Synthesis of oligo(ethylene glycol) substituted phosphatidylcholines: Secretory PLA2-targeted precursors of NSAID prodrugs

Article abstract of DOI:10.1016/j.chemphyslip.2009.10.001

A series of new phosphatidylcholine analogues with structurally modified sn-2-substituents have been prepared. The synthetic compounds include oligo(ethylene glycol) derivatives with chain-terminal pharmacophores that upon catalytic hydrolysis by phospholipase A₂ yielded a series of oligo(ethylene glycol)-conjugates of the respective drugs. The approach here outlined may open a new way to employ OEG derivatives of phospholipids for therapeutic applications as secretory PLA₂-targeted precursors of prodrugs.

Full text of DOI:10.1016/j.chemphyslip.2009.10.001

Chemistry and Physics of Lipids 163 (2010) 110–116
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Short communication
Synthesis of oligo(ethylene glycol) substituted phosphatidylcholines: Secretory
PLA2-targeted precursors of NSAID prodrugs
Renato Rosseto, Joseph Hajdu∗
Department of Chemistry and Biochemistry, California State University, 18111 Nordhoff Street, Northridge, CA 91330-8262, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2009
Received in revised form 6 October 2009
Accepted 8 October 2009
Available online 28 October 2009
A series of new phosphatidylcholine analogues with structurally modified sn-2-substituents have been
prepared. The synthetic compounds include oligo(ethylene glycol) derivatives with chain-terminal phar-
macophores that upon catalytic hydrolysis by phospholipase A2 yielded a series of oligo(ethylene
glycol)-conjugates of the respective drugs. The approach here outlined may open a new way to employ
OEG derivatives of phospholipids for therapeutic applications as secretory PLA2-targeted precursors of
prodrugs.
Keywords:
Oligo(ethylene glycol) substituted
phosphatidylcholines
© 2009 Elsevier Ireland Ltd. All rights reserved.
Phospholipase A2-targeted precursor of
prodrugs
Non-steroidal anti-inflammmatory drugs
Drug delivery methods
1. Introduction
proliferation, survival and migration (Rivera and Chun, 2008; Zhao
and Natarajan, 2009), while PAF is particularly involved in inflam-
Phospholipases A2 (PLA2’s, EC 3.1.1.4) comprise a large group
of intracellular and secreted enzymes that catalyze the hydrolysis
of the sn-2-ester bond of glycerophospholipids to yield free fatty
acids such as arachidonic acid, and lysophospholipids (Burke and
Dennis, 2009a, Eq. (1)):
matory processes (Prescott et al., 2000).
Secreted phospholipases A2 (sPLA2’s) are widespread in nature
(Six and Dennis, 2000). Early studies have focused on mem-
bers of the sPLA2 family isolated from insect and snake venoms;
more recently sPLA2’s have been found in plants, bacteria,
fungi, viruses, and mammals (Boyanovski and Webb, 2009).
The mammalian family of secreted phospholipase A2 enzymes
consists of 12 different members at the present (Rouault et
al., 2007). Isolated from the variety of sources, the sPLA2’s
share a series of common structural features. They are small,
secreted proteins (14–18 kDa), with a compact structure usually
containing 5–8 disulfide bonds (Fuentes et al., 2002). Stud-
ies focusing on their mechanism of action have shown that
Both products are precursors for signaling molecules with a wide
range of biological functions (Burke and Dennis, 2009b). Specifi-
cally, arachidonic acid is converted to eicosanoids that have been
shown to be involved in immune response, inflammation, pain per-
ception, and sleep regulation (Funk, 2001; Schaloske and Dennis,
2006), while lysophospholipids are precursors of lipid mediators
such as lysophosphatidic acid (LPA) and platelet activating factor
(PAF). Lysophosphatidic acid has been shown to be involved in cell
an active site histidine in close proximity to
a conserved
aspartate are the residues involved in the catalytic reaction,
with absolute dependence on Ca2+ for activation (Scott et al.,
1990).
∗
Corresponding author. Tel.: +1 818 677 3377; fax: +1 818 677 2912.
E-mail address: joseph.hajdu@csun.edu (J. Hajdu).
0009-3084/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2009.10.001

R. Rosseto, J. Hajdu / Chemistry and Physics of Lipids 163 (2010) 110–116
111
Fig. 1. The structures of NSAIDS’s used in the syntheses: indomethacin 4, sulindac 5, and diclofenac 6.
At the present there is a growing interest in elucidation of
the in vivo biological functions of mammalian sPLA2’s, since
used indomethacin 4, sulindac 5, and diclofenac 6, as these non-
steroidal anti-inflammatory drugs (NSAID) are subject of ongoing
biochemical and pharmacological investigation (Fig. 1).
they have been implicated in
a series of physiological and
pathophysiological functions including lipid digestion, signal
transduction, prostaglandin biosynthesis, cell proliferation, neu-
rosecretion (Burke and Dennis, 2009b), antibacterial defense
(Nevalainen et al., 2008), inflammatory diseases (Nevalainen et
al., 2000) and cancer (Dong et al., 2006). In this context, secre-
tory phospholipase A2 enzymes have recently been targets for a
therapeutic application, as elevated levels of one subtype, sPLA2-
IIA, were observed in the microenvironment surrounding tumors
in human colorectal adenocarcinomas (Abe et al., 1997; Kennedy
et al., 1998; Mounier et al., 2008), and in neoplastic prostate tissue
(Graff et al., 2001; Menschikowski et al., 2008). The high levels of
sPLA2 in tumors prompted the design of a tumor-activated drug
delivery system in the form of sPLA2 degradable liposomes, trans-
porting conventional therapeutics that were released at the tumor
site using sPLA2 as a tumor-specific trigger (Davidsen et al., 2003).
In this communication we report a new approach to develop
sPLA2-directed therapeutically applicable synthetic phospholipid
analogues. We have designed a series of structurally modi-
fied phosphatidylcholine analogues incorporating oligoethylene
glycol-substituted sn-2-ester functions. Relying on the minimum
structural requirements for catalytic hydrolysis by the enzyme
(Kuipers et al., 1990), including the presence of an essential
␣-methylene group adjacent to the sn-2-ester carbonyl of the
substrate, replacement of the hydrocarbon portion of the natu-
rally occurring fatty acid chain with an oligoethylene glycol group
appeared to be a promising modification to qualify for catalytic
cleavage by sPLA2 enzymes.
Specifically, in addition to their well-established inhibitory
profiles against cyclooxygenases (Prusakiewicz et al., 2004; Felts
et al., 2007; Blobaum and Marnett, 2007) non-steroidal anti-
inflammatory drugs have been found both in epidemiological and
clinical studies to reduce the prevalence and severity of Alzheimer’s
disease (Veld et al., 2001; Gasparini et al., 2004), as inflammation
exacerbates the pathology of that disease and other neurodegen-
erative diseases (Klegeris and McGeer, 2005). However, long-term
use of NSAID’s is limited by gastrointestinal and renal toxicity of
these drugs. Attempts to mitigate the toxic side effects include
development of improved delivery systems such as sustained
release strategies with the use of prodrugs (Dahan and Hoffman,
2007).
2.2. Syntheses
The synthesis of the target compounds shown in Scheme 1 relied
on the following key elements: (1) introduction of the oligoethy-
lene glycol ester at the sn-2-position incorporating a chain-terminal
protected amino group, (2) activation of the NSAID’s by preparation
of the corresponding p-nitrophenyl esters, and (3) deprotection
of the amino group, followed by attachment of the desired anti-
inflammatory drug. In implementing the synthesis we used a
two-prong approach: of the commercially available orthogonally
functionalized oligoethylene glycol building blocks we selected
OEG-carboxylic acids using both acid-labile t-BOC vs. base-labile
FMOC-protected amino groups to develop the best way for prepa-
ration of the target phospholipids.
For acylation of sn-1-palmitoyl lysophosphatidylcholine 7 we
used the method that we recently developed for acylation of
lysophospholipids: (1) increasing the surface of the reaction ves-
sel, where the reaction is believed to take place, by addition of
glass beads, and (2) preventing intramolecular acyl migration by
keeping the temperature at 25 ◦C. Thus, we used sonication rather
than stirring the reaction mixture and maintained the temper-
ature at 25 ◦C by cooling the sonicated vessel in a circulatory
water bath (Rosseto and Hajdu, 2005). Under these conditions
the reactions reached completion in 6–8 h and the products were
obtained in good yield, with high regioselectivity. Specifically,
reaction of compound 7 with t-BOC protected 11-amino-3,6,9-
trioxaundecanoic acid with dicyclohexylcarbodiimide (DCC), in the
presence of 4-dimethylaminopyridine (DMAP) as catalyst, pro-
duced the target compound 8 in 59% yield after purification on silica
gel chromatography, while a similar reaction using the base-labile
FMOC-protected 8-amino-3,6-dioxaoctanoic acid as acyl donor
resulted in formation of compound 11 in 75% yield.
2. Results and discussion
2.1. Design of the synthetic target compounds
In designing the synthetic phospholipid analogues we sought
to develop PLA2-directed precursors of oligoethylene glycol-
conjugated pharmacophores to be released as prodrugs upon
catalytic hydrolysis by the enzyme. Specifically, it has been shown
recently, that oligoethylene modification of therapeutic agents,
much like PEGylation using their high-molecular weight poly-
meric counterparts (Veronese and Pasut, 2005), greatly improves
the pharmacokinetic profiles and targeting of drugs (Marsac et al.,
2006). These benefits include: (1) increase in solubility to achieve
better bioavailability, (2) resistance to proteolytic degradation, (3)
reduction in immunogenicity and antigenicity, and (4) prolonged
plasma circulation time (Harris and Chess, 2003; Juillerat-Jeanneret
and Schmitt, 2007; Marcus et al., 2008). Furthermore, oligomeric
PEG analogues (OEGs) are readily derivatized for site-selective tar-
geting, and they are also amenable to a wide range of structural
modification including construction of linear or branched scaffolds
(Bowen et al., 2007). To explore the feasibility of the approach we
Acid-catalyzed deprotection of the chain-terminal amino group
of compound 8 was carried out using 1.0 M anhydrous HCl solution
in dioxane at room temperature for 2.5 h, and the resulting amine
hydrochloride was isolated from the reaction mixture by freeze-

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R. Rosseto, J. Hajdu / Chemistry and Physics of Lipids 163 (2010) 110–116
Scheme 1. Reagents and conditions: (a) (CH3)3OCONH(CH2CH2O)3COOH/DCC/DMAP, CHCl3, 25 ◦C; (b) FMOC-NH(CH2CH2O)2COOH/DCC/DMAP, CHCl3, 25 ◦C; (c) (i) 1.0 M
HCl/dioxane, (ii) Et3N/p-nitrophenyl indomethacin, CHCl3, rt, 3 h; (d) (i) 1.0 M HCl/dioxane, (ii) Et3N/p-nitrophenyl sulindac, CHCl3, rt, 3 h; (e) (i) DBU/CHCl3, rt, 1 h, (ii) dansyl
chloride/Et3N, rt, 1 h; (f) (i) DBU/CHCl3, rt, 1 h, (ii) p-nitrophenyl dichlofenac/DMAP, 5 h, rt.
drying. Deprotonation with anhydrous triethylamine, followed
by triethylamine catalyzed introduction of the desired pharma-
cophore via the corresponding p-nitrophenyl active-ester yielded
the NSAID substituted phosphatidylcholines of indomethacin 9 and
sulindac 10 in 92% and 97% yields, respectively.
In the second series, the FMOC protecting group of compound 11
was removed by DBU-catalyzed elimination reaction in chloroform
at room temperature for 1 h, followed by acylation of the amino
group in the same pot, without isolating the intermediate, using
the p-nitrophenyl ester of diclofenac in the presence of DMAP as
catalyst, to obtain the desired phosphatidylcholine 12 in 51% overall
yield.
In an assay system (Roodsari et al., 1999) containing Triton X-
100-phospholipid mixed micelles, in the presence of catalytically
essential Ca2+, each one of the synthetic phosphatidylcholine com-
pounds was completely hydrolyzed by the enzyme, yielding the
corresponding lysophosphatidylcholine 7 and the series of oli-
goethylene substituted long-chain fatty acid analogues, including
OEG-conjugated prodrugs of the NSAID’s indomethacin, sulindac,
and diclofenac (Fig. 2).
2.4. Discussion
The synthesis of the phospholipid analogues here reported pro-
vides a new way of employing oligoethylene glycol-substituted
phosphatidylcholines as secretory phospholipase A2 targeted
precursors of NSAID prodrugs. Specifically, polyethylene glycol
modification (PEGylation) of biomolecules such as proteins, pep-
tides as well as other small-molecular therapeutic agents continues
to be a method of growing interest for development of new strate-
gies in drug delivery, diagnostics, and for modification of the
physicochemical properties of the compounds. While it has been
well established, that PEGylation of a therapeutic agent extends
its half-life in circulation, decreases its immunogenicity and anti-
genicity, and alters the pattern of drug distribution, with the
availability of functionalized short-chain oligomers of ethylene
glycol (OPE’s) new design strategies have become possible. Specif-
ically, OPE’s have been shown to be useful not only for improving
drug delivery (Warnecke and Kratz, 2003), but also for preparation
of membrane biosensors (Chen et al., 2000), and for modula-
Finally, we have explored the use of the synthetic method
here developed for preparation an oligoethylene glycol-substituted
phosphatidylcholine analogue 13, carrying a spectroscopically
active reporter group at the sn-2-chain-terminal, shown in
Scheme 1. Incorporation of fluorescent reporter groups into phos-
pholipid analogues have been shown to be useful for both kinetic
studies of lipolytic enzymes, as well as for providing a convenient
way for in vivo tracking the fate of the carboxylic acid produced on
the catalytic hydrolysis by PLA2 enzymes (Feng et al., 2002).
2.3. Enzymatic hydrolysis
Catalytic hydrolysis of the synthetic phosphatidylcholine ana-
logues 8–13 was carried out with bee-venom phospholipase
A2, a widely used, readily available representative of the low-
molecular weight secretory PLA2 enzymes (Valentin et al., 2000).

R. Rosseto, J. Hajdu / Chemistry and Physics of Lipids 163 (2010) 110–116
113
Fig. 2. The concept of using PLA2-targeted phospholipids as precursors of prodrugs.
tion of the physicochemical properties of membrane-targeted
lipoproteins (Malolanarasimhan et al., 2007), opening the way
toward the design of biomolecule analogues with targeted phys-
ical behaviors (Grogan et al., 2005). In addition, branched-chain
OPE conjugates of peptides were used to incorporate fluorescent
reporter groups to study receptor–ligand interactions, showing
retention of the biological potency of the ligand (Bowen et al.,
2007).
and approximately 1 g of glass beads. The reaction was sonicated
for 8 h at 25 ◦C. The solvent was then evaporated and the residue
was purified on silica gel chromatography with CHCl3/MeOH
(4:1) to elute the impurities, followed by CHCl3/MeOH/H2O
(65:25:4) to isolate the phospholipid. The fractions corresponding
to the product were combined and freeze-dried from benzene
to give 8 (0.5721 g, 59%) as white solid. IR (CHCl3): 3342, 1741
br, 1248 cm−1 1H NMR (CDCl3, 200 MHz) ı 0.84 (br t, 3H), 1.22
.
Preparation and phospholipase A2 hydrolysis of compounds
present a new approach to design of PLA2-targeted oligoethylene
glycol conjugates of pharmacophores and other biomolecules. In
contrast to earlier applications of PEGylated phospholipid deriva-
tives with polymeric ethylene glycol substituents introduced at
the polar headgroup (Zalipsky et al., 1999), the use of struc-
turally well-defined oligoethylene glycol-substituted sn-2-chain
here reported, offers improved targeting, flexibility in design of
oligomeric linkers, and applicability for introduction of various
different therapeutically active pharmacophores as well. Specifi-
cally, while NSAID’s have been shown to reduce the prevalence
and severity of Alzheimer’s disease, as well as cyclooxygenase-
dependent anti-inflammatory and neuroprotecting effects, their
long-term use has been limited by the gastrointestinal and renal
toxicity exhibited by the drugs (Dahan and Hoffman, 2007). Avail-
ability of compounds such as 9, 10, and 12 may lead to development
of drug delivery systems relying on sPLA2-targeted release of
the respective OEG-prodrug conjugates to circumvent the prob-
lem.
Finally, the approach of sPLA2-directed drug delivery systems
here illustrated may lead to design of precursors of OEG-prodrugs
targeted at tissues with high levels of sPLA2 enzymes. Signifi-
cantly, elevated levels of secretory phospholipase A2’s have been
found in a number of tissues under pathological conditions such
as prostate and colorectal cancers (Menschikowski et al., 2008;
Mounier et al., 2008), atherosclerosis (Bostrom et al., 2007; Kimura-
Matsumoto et al., 2008), rheumatoid arthritis, and coronary heart
disease (Khuseyinova et al., 2005; Nijmeijer et al., 2008). Thus,
the approach here presented may open the way to employ new
strategies involving PLA2-aided tissue specific drug delivery for
the development of improved treatments of specific pathological
conditions.
(br s, 24H), 1.40 (s, 9H), 1.56 (m, 2H), 2.24 (t, 2H, J = 7.4 Hz), 3.27
(m, 2H), 3.33 (br s, 9H), 3.42 (t, 2H, J = 9.9 Hz), 3.65 (s, 4H), 3.67
(m, 4H), 3.75 (m, 2H), 3.88 (m, 2H), 4.13 (m, 2H), 4.30 (m, 4H),
5.03–5.30 (br m, 2H). 13C NMR (CDCl3, 50 MHz) ı 14.03, 22.57,
24.76, 28.35, 29.08, 29.25, 29.44, 31.82, 33.94, 40.24, 54.26, 59.43,
62.51, 63.56, 66.21, 68.26, 70.09, 70.37, 70.69, 71.36, 80.73, 155.91,
169.93, 173.38. 31P NMR (CDCl3, 160 MHz, pyrophosphate ref. ext.)
ı −1.28. Rf (CHCl3/MeOH/H2O 65:25:4) 0.41. [␣]D20 + 4.7◦ (c 0.98,
CHCl3/MeOH 4:1). Anal. Calcd for C37H73N2O13P·H2O·1/3CHCl3: C,
52.09; H, 9.06; N, 3.25; Found: C, 52.00; H, 8.82; N, 3.39. MS MH+
C37H73N2O13PH Calcd: 785.4923, Found: 785.4916.
3.2. 1-Palmitoyl-2-(11ꢀ-N-indomethacincarboxylamino-3,6,9-
trioxaundecanoyl)-sn-glycerophosphocholine
(9)
(i) Indomethacin p-nitrophenyl ester. To
a
solution of
indomethacin (0.5011 g, 1.4 mmol) in 30 mL CHCl3 were added
p-nitrophenol (0.2102 g, 1.54 mmol), DCC (0.3203 g, 1.54 mmol)
and DMAP (40 mg, 0.3 mmol). The reaction mixture was stirred
for 1 h at room temperature. The DCC-urea was then filtered and
the filtrate was evaporated to give the p-nitrophenyl ester of the
drug; Rf (CHCl3) 0.43, which was suitable for use in the subsequent
acylation step without further purification. (ii) Deprotection of the
amino group of compound 2. To a solution of 2 (0.2351 g, 0.24 mmol)
in 20 mL 1,4-dioxane was added 4 M HCl (6 mL) in dioxane, and the
mixture was stirred for 2.5 h at room temperature. A positive nin-
hydrin test confirmed the presence of the free amino group in the
product near the origin on a TLC plate. The amine hydrochloride was
freeze-dried from benzene to give a white solid. (iii) Coupling of the
indomethacin active-ester with the chain-terminal amino group. The
amine hydrochloride just obtained was dissolved in 10 mL CHCl3
and the mixture was treated with excess triethylamine (0.3 mL,
2.1 mmol) to adjust the pH of the solution to about 9, followed by
addition of indomethacin p-nitrophenyl ester (0.3 g, 0.63 mmol).
The mixture was kept at room temperature for 3 h, when a negative
ninhydrin test indicated that the reaction reached completion. The
solution was loaded onto a silica gel column and chromatographed
using first CHCl3/MeOH (4:1), followed by CHCl3/MeOH/H2O
(65:25:4). The fractions corresponding to the product were com-
bined, evaporated, dispersed in benzene and freeze-dried to give
9 (0.2231 g, 92%) as pale-yellow solid. IR (CHCl3): 3347, 1740 br,
3. Experimental procedures
3.1. 1-Palmitoyl-2-(11ꢀ-N-t-BOC-amino-3,6,9-
trioxaundecanoyl)-sn-glycerophosphocholine
(8)
To
a
suspension of 1-palmitoyl-2-hydroxy-sn-glycero-3-
phosphocholine (0.4987 g, 1 mmol) in 25 mL of CHCl3 were
added 11-N-t-BOC-amino-3,6,9-trioxaundecanoic acid (0.7011 g,
1.43 mmol), DCC (0.5218 g, 2.5 mmol), DMAP (0.3120 g, 2.5 mmol)
1690 cm−1 1H NMR (CDCl3, 200 MHz) ı 0.82 (br t, 3H), 1.19 (br
;

114
R. Rosseto, J. Hajdu / Chemistry and Physics of Lipids 163 (2010) 110–116
s, 24H), 1.50 (m, 2H), 2.21 (t, 2H, J = 7.4 Hz), 2.31 (s, 3H), 3.28 (br
s, 11H), 3.43 (br s, 6H), 3.54 (m, 6H), 3.76 (br s, 5H), 3.94 (m, 2H),
3.98 (m, 3H), 4.53 (m, 3H), 4.81 (br m, 1H), 5.27 (m, 1H), 6.62–6.92
(m, 3H), 7.42 (d, 2H, 8.4 Hz), 7.60 (d, 2H, 8.4 Hz). 13C NMR (CDCl3,
50 MHz) ı 13.18, 13.93, 22.48, 24.64, 28.95, 29.11, 29.14, 29.31,
29.45, 29.49, 31.70, 31.84, 33.81, 39.06, 54.11, 55.56, 59.22, 62.37,
63.29, 65.99, 68.14, 69.86, 70.13, 70.51, 71.19, 71.32, 101.25, 111.53,
113.22, 114.74, 128.97, 130.46, 130.69, 130.99, 133.56, 135.90,
139.14, 155.91, 168.07, 169.77, 169.95, 173.26. 31P NMR (CDCl3,
160 MHz, pyrophosphate ref. ext.) ı −0.68. Rf (CHCl3/MeOH/H2O
3.5. 1-Palmitoyl-2-(8ꢀ-N-diclofenaccarboxylamino-
3,6-dioxaoctanoyl)-sn-glycerophosphocholine (12)
(i) Diclofenac p-nitrophenyl ester. To a suspension of diclofenac
sodium salt (0.6362 g, 2 mmol) in 50 mL acetone was added Dowex-
H+ (25 mL). The mixture was stirred for 10 min, and then it was
filtered and the filtrate was evaporated to give the correspond-
ing free acid as a white solid. This acid was dissolved in 50 mL
CHCl3 and to the solution were added p-nitrophenol (0.2546 g,
1.83 mmol), DCC (0.4126 g, 2 mmol) and DMAP (52 mg, 0.43 mmol).
The reaction mixture was stirred for 1 h at room temperature. The
DCC-urea was removed by filtration, and the solvent was evap-
orated to give the p-nitrophenyl active-ester of diclofenac as a
yellow solid; Rf (CHCl3/EtOAc, 5:1) 0.92. It was suitable to be
used for the subsequent acylation step without further purifica-
tion. (ii) Elimination of the FMOC protecting group of compound
11, and N-acylation at the chain-terminal. To a solution of com-
pound 11 (0.2107 g, 0.24 mmol) in 5 mL CHCl3 was added DBU
(0.1852 g, 1.2 mmol) and the mixture was kept at room temper-
ature for 1 h. To this solution were added diclofenac p-nitrophenyl
ester (0.4 g, 0.96 mmol), DMAP (42 mg, 0.34 mmol). The reaction
was stopped after 5 h, when a ninhydrin test gave negative result
on the spot near the origin on the TLC plate. The reaction mix-
ture was loaded directly onto a silica gel column, eluted first
with CHCl3/MeOH (4:1) to remove the impurities followed by
CHCl3/MeOH/H2O (65:25:4) to elute the pure phospholipid. The
fractions corresponding to the product were combined, evapo-
rated, dispersed in benzene and freeze-dried to give 12 (0.1125 g,
65:25:4) 0.54. Anal. Calcd for
C
51H79ClN3O14P·1/3·H2O: C,
59.43; H, 7.79; N, 4.08; Found: C, 59.26; H, 7.80; N, 4.07. MS
MH+ C51H79ClN3O14PH Calcd: 1024.5061, Found: 1024.5075.
[␣]D20 + 5.8◦ (c 1.09, CHCl3/MeOH 4:1).
3.3. 1-Palmitoyl-2-(11ꢀ-N-sulindaccarboxylamino-3,6,9-
trioxaundecanoyl)-sn-glycerophosphocholine
(10)
This compound was prepared following the same activa-
tion/deprotection/acylation protocol outlined for compound 9. The
product 10 was obtained from the t-BOC protected precursor 8 in
an overall yield of 97%. IR (CHCl3): 3355, 1739 br cm−1 1H NMR
;
(CDCl3, 200 MHz) ı 0.86 (br t, 3H), 1.23 (br s, 24H), 1.54 (m, 2H),
2.24 (m, 5H), 2.80 (s, 3H), 3.34 (br s, 11H), 3.51 (br s, 8H), 3.65 (m,
6H), 3.83 (m, 2H), 4.04 (m, 2H), 4.17 (m, 2H), 4.34 (m, 2H), 5.28
(m, 1H), 6.51 (br t, 1H), 6.97–7.27 (m, 3H), 7.69 (dd, 4H, J = 5.5 Hz
and J = 8.9 Hz). 13C NMR (CDCl3, 50 MHz) ı 10.54, 14.06, 22.61,
24.76, 29.08, 29.24, 29.28, 29.45, 29.59, 29.63, 31.84, 33.48, 33.93,
39.30, 43.81, 54.37, 59.52, 62.35, 63.76, 66.23, 68.27, 69.59, 69.99,
70.29, 70.66, 71.19, 105.89, 106.36, 110.46, 110.91, 123.47, 123.79,
128.23, 129.62, 130.18, 133.06, 138.40, 139.55, 141.58, 145.42,
146.90, 160.77, 165.66, 169.47, 169.94, 173.40. 31P NMR (CDCl3,
160 MHz, pyrophosphate ref. ext.) ı −1.47. Rf (CHCl3/MeOH/H2O
65:25:4) 0.56. Anal. Calcd for C52H80FN2O13P·3/2·H2O·1/3CHCl3:
C, 56.81; H, 7.58; N, 2.52; Found: C, 56.92; H, 7.86; N, 2.34.
MS MH+ C52H80FN2O13PSH Calcd: 1023.5176, Found: 1023.5147.
[␣]D20 + 5.7 (c 1.14, CHCl3/MeOH 4:1).
51%) as pale-yellow solid. IR (CHCl3): 3330, 1737 br, 1532 cm−1
;
1H NMR (CDCl3, 200 MHz) ı 0.86 (br t, 3H), 1.23 (br s, 24H), 1.54
(m, 2H), 2.25 (t, 2H, J = 7.4 Hz), 3.24 (br s, 11H), 3.42 (m, 2H),
3.54 (br m, 6H), 3.73 (m, 4H), 4.04–4.17 (br m, 4H), 4.29 (m, 2H),
4.62 (m, 1H), 5.44 (m, 1H), 6.46 (d, 1H, J = 7.5 Hz), 6.85–6.98 (m,
3H), 7.24–7.32 (m, 3H), 8.08 (br s, 1H). 13C NMR (CDCl3, 50 MHz)
ı 14.05, 22.61, 24.77, 29.07, 29.27, 29.44, 29.62, 31.84, 33.95,
39.33, 40.37, 54.24, 59.37, 62.41, 63.77, 66.11, 68.31, 69.52, 70.01,
70.71, 71.48, 117.19, 121.06, 123.86, 125.49, 127.45, 128.78, 129.75,
130.67, 137.76, 143.24, 170.20, 172.44, 173.45. 31P NMR (CDCl3,
160 MHz, pyrophosphate ref. ext.) ı −1.28. Rf (CHCl3/MeOH/H2O
65:25:4) 0.56. Anal. Calcd for C44H70Cl2N3O11P·1/3CHCl3: C, 55.54;
H, 7.39; N, 4.38; Found: C, 55.58; H, 7.61; N, 4.40. MS MH+
C44H70Cl2N3O11PH Calcd: 918.4198, Found: 918.4232. [␣]D20 + 4.3◦
(c 0.87, CHCl3/MeOH 4:1).
3.4. 1-Palmitoyl-2-(8ꢀ-N-FMOC-amino-3,6-dioxaoctanoyl)-
sn-glycerophosphocholine (11)
To
a
suspension of 1-palmitoyl-2-hydroxy-sn-glycero-3-
phosphocholine (0.4012 g, 0.8 mmol) in 25 mL CHCl3 were added
8-N-FMOC-amino-3,6-dioxaoctanoic acid (0.3305 g, 0.86 mmol),
DCC (0.2482 g, 1.2 mmol), DMAP (0.1504 g, 1.2 mmol) and 1 g
of glass beads. The reaction was sonicated for 6 h at 25 ◦C. The
solvent was then evaporated and the residue was purified by silica
gel chromatography, first with CHCl3/MeOH (4:1), followed by
CHCl3/MeOH/H2O (65:25:4). The fractions of the product were
combined and freeze-dried from benzene to yield compound 11
3.6. 1-Palmitoyl-2-(8ꢀ-N-[5ꢀꢀ-dimethylaminonaphthalene-1ꢀꢀ-
sulfonyl]-amino-3,6-dioxaoctanoyl)-sn-glycerophosphocholine
(13)
To a solution of compound 11 (0.2115 g, 0.24 mmol) in 5 mL
CHCl3 was added DBU (0.1922 g, 1.2 mmol). The reaction mixture
was kept at room temperature for 1 h. To this solution were then
added dansyl chloride (0.1295 g, 0.48 mmol) and NEt3 (0.17 mL,
1.2 mmol), followed by 30 min later a second portion of dansyl
chloride (0.1287 g, 0.48 mmol). The reaction was over after 1 h, as
shown by a negative ninhydrin test on a TLC plate. The mixture was
purified directly by silica gel chromatography using CHCl3/MeOH
(4:1), to remove the impurities, followed by CHCl3/MeOH/H2O
(65:25:4) to elute the target phospholipid. The fractions corre-
sponding to the product were combined, evaporated, dispersed in
benzene and freeze-dried to give compound 13 (0.1028 g, 49%) as
(0.5134 g, 75%) as white solid. IR (CHCl3): 3330, 1732 br, 1690 cm−1
.
1H NMR (CDCl3, 200 MHz) ı 0.87 (br t, 3H), 1.24 (br s, 24H), 1.54 (m,
2H), 2.24 (t, 2H, J = 7.4 Hz), 3.30 (br s, 11H), 3.55–3.75 (br m, 6H),
3.98 (m, 2H), 4.15 (m, 4H), 4.24–4.50 (br m, 6H), 4.67 (m, 1H), 5.28
(m, 1H), 5.86 (m, 1H), 7.25–7.37 (m, 4H), 7.60 (d, 2H, J = 7.4 Hz),
7.73 (d, 2H, J = 7.4 Hz).13C NMR (CDCl3, 50 MHz) ı 13.93, 22.48,
24.63, 28.95, 29.11, 29.15, 29.32, 29.46, 31.71, 33.81, 40.60, 47.02,
54.03, 59.27, 62.33, 63.45, 65.97, 66.42, 68.15, 69.79, 70.59, 71.35,
119.75, 124.98, 126.87, 127.48, 141.04, 143.79, 156.42, 169.85,
173.26. Anal. Calcd for C45H71N2O12P·1/4CHCl3: C, 60.87; H, 8.04;
N, 3.14; Found: C, 60.64; H, 8.26; N, 3.03. MS MH+ C45H71N2O12PH
Calcd: 863.4817, Found: 863.4836. 31P NMR (CDCl3, 160 MHz,
pyrophosphate ref. ext.) ı −0.85. Rf (CHCl3/MeOH/H2O 65:25:4)
0.57. [␣]D20 + 5.2◦ (c 0.97, CHCl3/MeOH 4:1).
a white solid. IR (CHCl3): 3340, 1742 br cm−1 1H NMR (CDCl3,
;
200 MHz) ı 0.87 (br t, 3H), 1.24 (br s, 24H), 1.54 (m, 2H), 2.25
(t, 2H, J = 7.4 Hz), 2.85 (s, 6H), 2.97 (m, 2H), 3.32 (br s, 11H), 3.42
(m, 4H), 3.57 (m, 2H), 3.83 (m, 2H), 4.15 (br m, 4H), 4.30 (m, 2H),
4.67 (m, 1H), 5.32 (m, 1H), 7.15 (d, 1H, J = 7.4 Hz), 7.49–7.69 (m,
3H), 8.20 (d, 1H, 7.4 Hz), 8.38 (d, 1H, J = 7.4 Hz). 13C NMR (CDCl3,

R. Rosseto, J. Hajdu / Chemistry and Physics of Lipids 163 (2010) 110–116
115
50 MHz) ı 14.08, 22.64, 24.77, 29.10, 29.27, 29.31, 29.47, 29.66,
31.87, 33.95, 42.73, 45.38, 54.38, 59.65, 62.33, 64.06, 66.21, 68.40,
69.41, 69.99, 70.67, 71.50, 115.23, 119.41, 123.21, 128.26, 128.78,
129.65, 129.94, 135.59, 151.64, 170.24, 173.38. 31P NMR (CDCl3,
160 MHz, pyrophosphate ref. ext.) ı −1.72. Rf (CHCl3/MeOH/H2O
65:25:4) 0.40. Anal. Calcd for C42H72N3O12PS·CHCl3·C6H6: C, 54.92;
H, 7.43; N, 3.93; Found: C, 55.27; H, 7.71; N, 3.83. MS MH+
C42H72N3O12PSH Calcd: 874.4667, Found: 874.4602. [␣]D20 + 5.7◦
(c 1.04, CHCl3/MeOH 4:1).
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In a typical experiment, to a sample of phosphatidylcholine 9
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Acknowledgment
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We are grateful to the National Institutes of Health, grant 2S06
GM/HD48680 for financial support.
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