Synthetic phospholipids as specific substrates for plasma endothelial lipase

Article abstract of DOI:10.1016/j.bmcl.2016.06.032

We designed and prepared synthetic phospholipids that generate lyso-phosphatidylcholine products with a unique mass for convenient detection by LC–MS in complex biological matrices. We demonstrated that compound 4, formulated either as a Triton X-100 emulsion or incorporated in synthetic HDL particles can serve as a substrate for plasma EL with useful specificity.

Full text of DOI:10.1016/j.bmcl.2016.06.032

Bioorganic & Medicinal Chemistry Letters 26 (2016) 3514–3517
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthetic phospholipids as specific substrates for plasma endothelial
lipase
b,
b
b
a
Julien P. N. Papillon a, , Meihui Pan , Margaret E. Brousseau , Mark A. Gilchrist , Changgang Lou ,
⇑
⇑
Alok K. Singh a, Todd Stawicki b, James E. Thompson b
a Global Discovery Chemistry, Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, United States
b Cardiovascular and Metabolism, Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and prepared synthetic phospholipids that generate lyso-phosphatidylcholine products
with a unique mass for convenient detection by LC–MS in complex biological matrices. We demonstrated
that compound 4, formulated either as a Triton X-100 emulsion or incorporated in synthetic HDL parti-
cles can serve as a substrate for plasma EL with useful specificity.
Received 17 April 2016
Revised 10 June 2016
Accepted 11 June 2016
Available online 14 June 2016
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Synthetic phospholipids
Endothelial lipase
Plasma assay
Tandem mass spectrometry
HPLC–MS/MS
Lipid analysis
Lipid mass spectrometry
Low levels of plasma high density lipoprotein cholesterol (HDL-
C) are a risk factor for atherosclerotic cardiovascular disease, inde-
pendent of the benefit statins provide by reducing low density
lipoprotein cholesterol (LDL-C) levels.1 Endothelial lipase (EL), a
member of the triglyceride lipase gene family that includes hepatic
lipase (HL) and lipoprotein lipase (LPL), has been shown to be a
negative regulator of plasma HDL-C levels in both mice and
humans,2 and multiple lines of evidence suggest that pharmaco-
logical inhibition of EL enzymatic activity might be beneficial for
the treatment of atherosclerosis.3 In order to evaluate the efficacy
of EL inhibitors in vivo, the ability to correlate plasma EL enzymatic
activity with plasma lipoprotein levels is desirable. While EL, LPL
and HL have different lipoprotein substrate preferences, they are
all competent to hydrolyze phospholipids (PL), as well as triglyc-
erides.4 Moreover, their lipolysis products, free fatty acids (FFA)
and lyso-phosphatidylcholines (lysoPC or LPC) are abundant in
plasma. Therefore, developing a sensitive and specific assay to
measure EL activity with minimal processing of plasma samples
to support characterization of EL inhibitors represents a significant
challenge. Although multiple screening assays using fluorophore-
labeled phospholipid substrates have been reported,5 there are
few reports addressing the specific measure of plasma EL activity.
Radiometric assays using [1,2-14C]-dipalmitoylphosphatidyl-
choline (DPPC) have historically been used to measure the general
phospholipase activities in plasma or cell culture samples.6 This
method requires large volume of samples, laborious multiple-step
processing of radioactive material, but more importantly, DPPC is
not a specific substrate for the phospholipases. The application of
more phospholipase A1 (PL-A1) specific fluorescent substrates in
plasma has been the object of several reports.7 We envisioned that
using synthetic phospholipids as substrates to determine EL activ-
ity in plasma samples should have two advantages: they could be
optimized to be specific substrates for EL, and the lysoPC product
could be chosen so that its mass was distinct from endogenous
lipids, and could be readily detected by LC–MS. Progress towards
this goal is reported herein.
EL is a PL-A1, i.e., it catalyzes the hydrolysis of the ester on the
first carbon of the glycerol molecule (denoted sn-1) preferentially
over the ester on the second carbon of the glycerol molecule
(denoted sn-2). We surmised that the majority of background
hydrolysis could be suppressed by replacing the sn-2 ester with
functionalities that would not be readily hydrolyzed. As shown in
Table 1, we designed ethers (1–3) and carbamates (4–5) whose
lysoPC mass would be distinct from circulating phospholipids.
⇑
Corresponding authors. Tel.: +1 871 7653 (J.P.N.P.), +1 871 7336 (M.P.).
E-mail addresses: julien.papillon@novartis.com (J.P.N. Papillon), meihui.pan@
novartis.com (M. Pan).
http://dx.doi.org/10.1016/j.bmcl.2016.06.032
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

J. P. N. Papillon et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3514–3517
3515
Table 1
O
a
b
Synthetic phospholipids investigated in these studies
OTr
OTr
O
C15H31
O
sn-1
OH
15
O
16
O
P
O
O
O
O
N+
P
N+
R
d
O
O
OR
O
O
O
O
O
O
C15H31
C15H31
O
sn-2
O
O
O
R'
6
17 R=Tr
18 R=H
c
C17H33
C17H33
R
R0
Formula
Exact mass
LPC
Scheme 2. Synthesis of ester 6. Reagents and conditions: (a) palmitic acid,
tetraethylammonium bromide, 100 °C, 3 h, 65%. (b) Oleic acid, DCC, DMAP,
chloroform, rt, 2 days, 88%. (c) TFA, DCM, rt, 3 days, 42%. (d) 18, 2-chloro-2-oxo-
1,3,2-dioxaphospholane, TEA, toluene, 2 days, rt, then switch solvent to acetonitrile,
trimethylamine, ꢀ78 °C to 65 °C, 36 h, 28%.
PC
1
2
3
4
5
6
Myristate
Palmitate
Myristate
Palmitate
Myristate
Palmitate
–C12H25
–C12H25
–C6H13
–C(O)NHC14H29
–C(O)NHC14H29
Oleatea
C34H70NO7P
C36H74NO7P
C28H58NO7P
C39H79N2O8P
C37H75N2O8P
C42H83NO8P
635.5
663.5
551.4
734.5
706.5
760.5
425.3
425.3
341.2
496.3
496.3
522.3
a
Inverse configuration at sn-2.
We also investigated inversion of configuration at the glycerol chi-
ral center, as in 6. Syntheses of 1–5 started from glycerophospho-
choline (7), which was tritylated at the less hindered hydroxyl in
good yields (Scheme 1). Alkylation of the secondary alcohol in 8
to give 9 and 10 was accomplished using dimsyl sodium, freshly
prepared using sodium hydride in DMSO, and the appropriate
iodoalkane. Carbamate 11 was prepared by microwave heating 8
with the corresponding isocyanate. TFA was employed to cleave
the trityl group in 9–11. Finally sn-1 ester formation was accom-
plished with DCC/DMAP and the appropriate acid to give 1–5. In
order to prepare a PC with inverted configuration, we started with
enantiopure epoxide 15 (Scheme 2). Mixing 15 and palmitic acid
with tetraethylammonium bromide and heating to 100 °C gave
secondary alcohol 16 in 65% yield.8 DCC/DMAP esterification, fol-
lowed with TFA treatment afforded 18. Conversion of 18 to PC 6
was achieved according to a published protocol.9
Figure 1. LysoPC production by recombinant EL, HL and purified LPL in assay buffer
for PL 1–6 after a 60 min incubation, expressed as a percentage of lyso-DPPC control
(n = 2).
All PLs were formulated as Triton X-100 emulsions, based on a
published protocol,6 and after 60 min of incubation with the lipase
preparations, lysoPC products were quantified by LC–MS. We
found that all synthetic PLs could serve as substrates for recombi-
nant human EL (Fig. 1, black bars). However, ethers 1–3 and ester 6
were relatively poor substrates, with only 7–20% lysoPC produced
compared to DPPC. In contrast, the carbamates 4 and 5 were com-
parable to DPPC, with amounts of lysoPC generated as 52% and 58%
that of DPPC, respectively. All six synthetic PLs were poor sub-
strates for recombinant HL (Fig. 1, red bars), relative to DPPC,
and in contrast to EL, HL showed no substrate preference. In the
case of LPL, only ester 6 showed any significant hydrolysis. Overall,
the two carbamate PLs 4 and 5, while serving as relatively poor
substrates for HL and LPL, retained fairly high lipolysis by EL, and
4 was selected for further study.
O
N+
N+
O
O
O
b or c
or d
e
P
P
TrO
O
O
RO
O
O
O
OH
R'
9
R'=C12H25
7 R=H
8 R=Tr
10 R'=C6H13
11 R'=C(O)NHC14H29
a
N+
N+
O
O
O
O
O
P
P
f or g
HO
O
O
R
O
O
O
O
O
1 R= C13H27, R'= C12H25
2 R= C15H31, R'= C12H25
3 R= C13H27, R'= C6H13
R'
12 R'=C12H25
13 R'=C6H13
14 R'=C(O)NHC14H29
R'
4 R= C15H31, R'=C(O)NHC14H29
5 R = C13H27, R'=C(O)NHC14H29
Scheme 1. Synthesis of ethers 1–3 and carbamate 4–5. Reagents and conditions: (a) ZnCl2, DMF, 4 °C, 30 min, then TrCl, 4 °C, overnight, 50%. (b) 8, 1-iododecane, dimsyl
sodium, DMSO, rt, 1 h, 3%. (c) 8, 1-iodohexane, dimsyl sodium, DMSO, rt, 1 h, 2%. (d) 8, 1-isocyanatotetradecane, DMSO, W, 125 °C, 45 min, 5%. (e) TFA, chloroform, rt, 4 h,
l
87% (12), 71% (13), 15% (14). (f) Myristic acid, DCC, DMAP, chloroform, rt, overnight, 29% (1), 57% (3), 9% (5). (g) Palmitic acid, DCC, DMAP, chloroform, rt, overnight, 30% (2),
37% (4).

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J. P. N. Papillon et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3514–3517
plasma, with no meaningful difference observed between wild-
100
80
60
40
20
0
type and EL KO samples. Additional 4-lysoPC formation was
observed in both wild-type and KO post-heparin plasma compared
to pre-heparin samples. As anticipated, the post-heparin over pre-
heparin 4-lysoPC ratio was greater in wild-type (2.8:1) versus EL
KO (2:1). Although the hydrolysis of 4 in post-heparin EL KO
plasma was substantial, the significant difference in HR activity
between wild-type and EL KO plasma suggested a window to mea-
sure specific plasma EL activity.
We also evaluated 4 as a substrate with a more physiologically
relevant formulation. Synthetic HDL particles (rHDL)11 with HDL-
like sizes (diameter = 7.6 nm) were produced using a 60:12:1
molar ratio of compound 4 to cholesterol oleate to apoA-I, respec-
tively. Using this 4-rHDL substrate, a 3.2:1 ratio of post-heparin to
pre-heparin 4-lysoPC was observed (Fig. 3), suggesting that 4
incorporated in synthetic HDL might offer better specificity com-
pared to its Triton-X100 emulsion. The relative specificity of 4-
rHDL for plasma EL was further assessed by treating mouse plasma
with anti-EL antibody. Using wild-type post-heparin mouse
plasma, an antibody specific for mouse EL inhibited 4-lysoPC pro-
duction significantly compared to a control Ab (Fig. 4).
EL
HR
HR
p
p
p
p
e
e
e
H
H
He
-
-
-H
-
t
e
st
s
r
o
o
Pre
P
WPildtype
EPL KO
Figure 2. Amount of 4-lysoPC produced by incubating a Triton X-100 emulsion of 4
with wild-type and EL KO plasma pre- and post-heparin for 60 min (n = 3).
80
60
In conclusion, we designed and prepared synthetic phospho-
lipids that generate lysoPC products with a unique mass for conve-
nient detection by LC–MS in complex biological matrices. We
demonstrated that compound 4, formulated either as a Triton X-
100 emulsion or incorporated in synthetic HDL particles can serve
as a substrate for plasma EL with useful specificity.
HR
40
20
0
Acknowledgment
p
p
We thank Prof. Daniel J. Rader for providing EL KO mouse
plasma and rabbit Ab for mouse EL.
e
e
H
H
-
-
t
s
o
Pre
P
Supplementary data
Figure 3. Production of 4-lysoPC from 4-rHDL incubated with pre- and post-
heparin wild-type mouse plasma (n = 3).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.06.
032.
125
100
References and notes
1. Gordon, T.; Castelli, W. P.; Hjortland, M. C.; Kannel, W. B.; Dawber, T. R. Am. J.
Med. 1977, 62, 707; (b) Barter, P.; Gotto, A. M.; LaRosa, J. C.; Maroni, J.; Szarek,
M.; Grundy, S. M.; Kastelein, J. J. P.; Bittner, V.; Fruchart, J.-C. N. Eng. J. Med.
2007, 357, 1301; (c) Ng, D. S.; Wong, N. C. W.; Hegele, Robert A. Nat. Rev.
Endocrinol. 2013, 9, 308.
2. (a) Maugeais, C.; Tietge, U. J.; Broedl, U. C.; Marchadier, D.; Cain, W.; McCoy, M.
G.; Lund-Katz, S.; Glick, J. M.; Rader, D. J. Circulation 2003, 108, 2121; (b) Jin, W.;
Millar, J. S.; Broedl, U.; Glick, J. M.; Rader, D. J. J. Clin. Invest. 2003, 11, 357.
3. (a) Edmondson, A. C.; Brown, R. J.; Kathiresan, S.; Cupples, L. A.; Demissie, S.;
Manning, A. K.; Jensen, M. K.; Rimm, E. B.; Wang, J.; Rodrigues, A.; Bamba, V.;
Khetarpal, S. A.; Wolfe, M. L.; Derohannessian, S.; Li, M.; Reilly, M. P.; Aberle, J.;
Evans, D.; Hegele, R. A.; Rader, D. J. J. Clin. Invest. 2009, 119, 1042; (b) Larach, D.
B.; Cuchel, M.; Rader, D. J. Clin. Lipidol. 2013, 8, 635; (c) Yasuda, T.; Ishida, T.;
Rader, D. J. Circ. J. 2010, 74, 2263.
75
EL
50
25
0
ctr Ab
Saline
Anti-EL Ab
4. (a) McCoy, M. G.; Sun, G.-S.; Marchadier, D.; Maugeais, C.; Glick, J. M.; Rader, D.
J. J. Lipid Res. 2002, 43, 921; (b) Duong, M.; Psaltis, M.; Rader, D. J.; Marchadier,
D.; Barter, P. J.; Rye, K.-A. Biochemistry 2003, 42, 13778.
Figure 4. Production of 4-lysoPC from 4-rHDL incubated with post-heparin wild-
type mouse plasma in the presence of a control Ab or anti-EL Ab (n = 1).
5. (a) Mitnaul, L. J.; Tian, J.; Burton, C.; Lam, M. H.; Zhu, Y.; Olson, S. H.;
Schneeweis, J. E.; Zuck, P.; Pandit, S.; Anderson, M.; Maletic, M. M.; Waddell, S.
T.; Wright, S. D.; Sparrow, C. P.; Lund, E. G. J. Lipid Res. 2007, 48, 472; (b) Keller,
P. M.; Rust, T.; Murphy, D. J.; Matico, R.; Trill, J. J.; Krawiec, J. A.; Jurewicz, A.;
Jaye, M.; Harpel, M.; Thrall, S.; Schwartz, B. J. Biomol. Screening 2008, 13, 468;
(c) Darrow, A. L.; Olson, M. W.; Xin, H.; Burke, S. L.; Smith, C.; Schalk-Hihi, C.;
Williams, R.; Bayoumy, S. S.; Deckman, I. C.; Todd, M. J.; Damiano, B. P.;
Connelly, M. A. J. Lipid Res. 2011, 52, 374.
6. (a) Ishida, T.; Choi, S.; Kundu, R. K.; Hirata, K.-I.; Rubin, E. M.; Cooper, A. D.;
Quertermous, T. J. Clin. Invest. 2003, 111, 347; (b) Maugeais, C.; Tietge, U. J. F.;
Broedl, U. C.; Machadier, D.; Cain, W.; McCoy, M. G.; Lund-Katz, S.; Glick, J. M.;
Rader, D. J. Circulation 2003, 108, 2121.
EL is a secretory protein which binds to the endothelial surface
of blood/lymphatic vessels via cell-surface proteoglycans and is
thus heparin-releasable.10 Therefore, the hydrolytic activity of
mouse plasma on PL 4 was evaluated in pre- and post-heparin
samples, affording a measure of heparin-releasable (HR) activity.
In order to evaluate the specific contribution of EL, comparison of
activity was made between wild-type and EL knock-out (KO)
plasma. The results for PL 4 formulated as a Triton X-100 emulsion
are shown in Figure 2. Some hydrolysis of 4 occured in pre-heparin
7. (a) Sun, L.; Ishida, T.; Miyashita, K.; Kinoshita, N.; Mori, K.; Yasuda, T.; Toh, R.;
Nakajima, K.; Imamura, S.; Hirata, K.-I. J. Atheroscler. Thromb. 2014, 21, 313; (b)

J. P. N. Papillon et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3514–3517
3517
Basu, D.; Lei, X.; Josekutty, J.; Hussain, M. M.; Jin, W. J. Lipid Res. 2013, 54, 282;
(c) Miksztowicz, V.; McCoy, M. G.; Schreier, L.; Cacciagiú, L.; Elbert, A.;
Gonzalez, A. I.; Billheimer, J.; Eacho, P.; Rader, D. J.; Berg, G. Arterioscler. Thromb.
Vasc. Biol. 2012, 32, 3033.
9. Menger, F. M.; Chen, X. Y.; Brocchini, S.; Hopkins, H. P.; Hamilton, D. J. Am.
Chem. Soc. 1993, 115, 6600.
10. Fuki, I. V.; Blanchard, N.; Jin, W.; Marchadier, D. H.; Millar, J. S.; Glick, J. M.;
Rader, D. J. J. Biol. Chem. 2003, 278, 34331.
8. Lok, C. M. Chem. Phys. Lipids 1978, 22, 323.
11. Pittman, R. C.; Glass, C. K.; Atkinson, D.; Small, D. M. J. Biol. Chem. 1987, 262,
2435.