Design and synthesis of a stable oxidized phospholipid mimic with specific binding recognition for macrophage scavenger receptors

Article abstract of DOI:10.1021/jm300685s

Macrophage scavenger receptors appear to play a major role in the clearance of oxidized phospholipid (OxPL) products. Discrete peptide-phospholipid conjugates with the phosphatidylcholine headgroup have been shown to exhibit binding affinity for these receptors. We report the preparation of a water-soluble, stable peptide-phospholipid conjugate (9) that possesses the necessary physical properties to enable more detailed study of the role(s) of OxPL in metabolic disease.

Full text of DOI:10.1021/jm300685s

Brief Article
pubs.acs.org/jmc
Design and Synthesis of a Stable Oxidized Phospholipid Mimic with
Specific Binding Recognition for Macrophage Scavenger Receptors
William W. Turner,† Karsten Hartvigsen,‡,§ Agnes Boullier,∥ Erica N. Montano,‡ Joseph L. Witztum,*,‡
and Michael S. VanNieuwenhze*,†
†Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States
‡Department of Medicine, University of California, San Diego, La Jolla, California 92093-0682, United States
§Department of Biomedical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark
∥INSERM U1088, UFR de Med
ecine, Universite Picardie Jules Verne and CHU Amiens, FR-80054 Amiens, France
́ ́
S
* Supporting Information
ABSTRACT: Macrophage scavenger receptors appear to play a major role in the clearance of oxidized phospholipid (OxPL)
products. Discrete peptide−phospholipid conjugates with the phosphatidylcholine headgroup have been shown to exhibit
binding affinity for these receptors. We report the preparation of a water-soluble, stable peptide−phospholipid conjugate (9) that
possesses the necessary physical properties to enable more detailed study of the role(s) of OxPL in metabolic disease.
INTRODUCTION
■
Oxidized low-density lipoprotein (OxLDL) is believed to play
an important role in the pathogenesis of atherosclerosis.1
Unregulated uptake of OxLDL by macrophages within the
arterial wall leads to foam cell formation followed by
development of the fatty streak that is typical of early
atherosclerotic lesions.1,2 Macrophages express a number of
scavenger receptors that bind OxLDL.3 Among these is CD36,4
which recent evidence suggests is important in the uptake of
OxLDL by macrophages and may have a significant role in
inflammation and in foam cell formation in vivo.5 The
epitope(s) responsible for recognition of OxLDL by CD36,
and all of the other scavenger receptors, have been only
partially defined. Identification of the detailed structural
features on OxLDL that are responsible for recognition by
CD36 could provide a template for the design of compounds
with highly specific interactions with these macrophages and
other immune system components with the eventual goal of
developing new strategies for the treatment of inflammation
and atherosclerosis.
A wide variety of biologically active phospholipid oxidation
products can be formed upon oxidation of phospholipids.6 For
example, oxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-
phosphorylcholine (PAPC, 1) yields an oxidized phospholipid
(OxPL) byproduct, 1-palmitoyl-2-(5′-oxo)valeroyl-sn-glycero-
3-phosphoryl-choline (POVPC, 2), with a reactive aldehyde at
the ε-carbon (Figure 1).7 This reactive “phospholipid core
aldehyde” in turn forms adducts with lysine residues of apoB, or
other proteins, as well as with other amine containing
phospholipids.8
Figure 1. Biological oxidation of PAPC to POVPC.
α,β-unsaturated carbonyl groups.9c,d Our laboratory has shown
that the phosphocholine (PC) headgroup of oxidized
phosphatidylcholines (but not native PL) forms a different
and sufficient ligand. Detailed studies of POVPC and variously
modified conjugates revealed that the PC headgroup is
sufficient for binding to CD36, and furthermore, this activity
was retained even after the conjugation of the OxPL to a
peptide or protein, mediated by the sn-2 aldehyde to yield a
Schiff’s base.10 Initial experiments demonstrated that Schiff’s
base conjugates of POVPC with bovine serum albumin (BSA)
inhibited binding of OxLDL to CD36. In a similar manner,
POVPC−BSA inhibited the binding of the monoclonal
antibody (mAb) E06, which specifically binds the PC
headgroup of OxPL but not native PL. Thus, both CD36 and
E06 specifically bind the PC of OxPL. A POVPC conjugate
with a short peptide chain (3, Figure 2) containing a single
lysine residue for imine formation also inhibited binding of
OxLDL to both CD36 and E06.9
The aqueous solution of 3 slowly lost its activity in the assay
as the imine hydrolyzed. In an effort to improve the aqueous
stability of the Schiff’s base conjugate, the imine intermediate
was reduced to the corresponding amine (4) with sodium
cyanoborohydride. This compound retained its competitive
Previous work has shown that binding of OxLDL to CD36 is
mediated by oxidized phospholipids (OxPLs).8a,9 Several
different epitopes on OxPLs have been described as ligands
for CD36. One set of ligands results from oxidized moieties on
sn-2 side chain that have the common motif of oxidized and
truncated sn-2 fatty acids that terminate in γ-hydroxy (or oxo)-
Received: May 16, 2012
Published: August 30, 2012
© 2012 American Chemical Society
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Brief Article
Scheme 1. Synthetic Route to 9
Figure 2. Imine and amine adducts of POVPC.
binding activity but was still prone to a slow decomposition in
aqueous solution, presumably the result of delipidation arising
from intramolecular O′N-acyl transfer (Figure 3).
Figure 3. Proposed mechanism for delipidation.
We desired a stable compound with high selectivity and
suitable aqueous solubility which would bind to CD36 and to
mAb E06 and could be used as a probe in future biological
studies and models of atherosclerosis. Instability of both the
imine and amine adducts meant that the compounds generated
to this point did not meet this goal. Therefore, we set out to
find a water-soluble, more stable analogue that maintained the
binding specificity of the original POVPC conjugates.
predominately monoalkylation followed by the addition of
excess 37% aqueous formaldehyde solution to provide the
second alkylation and the tertiary amine. Using this technique,
mixtures with about 80% of the appropriate mixed dialkylated
amine could be generated with only minor amounts of the
homo-bis-dialkylation product(s). After purification, the benzyl
ester of 7 was removed by hydrogenation to unmask the acid
group needed for coupling to lyso-PC. A survey of the literature
uncovered a carbodiimide ester coupling technique using
sonication that greatly improved reaction yields and shortened
reaction times.11 The carboxylic acid was converted to the
corresponding ester using lyso-PC (3 equiv), diisopropylcarbo-
diimide (DIC) (2 equiv), and 4-dimethylaminopyridine
(DMAP) (2 equiv) in chloroform in only 2 h with a yield of
68% using sonication in the presence of glass beads to increase
the glass surface area. Couplings done using more traditional
solution methodology took several days with yields of 20−30%.
The product was globally deprotected (TFA, 0 °C, 2 h) to give
8, and the amino terminus of the peptide was acylated (1-
acetylimidazole, DIPEA, CH2Cl2) to give target compound 9.
The competition for binding of 9 was measured as shown in
Figure 4. Compound 9 efficiently competes for binding of
biotinylated CuOxLDL to J774 murine macrophages. Com-
pound 9 as well as the positive control, unlabeled CuOxLDL,
compete >99% and >90%, respectively, with biotinylated
CuOxLDL binding to macrophages, whereas the control
peptide TGTKGG and native LDL do not compete. The
fixed concentration of biotinylated CuOxLDL (1.8 pmol apoB/
mL, corresponding to 1 μg apoB/mL or 0.14 nmol PC-
RESULTS
■
To achieve the desired stability, we felt that modification of the
ε-amine of lysine would provide the simplest solution.
Conversion of the secondary amine to a tertiary amine should
eliminate the acyl transfer reaction that led to the delipidated
compound. Our previous work suggested that the amino acid
sequence or length of the peptide portion of the mimic was not
critical for activity, so we chose to slightly alter the peptide
sequence of 4 by replacing the tyrosine with smaller, less-
lipophilic glycine to optimize water solubility. These changes
produced a new target compound 9, which was synthesized by
the route shown Scheme 1.
The appropriately protected hexapeptide (5) was prepared
using a manual Fmoc solid phase peptide synthesis followed by
esterification of the acid terminus (see Supporting Information
(SI)). The ε-amine of the lysine unit of 5 was revealed by
cleavage of the carbonyl benzyloxy (Cbz) protecting group, and
a double reductive alkylation was performed to generate the
tertiary amino compound (7). This single-pot double alkylation
was conducted by first treating the amine with 1.3 equiv of
aldehyde 6 (NaCNBH3, AcOH, CH3CN, H2O, 1 h), giving
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Figure 4. Compound 9 efficiently competes with binding of OxLDL to macrophages. (A) In vitro binding experiment showing that biotinylated
CuOxLDL (diamonds) bound in a dose-dependent and saturable manner to J774 macrophages, whereas native LDL (circles) did not. Data are given
as relative light units (RLU) per 100 ms. Plot is a representative experiment of >4. (B) CuOxLDL (diamonds), but not native LDL (circles),
competes for Bt-CuOxLDL (1.5 μg/mL) binding to J774 macrophages. Data are shown as mean SD of B/B0 from two independent experiments
with triplicate determinations. (C) Compound 9 (triangles), but not control peptide TGTKGG (squares), competes for Bt-CuOxLDL (1.5 μg/mL)
binding to J774 macrophages. Data are shown as mean SD of B/B0 from two independent experiments with triplicate determinations. Data in (B)
and (C) were produced in parallel experiments.
epitopes/mL) was preincubated with compound 9, the control
peptide TGTKGG, CuOxLDL, or native LDL, added at the
concentrations indicated on the graphs, and then tested for
binding as described in the Experimental Section. The highest
CuOxLDL concentration tested was 200 μg/mL, which
corresponds approximately 28.3 nmoles of PC-epitopes/mL,
as estimated from our previous observation that 1 mol of apoB-
EXPERIMENTAL SECTION
■
General. See SI for details. All final compounds were confirmed to
be of >95% purity based on HPLC analysis.
(6S,12S,15S)-tert-Butyl-15-(4-((5-(benzyloxy)-5-oxopentyl)-
(methyl)amino)butyl)-6,12-bis((R)-1-(tert-butoxy)ethyl)-2,2-di-
methyl-4,7,10,13,16,19-hexaoxo-3-oxa-5,8,11,14,17,20 hexaa-
zadocosan-22-oate 2,2,2-trifluoroacetate Salt (7). A solution of
5 (2.5 g, 2.7 mmol) in ethanol (60 mL) to a slurry of 10% Pd/C (660
mg) in 20 mL of ethanol. Acetic acid (185 μL) was added, and the
solution was put under a balloon of hydrogen gas for 3 h. Completion
of the Cbz removal was confirmed by HPLC. The catalyst was
removed by filtration through a filter disk, and the solvent was
removed in vacuo to give the acetic acid salt of the amine intermediate
as a foam (2.07 g, 2.44 mmol, 90% yield). The foam was dissolved in
acetonitrile (15 mL) and water (2 mL). Benzyl 5-oxopentanoate (6)
(0.654 g, 3.17 mmol, 1.3 equiv) was dissolved in acetonitrile and
added to the reaction mixture (total volume 20 mL). Acetic acid
(0.559 mL, 9.76 mmol, 4 equiv) was added next, followed by a
solution of sodium cyanoborohydride (0.92 g, 14.65 mmol, 6 equiv) in
1 mL of water. The reaction was stirred at room temperature, and the
pH was checked (pH 5−6). After 1 h, a 37% formaldehyde solution in
water (0.5 mL, ∼6 mmol) was added and stirring was continued for an
additional hour. The reaction solution was made basic with 2N NaOH
solution, brine was added, and the mixture was extracted with ether
(3×). The aqueous layer was next extracted with dichloromethane
(2×). The combined organic extracts were dried over magnesium
sulfate and reduced in vacuo to give 1.13 g of crude product as a white
foam. HPLC analysis (Phenomenex C18(2) reverse-phase column,
50−90% acetonitrile/water over 10 min, buffer 0.1% trifluoroacetic
acid, product retention time 7.25 min) indicated that 79% of the crude
material was the desired product. The crude product was dissolved in
50% ACN/water with 0.1% TFA (20 mL), and trifluoroacetic acid
(181 μL) was added to protonate the amine. Purification over a
preparative C18 reverse-phase column, using the same conditions as
used for the analytical runs, and lyophilization gave 7 (1.24 g, 46%
100 from CuOxLDL contains 78
15 mol of covalently
attached phosphorus,10 which we assume are OxPL-PC-
epitopes only. The concentration of native LDL is plotted at
equal protein concentration to CuOxLDL (and thus not as
molar concentration of PC-epitopes). Each data point
represents the mean and SD of two independent experiments
of triplicate wells. In data provided in the SI (Figure S1), we
specifically demonstrate that compound 9 competes with high
affinity for the binding of CuOxLDL to the CD36 scavenger
receptor. These data imply that compound 9 should provide a
practical and useful agent for further biological studies in this
area. In addition, we show that mAb E06 specifically binds to
compound 9 as expected (Figure S2, SI).
CONCLUSION
■
These data demonstrate that compound 9 represents a water-
soluble, stable POVPC−peptide adduct12 that binds to
macrophage scavenger receptors and is able to compete with
the binding of OxLDL with high affinity. This compound
appears to meet the requirements specified. Because increasing
data support a major proinflammatory role for OxPL in disease,
this stable model POVPC−peptide adduct should be highly
useful in future studies to understand many of the biological
roles of oxidized phospholipids and their adducts with proteins.
In addition, it may be used to target imaging agents or drugs to
macrophages, thus enabling macrophage-specific diagnostic and
therapeutic possibilities.
1
yield) as a white solid. H NMR (400 MHz, DMSO-d6) δ 0.99 (d,
3H), 1.02 (d, 3H), 1.10 (s, 9H), 1.13 (s, 9H), 1.20−1.35 (m, 4H), 1.39
(s, 18H), 1.52−1.67 (m, 5H), 1.67−1.79 (m, 1H), 2.43 (t, 2H), 2.70
(d, 3H), 2.87−3.15 (m, 4H), 3.75 (m, 4H), 3.83 (m, 2H), 3.88 (m,
2H), 3.96 (m, 1H), 4.30−4.36 (m, 2H), 5.10 (s, 2H), 7.30−7.40 (m,
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5H), 7.75 (d, 1H), 7.97 (d, 1H), 8.07 (t, 1H), 8.14−8.21 (m, 2H),
9.33 (br s, 1H). 13C NMR (400 MHz, DMSO-d6) δ 19.2, 20.1, 21.8,
23.2, 28.1, 28.3, 28.4, 31.9, 33.2, 41.6, 41.9, 42.6, 52.6, 54.9, 55.2, 57.6,
59.5, 65.9, 67.6, 68.0, 74.0, 74.4, 78.8, 81.1, 128.4, 128.5, 128.9, 136.6,
155.5, 158.4, 158.8, 168.9, 169.2, 169.3, 169.5, 170.65, 171.7, 172.8.
HRMS (ESI) C50H86N7O13 (M + H+) calcd 992.6284, found
992.6251.
18 h. Complete conversion to product was seen when the reaction was
checked by HPLC (Phenomenex C18(2) reverse-phase column, 30−
90% acetonitrile/water over 10 min, buffer 0.1% TFA, product
retention time 9.3 min). The solvent was removed in vacuo, and the
residual oil was dissolved in 50% acetonitrile/water and purified over a
preparative HPLC column using the same conditions as for the
analytical HPLC. The pure fraction was lyophilized overnight. The
sample was relyophilized from water to give 25.6 mg (60% yield) of 9
as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 0.71, (t, 3H), 0.89
(d, 3H), 0.92 (d, 3H), 1.05−1.70 (m, 34H), 1.78 (s, 3H), 2.10−2.20
(m, 3H), 2.25−2.40 (m, 6H), 2.80 (s, 4H), 2.99 (s, 9H), 3.42 (s, 3H),
3.54−3.70 (m, 7H), 3.70−3.90 (m, 4H), 3.95−4.05 (m, 4H), 4.05−
4.15 (4H), 4.90 (br s, 1H), 5.05 (s, 1H), 7.62 (d, 1H), 7.84 (d, 1H),
7.93 (s, 2H), 8.09 (t, 1H), 8.16 (s, 1H). 13C NMR (400 MHz, DMSO-
d6) δ 14.4, 20.0, 20.2, 22.5, 22.9, 23.2, 24.8, 28.8, 29.1, 29.3, 29.4, 29.5,
31.4, 31.7, 33.7, 33.8, 41.1, 42.2, 42.7, 52.8, 53.6, 55.3, 58.6, 59.7, 59.4,
62.5, 63.9, 65.8, 66.8, 67.2, 71.0, 169.4, 169.6, 170.5, 170.6, 171.4,
171.6, 172.1, 172.4, 173.1. HRMS (ESI) C52H98N8O18P (M + H+)
calcd 1153.6737, found 1153.6749.
CD36 Macrophage Receptor Binding Competition Assay.
Binding of biotinylated OxLDL ligands to murine macrophages plated
in microtiter wells was assessed by a chemiluminescent binding assay
as previously described with modifications.13 Isolated human LDL was
biotinylated according to manufacturer’s protocol (catalogue no.
21326; Pierce Biotechnology). Native and biotinylated native LDL
were subjected to copper sulfate oxidation8a in parallel to prepare
unlabeled and biotinylated CuOxLDL ligands, respectively. We have
previously shown that CuOxLDL contains approximately 78 mol of
PC-epitopes per mol of apoB-100 (the sole protein on LDL).8a The
biotinylated native LDL and CuOxLDL ligands of equal protein
concentration were serially diluted and tested for binding to adherent
macrophages. The specificity of the binding of biotinylated CuOxLDL
to macrophages was tested in competition experiments, where fixed
concentrations of biotinylated CuOxLDL were incubated with the
serially diluted competitor (9) and controls in PBS at concentrations
indicated in Figure 2. The ligand-competitor solutions were incubated
overnight at 4 °C. Murine macrophages from the J774 cell line were
cultured in 10% fetal bovine serum in DMEM (DMEM-10) and plated
in 100 μL of L929-fibroblast conditioned media at 100000 or 25000
cells/well, respectively, in sterile 96-well flat-bottom white plates
(Greiner Bio-One). The plating media consisted of 20% L929-
fibroblast conditioned DMEM-10 and 80% fresh DMEM-10 and
served as a source of growth factors, including macrophage colony-
stimulating factor. After 72 h, plates were washed gently 5 times with
PBS using a microtiter plate washer (Dynex Technologies, Chantilly,
VA), and wells were blocked with ice-cold 200 μL of DMEM for 30
min while plates were kept on ice. After washing, macrophages were
incubated with ice-cold ligand and ligand-competitor solutions (100
μL/well) for 2 h on ice, washed again, and fixed with ice-cold 3.7%
formaldehyde in PBS for 30 min in the dark. After fixing the
macrophages, the remaining of the assay was carried out at room
temperature. Macrophage-bound biotinylated OxLDL ligands were
detected with NeutrAvidin-conjugated alkaline phosphatase (Pierce
Biotechnology), LumiPhos 530 (Lumigen, Southfield, MI), and a
Dynex Luminometer (Dynex Technologies). Ligand binding was
recorded and expressed as relative light units counted per 100 ms
(RLU/100 ms) or in the case of inhibition of binding by competitors
as a ratio of binding in the presence of competitor (B) divided by
binding in the absence of competitor (B0). In preliminary studies, we
have shown that at the concentrations used, there was no impact of
compound 9 on viability of macrophages even at room temperature for
up to 72 h as judged by cell number or protein content at the end of
experiment or by detailed time course studies of cell function (data not
shown). The binding studies shown here, however, were conducted on
ice.
(2R,14S,17S,23S,24R)-23-Ammonio-14-((2-((carboxymeth-
yl)-amino)-2-oxoethyl)carbamoyl)-24-hydroxy-17-((R)-1-hy-
droxyethyl)-9-methyl-4,16,19,22-tetraoxo-2-((palmitoyl-oxy)-
methyl)-3-oxa-9,15,18,21-tetraazapentacosyl-(2-
(trimethylammonio)ethyl)phosphate 2,2,2-Trifluoroacetate
(8). A catalyst slurry was formed by placing 370 mg of 10% Pd/C
in a dry flask and adding ethanol (3 mL). A solution of 7 (1.24 g, 1.121
mmol) in ethanol (15 mL) was added to the slurry, and the mixture
was put under a balloon of hydrogen for 3 h. An aliquot was checked
by HPLC (Phenomenex C18(2) reverse-phase column, 50−90%
acetonitrile/water over 15 min, buffer 0.1% trifluoroacetic acid,
product retention time 4.6 min) and showed complete conversion
to product. The catalyst was removed by filtration, and the solution
was reduced in vacuo to give 1.07 g (94% yield) of the acid as a foam/
oil. (HRMS (ESI) C43H80N7O13 (M + H+) calcd 902.5814, found
902.5836). The acid (1.07 g, 1.053 mmol, 1 equiv) and lyso-PC (1.566
g, 3.16 mmol, 3 equiv) were dissolved in toluene with sonication in the
reaction flask. The solvent was removed in vacuo to eliminate any
residual ethanol (2×). The flask was put under high vacuum using a
vacuum pump for 2 days to complete solvent removal. The solid was
slurried in ethanol-free chloroform (40 mL), and diisopropyl
carbodiimide (DIC) (0.326 mL, 2.1 mmol, 2 equiv) was added
followed by DMAP (257 mg, 2.1 mmol, 2 equiv). Glass beads were
added to just below the solvent surface, and the reaction flask was
sonicated for 2 h. The solution was pipetted away from the glass beads
and the beads washed with additional chloroform. The combined
solutions were reduced in vacuo to give 4.24 g of crude product.
HPLC (Phenomenex C18(2) reverse-phase column, 50−90%
acetonitrile/water over 10 min, buffer 0.1% trifluoroacetic acid,
product retention time 13.8 min) showed complete conversion to
product. The material was dissolved in 50% acetonitrile/water and
purified by HPLC (Phenomenex C18(2) reverse-phase column, 10
μm, 250 mm × 30 mm, 40 mL/min, 60−90% acetonitrile/water over
15 min, buffer 0.1% TFA). The product fraction was partially reduced
in vacuo to remove most of the acetonitrile and lyophilized. The
lyophilate was collected in dichloromethane and reduced again to give
the product oil. Drying overnight under high vacuum gave 1.07 g (68%
yield) of the coupled product. This material was dissolved in
trifluoroacetic acid (5 mL) and stirred in an ice bath for 2.5 h. The
solvent was removed in vacuo and the residue dissolved in
dichloromethane and reduced in vacuo to give a product oil. HPLC
(Phenomenex C18(2) reverse-phase column, 30% acetonitrile/water
for 2 min, then to 90% acetonitrile at 10 min, buffer 0.1%
trifluoroacetic acid, product retention time 9.59 min (98% pure))
The oil was dissolved in water and lyophilized to give 846.2 mg (88%
yield) of 8 as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 0.85 (t,
3H), 1.04 (d, 3H), 1.19 (d, 3H), 1.19−1.80 (m, 34H), 2.28 (t, 2H),
2.30−2.45 (m, 2H), 2.66 (d, 3H), 2.80−3.08 (m, 5H), 3.13 (s, 9H),
3.57 (s, 2H), 3.62 (br s, 1H), 3.75 (m, 4H), 3.80−4.05 (m, 7H), 4.10−
4.35 (m, 7H), 5.18 (s, 1H), 5.5 (br s, 1H), 8.01 (m, 2H), 8.05−8.20
(m, 4H), 8.32 (m, 1H), 8.83 (t, 1H), 10.80 (s, 1H). 13C NMR (400
MHz, DMSO-d6) δ 14.4, 20.1, 20.2, 22.5, 22.9, 23.2, 24.8, 28.8, 29.1,
29.3, 29.4, 29.5, 31.7, 33.7, 41.0, 42.1, 42.3, 53.5, 58.6, 59.3, 62.4, 64.0,
65.6, 66.3, 67.0, 70.8, 158.3, 158.6, 167.6, 168.9, 169.4, 170.3, 171.5,
172.1, 173.1. HRMS (ESI) C50H96N8O17P (M + H+) calcd 1111.6631,
found 1111.6614.
(4S,10S,13S,25R)-13-((2-((Carboxymethyl)amino)-2-
oxoethyl)carbamoyl)-4,10-bis((R)-1-hydroxyethyl)-18-methyl-
2,5,8,11,23-pentaoxo-25-((palmitoyloxy)methyl)-24-oxa-
3,6,9,12,18-pentaazahexacosan-26-yl-(2-(trimethylammonio)-
ethyl)phosphate (9). Compound 8 (49.8 mg, 0.037 mmol, 1 equiv)
was dissolved in 2 mL of anhydrous dichloromethane. 1-
Acetylimidazole (6.1 mg, 0.056 mmol, 1.5 equiv) and DIPEA (25.9
μL, 0.149 mmol, 4 equiv) were added, and the reaction was stirred for
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(7) Watson, A. D.; Leitinger, N.; Navab, M.; Fauli, K. F.; Horkko, S.;
̈
̈
ASSOCIATED CONTENT
■
Friedman, P.; Dennis, E. A.; Witztum, J. L.; Palinksi, W.; Schwenke,
D.; Salomon, R. G.; Sha, W.; Subbanagounder, G.; Fogelman, A. M.;
Berliner, J. A. Structural Identification by Mass Spectrometry of
Oxidized Phospholipids in Minimally Oxidized Low Density Lip-
oprotein that Induce Monocyte/Endothelial Interactions and Evidence
for Their Presence in Vivo. J. Biol. Chem. 1997, 272, 13597−13607.
S
* Supporting Information
Supplemental figures and experimental data for compounds 5
and 6. This material is available free of charge via the Internet at
http://pubs.acs.org.
(8) (a) Gillotte, K. L.; Horkko, S.; Witztum, J. L.; Steinberg, D.
̈
̈
AUTHOR INFORMATION
■
Oxidized phospholipids, linked to apolipoprotein B of oxidized LDL,
are ligands for macrophage scavenger receptors. J. Lipid Res. 2000, 41,
824−833. (b) Kamido, H.; Kuksis, A.; Marai, L.; Myher, J. J. Lipids
1993, 28, 331−336. (c) Kamido, H.; Kuksis, A.; Marai, L.; Myher, J. J.
Lipid−ester bound aldehydes among copper-catalyzed peroxidation
products of human plasma lipoproteins. J. Lipid Res. 1995, 36, 1876−
1886.
Corresponding Author
*Phone: 1-812-856-7545. Fax: 1-812-855-8300. E-mail:
mvannieu@indiana.edu.
Notes
The authors declare no competing financial interest.
(9) (a) Bird, D. A.; Gillotte, K. L.; Horkko, S.; Friedman, P.; Dennis,
̈
̈
ACKNOWLEDGMENTS
E. A.; Witztum, J. L.; Steinberg, D. Receptors for Oxidized Low-
Density Lipoprotein on Elicited Mouse Peritoneal Macrophages Can
Recognize Both the Modified Lipid Moieties and the Modified Protein
Moieties: Implications with Respect to Macrophage Recognition of
Apoptotic Cells. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6347−6352.
■
This work was supported by NIH grant GM069338 (W.W.T.,
M.S.V., J.L.W.), HL088093 (J.L.W.), and by HL086559 (K.H.
and J.L.W.).
(b) Boullier, A.; Gillotte, K. L.; Horkko, S.; Green, S. R.; Friedman, P.;
̈
̈
ABBREVIATIONS USED
Dennis, E. A.; Witztum, J. L.; Steinberg, D.; Quehenberger, O. The
binding of oxidized low density lipoprotein to mouse CD36 Is
mediated in part by oxidized phospholipids that are associated with
both the lipid and protein moieties of the lipoprotein. J. Biol. Chem.
2000, 275 (13), 9163−9169. (c) Podrez, E. A.; Poliakov, E.; Shen, Z.;
Zhang, R.; Deng, Y.; Sun, M.; Finton, P. J.; Shan, L.; Febbraio, M.;
Hajjar, D. P.; Silverstein, R. L.; Hoff, H. H.; Salomon, R. G.; Hazen, S.
L. A novel family of atherogenic oxidized phospholipids promotes
macrophage foam cell formation via the scavenger receptor CD36 and
is enriched in atherosclerotic lesions. J. Biol. Chem. 2002, 277, 38517−
38523. (d) Podrez, E. A.; Poliakov, E.; Shen, Z.; Zhang, R.; Deng, Y.;
Sun, M.; Finton, P. J.; Shan, L.; Gugiu, B.; Fox, P. L.; Hoff, H. H.;
Salomon, R. G.; Hazen, S. L. Identification of a novel family of
oxidized phospholipids that serve as ligands for the macrophage
receptor CD36. J. Biol. Chem. 2002, 277, 38503−38516.
■
OxLDL, oxidized low-density lipoprotein; CuOxLDL, copper-
oxidized low-density lipoprotein; OxPL, oxidized phospholipid;
lyso-PC, 1-palmitoyl-sn-glycero-3-phosphorylcholine; DIC,
diiso-propylcarbodiimide; POVPC, 1-palmitoyl-2-(5′-oxo)-
valeroyl-sn-glycero-3-phosphorylcholine; PAPC, 1-palmitoyl-2-
arachidonyl-sn-glycero-3-phosphorylcholine
REFERENCES
■
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