from commercially available (S)-
1) using phosphoramidite chemistry. As part of this
R
-
2
catalyzed
(PgsA3), which catalyzes the phosphatidylation of glycerol-3-
phosphate by CDP-diacylglycerol to afford
by
phosphatidylglycerophosphate
synthetase
citronellyl bromide, and its incorporation into Mtb PG. Finally,
we demonstrate that collision-induced dissociation mass
spectrometry of chemically-synthesized PG regioisomers
results in the formation of diagnostic fragment ions that have
utility in the structural characterization of PGs.
phosphatidylglycerophosphate;7 and step 2 which involves the
dephosphorylation of phosphatidylglycerophosphate to afford
PG, by a phosphatidylglycerophosphatase. Mtb PG occurs as a
range of lipoforms including an abundant species that bears
(R)-tuberculostearic acid (R-TBSA) at the sn-1 position, and
palmitic acid at the sn-2 position,4 a decoration that matches
that of other phosphatidyl-containing lipids9 such as
School of Chemistry and Bio21 Molecular Science and Biotechnology Institute,
University of Melbourne, Parkville, Victoria, Australia 3010.
E-mail: sjwill@unimelb.edu.auAddress here.
Fig
1 Structure of Mycobacterium tuberculosis (Mtb) TBSA-containing
phosphatidylglycerol (PG).
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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DOI: 10.1039/C7OB01786C
Organic and Biomolecular Chemistry
ARTICLE
Table 1. Previous synthetic approaches to (R)-tuberculostearic acid.
Starting material/key
intermediate
Approach/steps
Ref.
Starting material/key
intermediate
Approach/steps
Ref.
Chiral resolution
● 10 stepsa
Chiral resolution
● 7 steps
Prout, 194814
Hahn, 201515
Catalytic
enantioselective
induction
Chiral resolution
● 9 steps
Feringa,
Minnaard, 201010
Linstead, 195116
Larsen, 200717
● 6 stepsb
Catalytic
Chiral pool
● 8 steps
enantioselective
induction
Minnaard, 201318
● 5 steps
Chiral pool
● 7 steps
Chiral pool
● 5 steps
Seeberger, 200619
Williams, 201321
Hung, 201520
Baird, 200622
Chiral auxiliary
● 7 steps
Chiral pool
● 4 steps
a (R)-2-Methyldecanol was synthesized from bromooctane in 5 steps by resolution of an intermediate brucine salt.
b Isopropyl 10-undecenoate can be synthesized in one step from commercially-available undecylenic acid.
mixture of cross-coupled alkenes. We next attempted a direct
tandem cross-metathesis/hydrogenation reaction. Use of a 1:1
Results and discussion
mixture of alkenes afforded a 48% yield of ethyl
which could be improved to 76% by using a 3:1 ratio of ethyl
heptenoate/ . Saponification of ethyl -TBSA afforded
TBSA ( ) in three steps. Encouraged by these results, we next
enacted a two-step synthesis of -TBSA by employing a 3:1
ratio of 6-heptenoic acid/ under the same conditions, which
directly afforded -TBSA in 73% yield.
R-TBSA,
Synthesis of (
R
)-tuberculostearic acid
3
R
R-
2
Numerous syntheses of
R
-TBSA have been reported, using
19, 20
chiral pool sources,17,
chiral auxiliaries,21 catalytic
and resolution methods14-16
R
enantioselective induction,10,
18
3
R
(Table 1). The most efficient synthesis reported to date is that
of Roberts and Baird who prepared TBSA in four steps from
(
S
)-citronellyl bromide, in
a
route involving stepwise
elongations using copper(I)-catalyzed cross-coupling and
Wittig extension.22 We were inspired by this use of citronellyl
bromide as a linchpin, and anticipated that diverting the cross-
coupled
intermediate
into
a
Grubbs-cross-
metathesis/hydrogenation process could provide further
synthetic abbreviation. Thus, according to the reported
method,22
heptylmagnesium bromide with
afforded the alkene (Scheme 1). Initially, a 1:1 mixture of
ethyl 6-heptenoate and were reacted in the presence of
copper(I)-catalyzed
cross-coupling
S)-citronellyl bromide
of
(
3
Scheme 1 Two-step synthesis of (R)-tuberculostearic acid.
3
Hoveyda-Grubbs II catalyst, affording a 58% yield of an
E/Z
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Synthesis of phosphatidylglycerols
ARTICLE
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DOI: 10.1039/C7OB01786C
Reported methods for the synthesis of PGs include
chemoenzymatic methods,23 and purely chemical approaches;24
however, the reported chemical methods typically employ
highly moisture sensitive POCl3 as a phosphorylation reagent
and do not allow regioselective introduction of two different
acyl groups. In devising an approach to Mtb PG, we elected to
utilize contemporary phosphoramidite chemistry, which has
been widely applied in the synthesis of oligonucleotides,25
phosphatidylinositols,26 and cardiolipin.27
We chose to prepare 1, and analogues lacking the 10-methyl
group with alternate arrangements of the fatty acyl groups. For
the construction of the requisite diacylglycerols, we chose to
employ the approach of Minnaard, which proceeds with high
regioselectivity and without acyl group migration.18
Accordingly, opening of benzyl (
palmitic acid, stearic acid, or -TBSA using Jacobsen pre-
catalyst C1 28
followed by esterification with a second fatty
R)-glycidyl ether (5) with
R
,
acid and DCC/DMAP, gave the diesters 6a-c. Hydrogenolysis
of the benzyl ether afforded the diacylglycerols 7a-c, which
were used immediately without purification in the next step.
Scheme 3 Preparation of Mtb PG 1 and analogues.
O
R1CO2H, C1
R2CO2H
R2
O
Collision-induced dissociation mass spectrometry of
phosphatidylglycerols
OH
O
O
R1
BnO
R1
BnO
BnO
O
iPr2NEt, THF
DCC/DMAP
heptane
5
O
O
R1CO2H R2CO2H
yield (%)
Early studies reported that negative-ion fast-atom
bombardment or electrospray-ionization with collision-induced
C16
C18
C18
C16
C16
66
64
77
6a
6b
6c
H
N
H
N
dissociation/tandem
mass
spectrometry
of
R-TBSA
Co
phosphatidylethanolamine (PE) and PG result in fragmentation
to provide high abundance fatty acid carboxylate (RCO2 )
fragment ions.30, 31 These studies revealed similar fragmentation
tBu
O
O
tBu
–
H2, Pd(OH)2-C
EtOH
tBu
tBu
C1
O
pathways for PE and PG, and showed that regioisomeric PEs
fragment in a characteristic way to produce RCO2 ions for
which those derived from the sn-2 position are of greater
7a (C16/C18), 98% (crude)
7b (C18/C16), 96% (crude)
7c (C16/TBSA), 99% (crude)
R2
–
O
HO
O
R1
O
intensity, thereby allowing structural characterization of fatty
Scheme 2 Preparation of diacylglycerols.
31
acyl regiochemistry.10,
Subsequently, it was shown that
CID/MS-MS of PG occurs with similar results, producing
RCO2 ions in which those from the sn-2 position are more
Assembly of the PG structure relied upon the use of a
phosphordiamidite linchpin, benzyl bis(
–
N,N-
abundant, in what has been described as a charge-driven
process.32, 33 In order to confirm these fragmentation patterns
we studied the chemically synthesized regioisomers 11a and
11b negative ion CID/MS2 experiments using electro-spray
diisopropyl)phosphordiamidite (Scheme 3).17, 29 Reaction of the
diacylglycerols 7a-c with this phosphordiamidite, catalyzed by
diisopropyl ammonium tetraazolide (DIPAT), a weak acid that
does not active the product,25 afforded the unstable
phosphoramidites 8a-c. Compounds 8a-c were purified by a
quick passage over silica gel to remove any residual reactant or
ionization. Fragmentation of the parent anions [M−H]−
(m/z
749.5) formed from the regioisomers 11a (C16/C18) and 11b
–
(C18/C16), yielded characteristic fatty acid carboxylate (RCO2 )
reagents, and then immediately subjected to
phosphitylation reaction with dibenzylglycerol , catalyzed by
-tetrazole. Upon consumption of the phosphoramidate, t-
a second
fragment ions at
m/z 255.2 and 283.3 (Figure 2a,b). In each
9
case the more abundant ion is that derived from the fatty acid
located in the sn-2 position. This work confirms that CID-
induced fragmentation patterns can be used to identify the
structures of asymmetrical PG molecules. Figure 2c shows the
1
H
BuOOH was added to oxidize the intermediate phosphite to the
protected PGs 10a-c. Finally, hydrogenolysis of the benzyl
groups provided Mtb PG 1, and analogues 11a and 11b.
CID-MS2 spectrum of
parent ion [M−H]−
255.2 and 297.3. Here, the most abundant fragment (
1
, revealing that fragmentation of the
763.5) affords daughter ions at
255.2)
(
m
/
z
m/z
m/z
is that derived from the palmitic acid located at the sn-2
position. We note that these and earlier observations for CID-
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ARTICLE
Organic Biomolecular Chemistry
MS of PGs differ from those for the structurally-related presented lipid antigen, other closely related PGs from
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phosphatidylinositols32 and phosphatidylinositolmannosides,11, mammals, Escherichia coli DOI: 10.1039/C7OB017a86nCd
, Corynebacterium glutamicum
17
which under similar conditions fragment by neutral loss of Listeria monocytogenes have been identified as CD1b-
fatty acids and fatty acid ketenes in a diagnostic way. While presented lipid antigens.3 Access to synthetic Mtb PG should
–
these systems also yield RCO2 fragment ions, their relative support studies to understand its immunological properties, and
intensities do not reflect the regiochemistry of their location on investigation of the effect of the branched-methyl group in the
the phosphatidyl group because other pathways also yield these fatty acyl chain. Collision-induced dissociation mass
ions in abundances that are sensitive to the applied collision spectrometry analysis confirms the desired acylation patterns in
energy.32
our synthetically acquired materials, and provides a foundation
for structural analyses of mycobacterial PGs.
Experimental
General methods
Proton (1H NMR, 400 or 500 MHz), proton-decoupled carbon-
13 (13C NMR, 100 or 125 MHz), and phosphorus-31 (31P NMR,
202 MHz) nuclear magnetic resonance spectra were obtained in
deuterochloroform, methanol-d4 (CD3OD) or DMSO-d6 with
residual protonated solvent or solvent carbon signals as internal
standards. Abbreviations for multiplicity are s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet. Flash
chromatography was carried out on silica gel 60 according to
the procedure of Still et al.34 Analytical thin layer
chromatography (t.l.c.) was conducted on aluminium-backed 2
mm thick silica gel 60 F254 and chromatograms were visualized
with ceric ammonium molybdate (Hanessian's stain), potassium
permanganate or 5% H2SO4/MeOH, with charring as necessary.
High resolution mass spectra (HRMS) were obtained using an
ESI-TOF-MS; all samples were run using 0.1% formic acid.
Dry CH2Cl2, THF, and Et2O were obtained from a dry solvent
apparatus (Glass Contour of SG Water, Nashua, U.S.A.) as per
the procedure of Pangborn et al.
35 Dry DMF was dried over 4 Å
molecular sieves. Pet. spirits refers to petroleum ether, boiling
range 40-60 °C.
1. Synthesis of (R)-tuberculostearic acid (2)
(R)-2,6-Dimethyltetradecene (4)
See: Roberts and Baird:22 Hexylmagnesium bromide (3.14 ml,
4.56 mmol) (2.0 M in Et2O) was added to a solution of (S)-
citronellyl bromide (0.23 ml, 1.14 mmol) in dry Et2O (2.3 ml)
ᵒ
at –78 C followed by dilithium tetrachlorocuprate (Li2CuCl4)
(0.1 M in THF) (1.94 ml, 0.12 mmol). The solution was
allowed to warm to room temperature and stirred for 36 h. The
reaction mixture was quenched with sat aq. NH4Cl, extracted
Fig 2 Multistage mass spectrometry (MS2) experiments involving the collision- with Et2O, and the organic phase washed with brine, dried
induced dissociation of negative ions formed by electro-spray ionization. (a)
MS2 spectrum of deprotonated 11a (C16/C18). (b) MS2 spectrum of deprotonated
11b (C18/C16). (c) MS2 spectrum of deprotonated
compound . See Experimental for details.
†
(MgSO4) and concentrated under reduced pressure. Flash
(d) Fragmentation of chromatography (0.5% Et2O:petroleum ether) of the residue
afforded 0.5
as a colorless oil (240 mg, 95%); [α]D22 +0.75 (
1.
1
4
c
in CHCl3) (lit.22 [α]D +1.5 in CHCl3); H NMR (400 MHz,
CDCl3) δ 0.88–0.83 (6 H, m, 2 × CH3), 1.15–1.06 (2 H, m,
CH2), 1.31–1.22 (20 H, m, 10 × CH2), 1.60 (3 H, s, CH3), 1.68
25
1
Conclusions
(3 H, s, CH3), 1.95 (2 H, dd,
J = 6.8, 13.7 Hz, CHCH2), 5.10 (1
In this work we disclose the first total synthesis of Mtb PG
, and analogues 11a and 11b. Our approach includes the most
concise preparation of R-TBSA reported to date. We note that
H, t,
J
= 7.0 Hz, CH=C); 13C NMR (101 MHz, CDCl3) δ 14.2,
1
17.7, 19.2, 22.8, 25.7, 27.1, 29.5, 29.8, 30.1, 32.0, 32.5, 37.1,
37.3, 125.2, 131.0; HRMS calcd for C16H32 [M+H]+ 225.2538,
found 225.2534.
while Mtb PG has only been studied in the context of a CD1d-
4 | Org. Biomol. Chem., 2017, 00, 1-7
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Organic Biomolecular Chemistry
ARTICLE
oxygen balloon for 10 min. The solvent was evaporated under
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vacuum to afford a brown residue. THFD(O0I.:210m.10l)39a/nCd7OHB0ü1n7i8g6’Cs
Ethyl (R)-10-methyloctadecanoate
Hoveyda-Grubbs 2nd generation catalyst (HG-II) (2.8 mg, 4
base (58 µl, 372 µmol) was added to the residue, and the
mixture was stirred for 5 min. (R)-Glycidyl benzyl ether (56.7
mol%) was added to a solution of
4 (250 mg, 0.11 mmol) and
µl, 372 µmol) was added and the resulting mixture was stirred
overnight at room temperature followed by removal of solvents
in vacuo. Heptane (800 µl), palmitic acid (114 mg, 446 µmol)
and DMAP (2.27 mg, 18.6 µmol, 5 mol%) were added to the
resulting residue. The mixture was cooled to 0 °C, followed by
addition of DCC (91.9 mg, 446 µmol) and stirred overnight.
The solvent was removed in vacuo and the residue was
subjected to flash chromatography (10% Et2O:pentane) to
afford the title compound as a colorless thick liquid (178 mg,
ethyl heptenoate (59 mg, 0.33 mmol) in dry dichloromethane
(1.14 ml) and stirred for 24 h under reflux. Upon consumption
of starting material, the reaction mixture was concentrated
under reduced pressure. EtOAc (5.0 ml) was added to the
residue and the solution was subjected to hydrogenation under
high pressure (37 bar) in a Buchi® hydrogenator overnight at
room temp after which the solvent was removed under reduced
pressure. Flash chromatography (2% Et2O/petroleum spirits) of
the crude mixture afforded the title compound as a colourless
25
23
77%); [α]D +6.2 (c = 0.5, CHCl3), [α]D lit.19 +5.45 (c = 3.9,
oil (28 mg, 76%); [α]22 –0.14 (
c
0.5 in CHCl3) (lit.14 [α]25
–
D
D
1
0.16 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.82 (3 H, d,
6.5 Hz, CHC 3), 0.87 (3 H, t,
H, m, CH2), 1.31–1.20 (28 H, m, 12 × CH2, 1 × CH,
COOCH2CH3), 1.61 (2 H, d, = 7.1 Hz, COCH2C 2), 2.27 (2
H, t, = 7.6 Hz, COCH2), 4.11 (2 H, d, = 7.1 Hz, C=OOCH2);
J
=
CHCl3); H NMR (400 MHz, CDCl3) δ 0.83 (3 H, d,
J
= 6.5
= 6.8 Hz, 2 × CH3), 1.07 (2
H, m, CH2 (TBSA)), 1.25 (48 H, m, 24 × CH2), 1.60 (4 H, m, 2
× COCH2C 2), 2.25–2.34 (4 H, m, 2 × COCH2), 3.59 (2 H, d,
= 5.2 Hz, H3a,3b), 4.19 (1 H, dd, = 11.9, 6.4 Hz, H1a), 4.34
(1 H, dd, = 11.9, 3.8 Hz, H1b), 4.50-4.58 (2 H, m, C 2Ph),
Hz, CCH3 (TBSA)), 0.88 (6 H, t,
J
H
J = 6.8 Hz, CH3), 1.10–1.03 (2
H
J
J
H
J
J
J
13C NMR (101 MHz, CDCl3) δ 14.2, 14.4, 19.8, 22.8, 25.1,
27.1, 27.2, 29.3, 29.4, 29.5, 29.6, 29.8, 30.0, 30.1, 32.0, 32.8,
32.8, 34.5, 37.2, 60.2, 174.0 (C=O); HRMS calcd for C21H42O2
[M+H]+ 327.3218, found 327.3220.
J
H
5.24 (1 H, m, H2), 7.27–7.37 (5 H, m, Ph); 13C NMR (125
MHz, CDCl3) δ 14.2, 19.8, 22.8, 25.0, 25.1, 27.2, 29.2, 29.2,
29.4, 29.5, 29.6, 29.6, 29.8, 30.1, 30.1, 32.0, 32.9, 34.2, 34.4,
37.2, 62.8, 68.4, 70.1, 73.4, 127.7, 127.9, 128.5, 137.8 (Ph),
173.2, 173.5 (C=O); HRMS: calcd for C45H80O5 [M + H]+
701.6039, found 701.6038.
(R)-Tuberculostearic acid ((R)-10-methyloctadecanoic acid) (2)
(a) From ethyl (R)-10-methyloctadecanoate: 2 M KOH (0.76
ml, 1.5 mmol) was added to a solution of the ethyl ester (50 mg,
0.153 mmol) in MeOH/THF (1:1, 0.8 ml) and stirred under
reflux overnight. The solution was acidified with aq citric acid
(1 M), extracted with EtOAc, and the organic phase washed
with brine, dried (MgSO4) and concentrated under reduced
3-O-Benzyl-1-O-palmitoyl-2-O-stearoyl-sn-glycerol (6a)
Synthesized as for 6c, 250 mg (0.36 mmol, 66%). [α]D25 +5.6 (c
1
= 0.5, CHCl3); H NMR (400 MHz, CDCl3) δ 0.88 (6 H, t,
J =
6.8 Hz, 2 × CH3), 1.27 (48 H, m, 24 × CH2), 1.60 (4 H, m, 2 ×
COCH2C 2), 2.25–2.34 (4 H, m, 2 × COCH2), 3.59 (2 H, d,
5.2 Hz, H3a,3b), 4.19 (1 H, dd, = 11.9, 6.4 Hz, H1a), 4.34 (1
H, dd, = 11.9, 3.8 Hz, H1b), 4.50–4.58 (2 H, m, C 2Ph), 5.24
pressure to afford
2
as a colorless oil (44 mg, 97%); [α]25D –0.9
H
J =
1
1.0 in CHCl3) (lit.22 [α]25 –0.34 in CHCl3); H NMR (400
J
(
c
D
J
H
MHz, CDCl3) δ 0.83 (3 H, d,
= 6.8 Hz, CH2C 3), 1.12–1.04 (2 H, m, CH2), 1.36–1.21 (25
H, m, 12 × CH2, 1 × CH), 1.67–1.59 (2 H, m, OCOCH2C 2),
2.35 (2 H, t,
= 7.5 Hz, OCOCH2); 13C NMR (101 MHz,
J = 6.5 Hz, CHCH3), 0.88 (3 H, t,
(1 H, m, H2), 7.28–7.37 (5 H, m, Ph); 13C NMR (101 MHz,
CDCl3) δ 14.2, 22.8, 25.0, 25.1, 29.2, 29.2, 29.4, 29.5, 29.6,
29.8, 29.8, 32.0, 34.2, 34.4, 62.8, 68.4, 70.1, 73.4, 127.7, 127.9,
128.5, 137.8, 173.2, 173.5; HRMS: calcd for C44H78O5 [M +
H]+ 687.5883, found 687.5884.
J
H
H
J
CDCl3) δ 14.2, 19.8, 22.8, 24.8, 27.1, 27.2, 29.2, 29.4, 29.5,
29.6, 29.8, 30.0, 30.1, 32.0, 32.8, 34.0, 37.2, 179.5; HRMS
calcd for C18H36O2 [M+H]+ 285.2749, found 285.2747.
3-O-Benzyl-1-O-stearoyl-2-O-palmitoyl-sn-glycerol (6b)
(b) From alkene 4: HG-II (2.80 mg, 4 mol%) was added to a
Synthesized as for 6c, 62 mg (0.093 mmol, 64%). [α]D25 +5.8 (c
solution of
4 (25 mg, 0.114 mmol) and heptenoic acid (59 mg,
= 0.5, CHCl3), lit.36 [α]D +5.4 (c = 1.63, CHCl3); H NMR
23
1
0.334 mmol) in dry CH2Cl2 (1.14 ml) and stirred for 24 h under
reflux. The reaction mixture was concentrated under reduced
pressure. EtOAc (5.0 ml) was added to the residue and the
solution was subjected to hydrogenation under high pressure
(37.5 bar) in a Buchi® hydrogenator overnight at room
temperature after which solvent was removed under reduced
pressure. Flash chromatography (2% Et2O/petroleum spirits) of
(400 MHz, CDCl3) δ 0.88 (6 H, t,
(48 H, m, 24 × CH2), 1.60 (4 H, m, 2 × COCH2C
(4 H, m, 2 × COCH2), 3.59 (2 H, d, = 5.2 Hz, H3a,3b), 4.19 (1
H, dd, = 11.9, 6.4 Hz, H1a), 4.34 (1 H, dd, = 11.9, 3.8 Hz,
H1b), 4.50-4.58 (2 H, m, C 2Ph), 5.24 (1 H, m, H2), 7.28–7.37
J
= 6.8 Hz, 2 × CH3), 1.27
H
2), 2.25–2.34
J
J
J
H
(5 H, m, Ph); 13C NMR (101 MHz, CDCl3) δ 14.2, 22.8, 25.0,
25.1, 29.2, 29.2, 29.4, 29.5, 29.6, 29.8, 29.8, 32.0, 34.2, 34.4,
62.8, 68.4, 70.1, 73.4, 127.7, 127.9, 128.5, 137.8 (Ph), 173.2,
173.5 (C=O); HRMS: calcd for C44H78O5 [M + H]+ 687.5883,
found 687.5884.
the crude mixture afforded
2 as a colorless oil (25 mg, 73%).
2. Synthesis of diacylglycerols
3-O-Benzyl-1-O-((R)-10-methyloctadecanoyl)-2-O-palmitoyl-sn-
glycerol (6c)
1-O-((R)-10-Methyloctadecanoyl)-2-O-palmitoyl-sn-glycerol (7c)
A solution of (R)-TBSA 2 (100 mg, 335 µmol) and Co[salen]
C1 (4.0 mg, 2 mol%) in Et2O (1 ml) was stirred under an
A mixture of 6c (65 mg, 0.089 mmol) and Pd(OH)2 on carbon
(20%, 40 mg, 0.285 mmol) in ethanol (3 ml) was stirred under
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem., 2017, 00, 1-7 | 5
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ARTICLE
Organic Biomolecular Chemistry
hydrogen at 10 bar for 2 h. The reaction mixture was filtered
through Celite and the solvent removed in vacuo to afford the phosphoramidite (42.0 mg, 0.049 mmol) and 2,3-di-
product as a colorless liquid (61 mg, 99%). The compound was sn-glycerol
38 (10.7 mg, 0.0392 mmol) in dry CH2Cl2 (2 ml) at
used directly without delay or purification. 1H NMR (400 MHz, 0 °C under nitrogen. The reaction mixture was stirred for 15
CDCl3) δ 0.83 (3 H, d, = 6.5 Hz, CC 3 (TBSA)), 0.88 (6 H, t, min at 0 °C and 2 hours at room temperature. The reaction
= 6.8 Hz, 2 × CH3), 1.08 (2 H, m, CH2 (TBSA)), 1.27 (48 H, mixture was cooled to 0 °C and -BuOOH (5 M in decane) (5
m, 24 × CH2), 1.62 (4 H, m, 2 × COCH2C 2), 2.30–2.37 (4 H, µl, 0.0582 mmol) was added and stirred for 2 h warming to
m, 2 × COCH2), 3.73 (2 H, d, = 5.2 Hz, H3a,3b), 4.24 (1 H, room temperature. Excess oxidant was quenched with sodium
dd, = 11.9, 5.6 Hz, H1a), 4.32 (1 H, dd,
H1b), 5.06–5.11 (1 H, m, H2).
1
H
-Tetrazole (4.8 mg, 0.068 mmol) was added toVaiewsoArltuictleioOnnlionef
DOI: 10.1039/CO7O-Bb0e1n7z8y6lC-
9
J
H
J
t
H
J
J
J
= 11.9, 4.6 Hz, metabisulfite (1M, 0.1 ml), the reaction mixture was diluted
with CH2Cl2, extracted, dried (MgSO4), filtered and
concentrated. The residue was purified by column
chromatography on silica gel (35% EtOAc/ petroleum ether,
1-O-Palmitoyl-2-O-stearoyl-sn-glycerol (7a)
1
0.5% Et3N) to afford the title compound as an oil (25 mg,
Synthesized as for 7c, 30.4 mg (98%). H NMR (500 MHz,
CDCl3) δ 0.88 (6 H, t, = 7.0 Hz, 2 × CH3), 1.27–1.34 (48 H,
m, 24 × CH2), 1.62 (4 H, m, 2 × COCH2C 2), 2.30–2.37 (4 H,
m, 2 × COCH2), 3.73 (2 H, dd, = 5.0, 1.4 Hz, H3a,3b), 4.23 (1
H, dd, = 11.9, 5.7 Hz, H1a), 4.32 (1 H, dd, = 11.9, 4.5 Hz,
24
1
55%); [α]D 6.3 (
c
0.25, CHCl3); H NMR (600 MHz, CDCl3)
3 (TBSA)), 0.88 (6 H, t, = 6.9
J
δ 0.83 (3 H, d, = 6.6 Hz, CC
J
H
J
H
Hz, 2 × CH3 (TBSA, palmitoyl)), 1.06–1.07 (2 H, m, CH2),
1.26 (49 H, m, 24 × CH2, 1 × CH), 1.57 (4 H, m, 2 ×
J
J
J
COCH2C
Hz, H3'a,3'b), 3.77 (1 H, m, H2'), 4.03–4.16 (4 H, m,
H3a,3b,1'a,1'b), 4.23 (2 H, m, H1a,1b), 4.51 (2 H, d, = 4.8 Hz,
2Ph), 5.02-5.05 (2 H, m,
2Ph), 5.16 (1 H, m, H2'), 7.32 (15 H, m, Ph); 13C NMR (126
MHz, CDCl3) δ 14.2, 19.8, 22.8, 24.9, 27.2, 29.2, 29.2, 29.4,
29.5, 29.6, 29.7, 29.8, 30.1, 30.2, 32.0, 32.9, 34.1, 34.2, 37.2,
61.8, 65.5, 67.3, 67.3, 69.1, 69.4, 69.4, 69.5, 69.6, 72.3, 73.6,
127.7, 127.8, 127.9, 128.0, 128.5, 128.5, 128.7 (Ph), 172.9,
173.3 (C=O); 31P NMR (202 MHz, CDCl3) δ –1.01, –0.98;
HRMS: [M + H]+ calcd for C62H99O10P, 1035.7009; found
1035.7006.
H2), 2.26 (4 H, m, 2 × COCH2), 3.56 (2 H, t, J = 5.6
H1b), 5.06–5.11 (1 H, m, H2).
1-O-Stearoyl-2-O-palmitoyl-sn-glycerol (7b)
J
1
C
C
H
H
2Ph), 4.60–4.67 (2 H, m, CH
Synthesized as for 7c, 40.2 mg (96%). H NMR (500 MHz,
CDCl3) δ 0.88 (6 H, t, = 7.0 Hz, 2 × CH3), 1.27–1.34 (48 H,
m, 24 × CH2), 1.62 (4 H, m, 2 × COCH2C 2), 2.30–2.37 (4 H,
m, 2 × COCH2), 3.73 (2 H, dd, = 5.0, 1.4 Hz, H3a,3b), 4.23 (1
H, dd, = 11.9, 5.7 Hz, H1a), 4.32 (1 H, dd, = 11.9, 4.5 Hz,
J
H
J
J
J
H1b), 5.06–5.11 (1 H, m, H2).
3. Synthesis of phosphatidylglycerols
O-Benzyl-O-(1-O-((R)-10-methyloctadecanoyl)-2-O-palmitoyl-sn-
glycer-3-yl) N,N-diisopropylphosphoramidite (8c)
Triethylammonium O-(1-O-(R)-10-methyloctadecanoyl-2-O-
palmitoyl-sn-glyceryl)-O-(sn-glycer-1-yl) phosphate (1)
Diisopropylammonium tetrazolide (34.1 mg, 0.199 mmol) was
added to a solution of alcohol 7c (58.0 mg, 0.094 mmol) and
benzyl bis(N,N
-diisopropyl)phosphoramidite37 (62.3 mg, 0.189
mmol) in dry CH2Cl2 at 0 °C under nitrogen. The reaction
mixture was stirred for 30 min at 0 °C and 25 min at room
temperature. The solvent was removed in vacuo and the residue
was purified by column chromatography on alumina (5%
EtOAc/petroleum ether) to afford the title compound as a
A mixture of Pd(OH)2 on carbon (20%, 5 mg, 0.035 mmol),
10c (10 mg, 9.65 µmol) in EtOAc:methanol (5 ml, 5:1) was
stirred under hydrogen (20 bar) for 4 h at room temperature.
The reaction mixture is filtered through a short plug of Celite to
remove the catalyst. Triethylamine (5 µl) was added to the
filtrate and solvent removed under reduced pressure. The
resulting residue was purified by flash chromatography (20%
23
1
colorless oil (50 mg, 85%); [α]D +5.4 (c = 0.5, CHCl3); H
methanol:chloroform) to afford
1
as a colorless oil (9 mg,
0.25, CHCl3); 1H NMR (500 MHz,
CD3OD:CDCl3 (1:2)) δ 0.80 (3 H, d, = 6.6 Hz, CCH3
(TBSA)), 0.85 (6 H, t, = 6.8 Hz, 2 × C 3 (TBSA, palmitoyl)),
1.03–1.06 (2 H, m, CH2), 1.17–1.36 (62 H, m, 26 × CH2, 1 ×
CH, HN(CH2C 3)3), 1.57 (4 H, m, 2 × COCH2C 2), 2.29 (4 H,
dd, = 15.6, 8.0 Hz, 2 × COCH2), 3.11 (4 H, q, = 7.3 Hz,
NH(C 2CH3)3) 3.58 (2 H, s, H3'a,3'b), 3.76 (1 H, s, H2'), 3.90
(2 H, s, H1'a,1'b), 3.98 (2 H, s, H3a,3b), 4.15 (1 H, dd, = 11.8,
6.5 Hz, H1b), 4.38 (1 H, d, = 12.0 Hz, H1a), 5.20 (1 H, s,
NMR (500 MHz, CDCl3) δ 0.83 (3 H, d,
(TBSA)), 0.88 (6 H, t, = 6.9 Hz, 2 × C
CH2), 1.17–1.20 (12 H, m, 2 × CH(C
24 × CH2, 1 × CH), 1.57–1.62 (4 H, m, COCH2C
(4 H, m, COCH2), 3.60–3.81 (4 H, m, 2 × C (CH3)2, H3a,3b),
4.13–4.20 (1 H, m, H1a), 4.31–4.37 (1 H, m, H1b), 4.64–4.77
(2 H, m, C 2Ph),5.15–5.22 (1 H, m, H2), 7.30–7.41 (5 H, m,
J
H
= 6.6 Hz, CCH3
3), 1.18 (2 H, m,
24
98%); [α]D +5.8 (
c
J
J
H
3)2), 1.23–1.35 (49 H, m,
2), 2.21–2.31
J
H
H
H
H
H
J
J
H
H
Ph); 13C NMR (75 MHz, CDCl3) δ 14.2, 19.8 22.8, 24.6, 24.7,
24.8, 25.0, 29.2, 29.4, 29.5, 29.6, 29.8, 29.8, 32.0, 34.3, 43.1,
43.2, 61.6, 61.7, 61.9, 62.6, 63.2, 65.4, 65.6, 70.9 (N-CH),
127.0, 127.4, 128.3, 139.4 (Ph), 173.1, 173.5 (C=O); 31P NMR
(202 MHz, CDCl3) δ 148.8; HRMS-ESI: [M + H]+ calcd for
C51H95O6NP 848.6897; found 848.6885.
J
J
H2); 13C NMR (126 MHz, CD3OD:CDCl3 (1:2)) δ 14.2, 19.9,
23.0, 25.2, 25.2, 27.4, 29.5, 29.5, 29.7, 29.9, 29.9, 30.0, 30.3,
30.4, 32.3, 33.1, 34.4, 34.6, 37.4, 46.8, 62.9, 64.2, 67.1, 70.7,
71.4 (glycerol C), 174.0, 174.3 (C=O); 31P NMR (202 MHz,
CD3OD:CDCl3 (1:2)) δ 4.78; HRMS: [M + H]+ calcd for
C47H96NO10P 866.6805; found 866.6806.
O-Benzyl-O-(1-O-((R)-10-methyloctadecanoyl)-2-O-palmitoyl-sn-
glyceryl)-O-(2,3-di-O-benzyl-sn-glycer-1-yl) phosphate (10c)
6 | Org. Biomol. Chem., 2017, 00, 1-7
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Organic & Biomolecular Chemistry
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Organic Biomolecular Chemistry
ARTICLE
Synthesized as for 10c, 10.2 mg (34%). [α]D +6.2 (c = 0.25,
25
O-Benzyl-O-(1-O-(palmitoyl)-2-O-stearoyl-sn-glycer-3-yl) N,N-
diisopropylphosphoramidite (8a)
View Article Online
CHCl3); 1H NMR (600 MHz, CDCl3) δ 0D.8O8I:(160.H10,3t9,/C7OB01786C
2 × C 3), 1.27 (56 H, m, 28 × CH2), 1.57 (4 H, m, 2 ×
COCH2C 2), 2.26 (4 H, t, = 7.6 Hz, 2 × COCH2), 3.56 (2 H,
t, = 5.6 Hz, H3'a,3'b), 3.75–3.79 (1 H, m, H2'), 4.03–4.29 (6
H, m, H1'a,1'b,3a,3b,1a,1b), 4.50–4.54 (2 H, m, C 2Ph), 4.60–
4.68 (2 H, m, C 2Ph), 5.04 (2 H, m, C 2Ph), 5.13–5.20 (1 H,
J = 6.8 Hz,
H
Alcohol 7a (30 mg, 0.050 mmol) was treated as for the
24
H
J
preparation of 8c to afford 8a (30.4 mg, 72%). [α]D +5.8 (c =
0.5, CHCl3); 1H NMR (600 MHz, CDCl3) δ 0.88 (6 H, t,
J
= 6.8
3)2), 1.23–1.35
2), 1.57–1.62 (4 H, m, COCH2C 2), 2.21–
2.31 (4 H, m, COC 2), 3.60–3.67 (2 H, m, 2 × C (CH3)2),
3.69–3.75 (1 H, m, H3b), 3.76–3.81 (1 H, m, H3a), 4.13–4.20
(1 H, m, H1a), 4.34 (1 H, td, = 12.2, 3.8 Hz, H1b), 4.63–4.79
(2 H, m, C 2Ph), 5.18–5.19 (1 H, m, H2), 7.27–7.35 (5 H, m,
J
H
Hz, 2 × C
H3), 1.17–1.20 (12 H, m, 2 × CH(CH
H
H
(52 H, m, 26 × C
H
H
m, H2), 7.27–7.35 (15 H, m, Ph); 13C NMR (126 MHz, CDCl3)
δ 14.2, 22.8, 24.9, 27.2, 29.2, 29.2, 29.4, 29.5, 29.6, 29.7, 29.8,
30.1, 30.2, 32.0, 32.9, 34.1, 34.2, 37.2, 61.8, 65.5, 67.3, 67.3,
69.1, 69.4, 69.4, 69.5, 69.6, 72.3, 73.6, 127.7, 127.8, 127.9,
128.0, 128.5, 128.5, 128.7 (Ph), 172.9, 173.3 (C=O); 31P NMR
(202 MHz, CDCl3) δ –1.01, –0.98; HRMS: [M + H]+ calcd for
C61H97O10P 1021.6853; found 1021.6852.
H
H
J
H
Ph);13C NMR (101 MHz, CDCl3) δ 14.2, 22.8, 24.6, 24.7, 24.8,
25.0, 29.2, 29.4, 29.5, 29.6, 29.8, 29.8, 32.0, 34.3, 43.1, 43.2,
61.6, 61.7, 61.9, 62.6, 63.2, 65.4, 65.6, 70.9 (N-CH), 127.0,
127.4, 128.3, 139.4 (Ph), 173.1, 173.5 (C=O); 31P NMR (202
MHz, CDCl3) δ 148.8; HRMS-ESI: [M + H]+ calcd for
C50H92NO6P 834.6696; found 834.6696.
Triethylammonium O-(1-O-palmitoyl-2-O-stearoyl-sn-glyceryl)-O-
(sn-glycer-1-yl) phosphate (11a)
25
Synthesized as for 1, 6.2 mg (27% over 2 steps). [α]D +5.6 (c
0.25, CHCl3); 1H NMR (400 MHz, CD3OD:CDCl3 (1:2)) δ 0.84
O-Benzyl-O-(1-O-(stearoyl)-2-O-palmitoyl-sn-glycer-3-yl) N,N-
diisopropylphosphoramidite (8b)
(6 H, t,
HN(CH2C
× COCH2C
J
H
H
= 6.4 Hz, 2 × C
3)3), 1.17–1.36 (56 H, m, 28 × CH2), 1.52 (4 H, s, 2
2), 2.23 (4 H, dd, = 14.5, 7.2 Hz, 2 × COCH2),
3.46–3.61 (6 H, s, H3'a,3'b, HN(C 2CH3)3)), 3.65–3.73 (1 H,
m, H2'), 3.83–3.90 (4 H, m, H1'a,1'b,3a,3b), 3.95 (2 H, m,
H3a,3b), 4.11 (1 H, dd, = 12.0, 6.6 Hz, H1b), 5.16 (1 H, s,
H3), 1.10–1.14 (9 H, m,
Alcohol 8a (36.0 mg, 0.60 mmol) was treated as for the
preparation of 8c to afford 8b (29.8 mg, 58%). [α]D +5.5 (
24
J
c
0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 (6 H, t,
Hz, 2 × CH3), 1.17–1.20 (12 H, m, 2 × CH(C 3)2), 1.23–1.35
(52 H, m, 26 × CH2), 1.57–1.62 (4 H, m, COCH2C 2), 2.21–
2.31 (4 H, m, COCH2), 3.58–3.83 (4 H, m, 2 × C (CH3)2,
H3a,3b), 4.12–4.19 (1 H, m, H1a), 4.30–4.38 (1 H, m, H1b),
4.62–4.80 (2 H, m, C 2Ph), 5.17–5.24 (1 H, m, H2), 7.27–7.35
J
= 6.8
H
H
J
H
H
H2); 13C NMR (126 MHz, CD3OD:CDCl3 (1:2)) δ 14.2, 23.0,
25.2, 25.2, 29.5, 29.5, 29.7, 29.7, 29.9, 30.0, 30.0, 32.3, 34.4,
34.6, 46.5, 62.8, 63.0, 63.7, 66.8, 70.8, 70.9, 71.5, 72.7, 174.0,
174.4; 31P NMR (202 MHz, CDCl3) δ 4.78; HRMS: [M + H]+
calcd for C40H79O10P 751.5444; found 751.5444.
H
(5 H, m, Ph); 13C NMR (75 MHz, CDCl3) δ 14.2, 22.8, 24.6,
24.7, 24.8, 25.0, 29.2, 29.4, 29.5, 29.6, 29.8, 29.8, 32.0, 34.3,
43.1, 43.2, 61.6, 61.7, 61.9, 62.6, 63.2, 65.4, 65.6, 70.9 (N-CH),
127.0, 127.4, 128.3, 139.4 (Ph), 173.1, 173.5 (C=O); 31P NMR
(202 MHz, CDCl3) δ 148.0; HRMS-ESI: [M + H]+ calcd for
C50H92NO6P 834.6696; found 834.6696.
O-(1-O-Stearoyl-2-O-palmitoyl-sn-glyceryl)-O-(sn-glycer-1-yl)
phosphate (11b)
25
Synthesized as for
1, 9.1 mg (90%). [α]D +4.8 (c 0.25,
CHCl3); 1H NMR (400 MHz, CD3OD:CDCl3 (1:2)) δ 0.84 (6 H,
t, = 6.4 Hz, 2 × C 3), 1.17–1.36 (56 H, m, 28 × CH2), 1.57 (4
H, s, 2 × COCH2C 2), 2.29 (4 H, dd, = 14.5, 7.2 Hz, 2 ×
O-Benzyl-O-(1-O-(palmitoyl)-2-O-stearoyl-sn-glycer-3-yl)-O-(2,3-di-
O-benzyl-sn-glycer-1-yl) phosphate (10a)
J
H
H
J
COCH2), 3.57 (2 H, s, H3'a,3'b), 3.70–3.78 (1 H, m, H2'), 3.84–
3.90 (2 H, s, H1'a, H1'b), 3.95 (2 H, m, H3a,3b), 4.15 (1 H, dd,
1
H
-Tetrazole (2.7 mg, 0.039 mmol) was added to a solution of
phosphoramidite (24.0 mg, 0.028 mmol) and 2,3-di- -benzyl-
sn-glycerol
38 (9.4 mg, 0.034 mmol) in dry CH2Cl2 (2 ml) at 0
O
J
= 11.9, 6.7 Hz, H1b), 4.37 (1 H, m, H1a), 5.20 (1 H, s, H2);
9
13C NMR (126 MHz, CD3OD:CDCl3 (1:2)) δ 14.2, 23.0, 25.2,
25.2, 29.5, 29.5, 29.7, 29.7, 29.9, 30.0, 30.0, 32.3, 34.4, 34.6,
46.5, 62.8, 63.0, 63.7, 66.8, 70.8, 70.9, 71.5, 72.7, 174.0, 174.4;
31P NMR (202 MHz, CDCl3) δ 5.25; HRMS: [M + H]+ calcd
for C40H79O10P 751.5444; found 751.5444.
°C under nitrogen. The reaction mixture was stirred for 15 min
at 0 °C and 2 h at room temperature. The reaction mixture was
cooled to 0 °C and t-BuOOH (5 M in decane) (4.1 µl, 0.042
mmol) was added to the reaction mixture and stirred for 2 h
with warming to room temperature. Excess oxidant was
quenched with sodium metabisulfite (1 M, 0.1 ml), the reaction
mixture was diluted with CH2Cl2, extracted, dried (MgSO4),
filtered and concentrated. The solvent was removed in vacuo
Collision-induced dissociation mass spectrometry of
phosphatidylglycerols
and the residue was purified by column chromatography on Characterization of lipid ions, introduced to a Q Exactive Plus
silica gel (35% EtOAc/ petroleum ether, 0.5% Et3N) which Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen,
resulted in a 1:1 mixture the product and unreacted dibenzyl Germany) via nanoESI (nESI) using an Advion Triversa
glyercol, which was directly subjected to hydrogenolysis.
Nanomate (Advion, Ithaca, NY), was performed in negative
ionization mode by higher-energy collision induced
dissociation (HCD)-tandem mass spectrometry (MS/MS).
Deprotonated precursor ions were mono-isotopically isolated
O-Benzyl-O-(1-O-(stearoyl)-2-O-palmitoyl-sn-glycer-3-yl)-O-(2,3-di-
O-benzyl-sn-glycer-1-yl) phosphate (10b)
using a window of ± 0.5
m/z. Product ion spectra were acquired
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem., 2017, 00, 1-7 | 7
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Page 8 of 8
ARTICLE
Organic Biomolecular Chemistry
20.
21.
P. S. Patil, T.-J. R. Cheng, M. M. L. Zulueta, S.V-Tie.wYAarnticgle, LO.nSlin.e
Lico and S.-C. Hung, Nat. Commun.D,O2I0:1105.,160,397/2C379O.B01786C
B. Cao, X. Chen, Y. Yamaryo-Botte, M. B. Richardson, K. L.
Martin, G. N. Khairallah, T. W. Rupasinghe, R. M.
O'Flaherty, R. A. O'Hair, J. E. Ralton, P. K. Crellin, R. L.
Coppel, M. J. McConville and S. J. Williams, J. Org. Chem.,
2013, 78, 2175-2190.
using a mass resolving power of 70,000. Spectra shown are the
average of 100 scans.
Acknowledgements
We acknowledge the Australian Research Council for financial
support (DP160100597). Prof Gavin Reid and Mengxuan Fang
are thanked for assistance with the mass spectrometric analysis
of PGs.
22.
23.
I. O. Roberts and M. S. Baird, Chem. Phys. Lipids, 2006,
142, 111-117.
P. D'Arrigo, L. de Ferra, G. Pedrocchi-Fantoni, D. Scarcelli,
S. Servi and A. Strini, J. Chem. Soc. Perkin Trans. 1, 1996,
2657-2660; R. Sato, Y. Itabashi, H. Fujishima, H. Okuyama
and A. Kuksis, Lipids, 2004, 39, 1025-1030.
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