Synthesis of phosphatidylcholine analogues derived from glyceric acid: a new class of biologically active phospholipid compounds

Article abstract of DOI:10.1016/j.tetlet.2008.03.084

Synthesis of a new class of phosphatidylcholine analogues derived from glyceric acid is reported for spectroscopic studies of phospholipases and the conformation of phospholipid side-chains in biological membranes, using fluorescence resonance energy transfer (FRET) techniques.

Full text of DOI:10.1016/j.tetlet.2008.03.084

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3500–3503
Synthesis of phosphatidylcholine analogues derived from glyceric acid:
a new class of biologically active phospholipid compounds
Renato Rosseto a, Celize M. Tcacenco b, Radha Ranganathan b, Joseph Hajdu a,*
a Department of Chemistry and Biochemistry, California State University, Northridge, Northridge, CA 91330-8262, United States
b Department of Physics and Astronomy, California State University, Northridge, Northridge, CA 91330-8262, United States
Received 8 March 2008; revised 15 March 2008; accepted 18 March 2008
Available online 20 March 2008
Abstract
Synthesis of a new class of phosphatidylcholine analogues derived from glyceric acid is reported for spectroscopic studies of phospho-
lipases and the conformation of phospholipid side-chains in biological membranes, using fluorescence resonance energy transfer (FRET)
techniques.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Phosphatidylcholine analogue synthesis; Glyceric acid ester; Orthogonal O-protection; Fluorescence resonance energy transfer (FRET)
Biologically active synthetic phospholipid compounds
are required for structural and dynamic studies of biomem-
branes for the establishment of structure–activity relation-
ships with respect to phospholipid–phospholipid and
phospholipid–protein interactions, as well as for mechanis-
tic studies of phospholipid metabolizing enzymes.1–3 Spe-
cifically, with the discovery that phospholipases generate
a number of physiologically important lipid metabolites,
including second messengers that are involved in cell sig-
naling,1 the development of new synthetic methods for
the preparation of phospholipid derivatives became a key
step in advancing membrane biochemistry. Phospholipid
analogues incorporating spectroscopically active reporter
groups have been shown to be valuable structural probes
to study the conformation and the dynamics of phospho-
lipids in aggregates (e.g., micelles, bilayers, and vesicles)
as well as for the development of spectroscopic assay sys-
tems of lipolytic enzymes,4–6 and for in vivo monitoring
of the fate of products generated by phospholipids meta-
bolizing enzymes.7
As a part of our work in this area we focused on the
development of highly specific fluorescent phospholipase
A2 substrates for kinetic studies and for the detection of
in vivo activity of the enzyme. Phospholipases A2 (PLA2,
EC 3.1.1.4) comprise a large group of intracellular and
secreted enzymes that catalyze the hydrolysis of the sn-2
ester function of glycerophospholipids to produce free fatty
acids, such as arachidonic acid and lysophospholipids.8
Both products are the precursors of signaling molecules
with a multitude of biological functions.8–13 Specifically,
arachidonic acid is converted to eicosanoids that have been
shown to be involved in immune response, inflammation,
pain perception, and sleep regulation,8,9 while lysophos-
pholipids are the precursors of lipid mediators such as lyso-
phosphatidic acid (LPA) and platelet activating factor
(PAF). LPA is involved in cell proliferation, survival, and
migration, and PAF is particularly involved in inflamma-
tory processes.8 Thus, mammalian secreted phospholipase
A2s (sPLA2s) have been implicated in a series of physiolog-
ical and pathophysiological functions including lipid diges-
tion, cell proliferation, neurosecretion, antibacterial
defense, cancer, and inflammatory diseases.10 Kinetic char-
acterization and mechanistic elucidation of sPLA2 enzymes
should contribute to better understanding of their exact
*
Corresponding author. Tel.: +1 818 677 3377; fax: +1 818 677 2912.
E-mail address: joseph.hajdu@csun.edu (J. Hajdu).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.03.084

R. Rosseto et al. / Tetrahedron Letters 49 (2008) 3500–3503
3501
physiological role in these events, which remain to be elu-
cidated.11 In addition, fluorescence-based phospholipase
A2 assay methods should also be applicable for high-
throughput screening of potential PLA2 inhibitors.4 Signifi-
cantly, recent studies indicated therapeutic potential of
phospholipase A2 inhibitors in cardiovascular disease,12
and it has also been suggested that select isoforms of the
enzyme may be targets for drugs in the treatment of
cancer.13
In the present Letter, we report the synthesis of a new
series of phospholipid compounds derived from glyceric
acid (1), incorporating an ‘inverse ester’ function at the
sn-1-position preventing hydrolysis by PLA1, while keeping
the sn-2-ester group intact for cleavage by phospholipase
A2 enzymes (2).
Phospholipid analogues such as compound 2 should
also be applicable to detect the activity of PLA2 enzymes,
because the change in fluorescence depends on specific
cleavage of the ester linkage at the sn-2-position of the sub-
strate. In designing the target structure we selected two
photostable coumarin fluorophores with the requisite spec-
troscopic properties to provide a suitable donor–acceptor
pair.14 In addition, the chain-terminal reporter groups are
relatively small in size in order to minimize the impact on
the properties of the hydrocarbon chains. Using the
‘inverse ester’ strategy instead of relying on the sn-1-alkyl
ether substitution to achieve PLA2 selectivity brings the
compound closer to the naturally occurring glycerophos-
pholipid structure.1 Furthermore, the synthetic sequence
is designed in such a way that includes lysophospholipid
intermediates, to allow the incorporation of substituents
with chain-terminal functional groups, including those that
would not survive the reaction conditions involved in phos-
phorylation. Significantly, fluorescent lysophospholipid
compounds have been shown to be important biologically
active lipid analogues in their own right.1,15
O
O
O
O P
O
R1
N
HO
OH
O
m
O
n
OH
R2
O
O
2
1
Scheme 1. Reagents and conditions: (a) (i) 1.2 M NaOH, 80% aq dioxane, (ii) Bio-Rad AG 50-X (H+), (iii) 12-(70-mercapto-40methylcoumarin)dodec-
anol–DCC–DMAP, CHCl3; (b) 0.4 M HCl, aq dioxane, 2 h; (c) 1.4 equiv C6H5OCH2COCl, 2,4,6-trimethylpyridine, CHCl3, 0 °C, 3 h; (d) (i) DHP, PPTS,
CH2Cl2, rt, 3 h, (ii) 5 equiv tert-butylamine, CHCl3–MeOH (1:1), 0 °C, 14 h; (e) ethylene chlorophosphate, Et3N, benzene, (ii) (CH3)3N, MeCN, 65 °C; (iii)
0.3 M HCl, rt, 6 h; (f) (i) FMOC-12-aminododecanoic acid–DCC–DMAP, CHCl3, (ii) DBU, rt, 1 h, (iii) p-nitrophenyl-7-diethylaminocoumarin-3-
carboxylate, DMAP, CHCl3; (g) (i) and (ii) as in (f), then p-nitrophenyl-PROXYL-3-carboxylate, DMAP, CHCl3.

3502
R. Rosseto et al. / Tetrahedron Letters 49 (2008) 3500–3503
The synthetic strategy, outlined in Scheme 1, is based
was purified by silica gel chromatography using chloro-
form–ethyl acetate (5:1) as eluant, then freeze-dried from
benzene to give a colorless oil that turned to a white waxy
solid on standing (68%). The high-field 1H NMR spec-
trum of compound 6 shows baseline resonance in the d
5.00–5.09 region, confirming that the secondary hydroxyl
group remained intact in the isolated product.17 Next, the
tetrahydropyranyl function was elaborated at the sn-2-
glycerol position using five-fold excess of 3,4-dihydro-
2H-pyran with pyridinium p-toluenesulfonate catalysis in
CH2Cl2 at room temperature for 3 h, in 96% yield, and
base-catalyzed chemoselective cleavage of the phenoxyace-
tyl ester function was accomplished using 5 equiv of tert-
butylamine in chloroform–methanol (1:1) at 0 °C for 14 h.
Product 7 was purified by column chromatography on sil-
ica gel using a stepwise gradient of CHCl3–EtOAc (9:1
followed by 6:1) to obtain the primary alcohol 7 in 97%
yield.
Phosphorylation of the substituted glycerol 7 at the sn-3-
position was carried out using 2 equiv of 2-chloro-2-oxo-
1,3,2-dioxaphospholane and triethylamine in sodium-dried
benzene at room temperature for 4 h. The phosphorylated
intermediate was treated with 5 mL anhydrous trimethyl-
amine in acetonitrile at 65 °C (in a pressure bottle) for
24 h to afford the crude product that separated from the
reaction mixture on cooling. This intermediate was purified
by silica gel chromatography (CHCl3–CH3OH–H2O
65:25:4, 74% yield), and treated with a 0.3 M HCl in a
biphasic system of chloroform-water (4:0.1) for 2 h.
Freeze-drying from benzene yielded the crude fluorescent
lysophospholipid 8, which was purified by chromatography
(CHCl3–CH3OH–H2O 65:25:4) and isolated as a white
solid (57%). Next, compound 8 was acylated with 2.5 fold
excess of FMOC-12-aminododecanoic acid–DCC–DMAP
in CHCl3 in the presence of glass beads at 25 °C, with
sonication, for 5 h.18 Purification by silica gel chromato-
graphy, first with CHCl3–MeOH (4:1), followed by
CHCl3–MeOH–H2O (65:25:4) as eluant gave the
FMOC-protected phosphatidylcholine derivative (60%).
Base-catalyzed elimination of the FMOC protecting group
using 4 equiv of DBU in CHCl3 at room temperature for
1 h, followed by the treatment with p-nitrophenyl-7-
diethylaminocoumarin-3-carboxylate in the presence of
catalytic amount of DMAP for 4 h in the same reaction
mixture18 afforded the target phospholipid 2a. Purification
by silica gel chromatography using the same solvent
systems as mentioned above yielded the fluorescent
product 2a (66% from 8) as a yellow solid.19
upon two key elements. First, commercially available 2,3-
O-isopropylidene-L-methyl glycerate 3 is used to provide
the three-carbon skeleton to construct the target
phospholipid compound. Specifically, we have discovered
that while glyceric acid itself is rather intractable due to
its poor solubility in organic solvents, base-catalyzed
hydrolysis of acetonide 3 readily affords the correspond-
ing sodium glycerate in essentially quantitative yield.
Treatment of the sodium salt with ion exchange resin
provides the corresponding free acid, which in turn can
be converted to the long-chain fluorescent ester 4 in con-
densation with the desired alcohol.16 The second princi-
pal element involves the orthogonal protection of the
diol portion of the glyceric acid skeleton. Here, we
selected the phenoxyacetyl group to provide base-labile
protection at the sn-3-position, since we have found that
the acylation of compound 5 using a combination of
phenoxyacetyl chloride/collidine in CHCl3 proceeds with
high regioselectivity at the primary hydroxyl group, the
only byproduct being the diacyl derivative that is readily
separated from product 6 on column chromatography.
On the other hand, for acid labile protection at the sn-
2-alcohol function tetrahydropyranylation turned out to
be the most suitable choice. Specifically, both the intro-
duction of the tetrahydropyranyl function as well as its
cleavage later on in the sequence, can be carried out
under mild acidic conditions, and most importantly with-
out acyl group migration from the adjacent ester function.
Although the protection step introduces a second chiral
center, it does not complicate the synthesis because the
intermediate can easily be carried through the sequence
as a diastereoisomeric mixture up to the deprotection
step.
Thus, the hydrolysis of methyl 2,3-O-isopropylidene-L-
glycerate 3 with stoichiometric amount of 1.2 M NaOH
in 80% aqueous 1,4-dioxane at room temperature for 2 h
yielded the sodium salt of the protected glyceric acid as a
white powder (97%). The compound was isolated from
the reaction mixture by freeze-drying. The product was
treated with 30 mL Bio-Rad AG50-X (H+) ion exchange
resin in dioxane for 15 min at room temperature to obtain
the carboxylic acid as a colorless oil, which was converted
to the fluorescent ester 4 in reaction with 12-(70-mercapto-
40-methylcoumarin)dodecanol–DCC–DMAP in chloro-
form at room temperature for 6 h. The resulting ester 4
was purified by silica gel chromatography (CHCl3–EtOAc
9.5:0.5), and freeze-dried from benzene as a white solid
(88%). Acid-catalyzed hydrolysis of the isopropylidene
function with 0.4 M HCl in 97% aq dioxane at room tem-
perature for 2 h followed by freeze-drying and subsequent
chromatography (CHCl3–EtOAc 5:1) led to the pure diol
5 in 78% yield.
With the availability of compound 8 we were able to
extend the scope of reporter groups to include a nitroxide
spin label introduced at the sn-2-ester group’s chain-termi-
nal 2b. Conversion of compound 8 in the same sequence of
reactions as outlined for 2a afforded the double-labeled
phosphatidylcholine derivative 2b in 52% overall yield.
Both compounds 2a and 2b were completely hydrolyzed
by bee-venom phospholipase A2 yielding the corresponding
lysophospholipid 8 (Eq. 1).
Regioselective acylation at the primary hydroxyl group
of compound 5 was accomplished using 1.4 equiv of phen-
oxyacetyl chloride in the presence of 2.0 equiv of 2,4,6-tri-
methyl pyridine in chloroform at 0 °C for 3 h. Product 6

R. Rosseto et al. / Tetrahedron Letters 49 (2008) 3500–3503
3503
O
O
O
O
O
O P
O
O
PLA2
H2O
R1
N
R1
N
R2
O
R2
OH
O
O
O P O
O
OH
O
O
8
2
ð1Þ
O
NH(CH2)11
NH(CH2)11
R1 =
O
2a: R2 =
2b: R2 =
Et2N
O
O
O
S(CH2)12
N
O
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coumarin group (kem = 397 nm) shows substantial overlap with the
excitation spectrum of the acceptor, 7-dimethyl-aminocoumarin
(kexc = 405 nm), and its emission can be followed at kem = 462 nm.
These spectral parameters have been recorded in aqueous buffer at pH
8, in the presence of Triton X-100, conditions that have been used for
assaying phospholipase A2 enzymes.3
Specifically, the hydrolysis of 2a occurs with a decrease
in the fluorescence emission of the acceptor at 462 nm, and
an increase in the emission of the donor at 397 nm due to
loss of fluorescence resonance energy transfer. On the other
hand, cleavage of the sn-2-ester linkage of 2b leads to
enhanced fluorescence at 397 nm via de-quenching, due
to the release of the paramagnetically labeled fatty acid
from the molecule.20
In conclusion, the synthesis reported here provides a fac-
ile and efficient method for the preparation of a new class
of phospholipid analogues. The method should be applica-
ble for the preparation of a wide range of functionalized
phospholipid derivatives not only for the development of
new real-time spectrophotometric assays of phospholipases
and high-throughput screening of phospholipase inhibi-
tors, but also for the design and the development of new
membrane probes for the study of conformation and inter-
action of phospholipids in monolayers, bilayers, and
micelles. Work toward these goals is underway in our
laboratory.
Acknowledgment
We are grateful to the National Institutes of Health,
Grant 2S06 GM/HD48680 for financial support.
15. (a) Ishii, I.; Fukushima, N.; Ye, X.; Chun, J. Annu. Rev. Biochem.
2004, 73, 321–340; (b) Xu, Y. Biochim. Biophys. Acta 2002, 1582, 81–
88.
16. To the best of our knowledge this is the first synthetic method for the
preparation of glyceric acid esters.
Supplementary data
Characterization of the synthetic compounds by spec-
troscopic methods including IR, H, 13C NMR, HRMS,
1
17. Roodsari, F. S.; Wu, D.; Pum, G. S.; Hajdu, J. J. Org. Chem. 1999,
64, 7727–7737.
and elemental analyses. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2008.03.084.
18. Rosseto, R.; Hajdu, J. Tetrahedron Lett. 2005, 46, 2941–2944.
19. The new compounds were characterized by spectroscopic methods
including IR, 1H, 13C NMR, HRMS, and elemental analysis. In
addition, compounds 2a and 2b were completely hydrolyzed by bee-
venom phospholipase A2 yielding the corresponding sn-2-lysophos-
References and notes
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