G Model
CPL 4300 1–7
6
R. Rosseto, J. Hajdu / Chemistry and Physics of Lipids xxx (2014) xxx–xxx
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Cald for C40H70N3O11
P
ꢂ
0.5H2O C, 59.39; H, 8.85; N, 5.19; found: C,
127.86, 128.57, 129.63, 137.05, 141.30, 143.90, 156.17, 169.83, 172.39,
173.72. Rf (CHCl3/MeOH/H2O 65:25:4) 0.48. Anal. Cald for
59.07; H, 8.80; N, 5.33. FAB-MS MH+ C40H70N3O11PH Calcd:
800.4821, found: 800.4827. [a D
]
25 +1.44 (c 1.04, CHCl3/MeOH 4:1)
C50H72N3O11P 2.5H2O C, 62.09; H, 8.02; N, 4.34; found: C, 62.33;
ꢂ
H, 8.03; N, 4.04. FAB-MS MH+ C50H72N3O11PH Calcd: 922.4977,
25 ꢀC
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4.5. 1-Palmitoyl-2-(CBZ-
phosphocholine (13)
D-phe-phe-gly)-sn-glycero-3-
found: 922.4981. [a D
]
–6.73 (c 0.98, CHCl3/MeOH 4:1).
401
4.7. 1-palmitoyl-2-(N-BOC-glycyl)-sn-glycero-3-phosphocholine (14)
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To a solutionof9 (0.3850 g, 0.48 mmol) in 20 mL 1,4-dioxanewas
added4 MHCl in1,4-dioxane(7 mL)dropwiseatroomtemperature.
The reaction mixture was stirred for 2.5 h, followed by the addition
of 30 mL benzene and it was freeze-dried to give the deprotected
amine 10 as a white solid. The 1H NMR (CD3OD, 200 MHz) spectrum
of the compound 12 showed the same pattern as the spectrum of
compound 11, except for the absence of the signal assigned to the
protons at d1.40 (s, 9H) of the removedBOC protecting group. To the
whiteprecipitateof12 dissolvedin20 mLofCHCl3 was addedDMAP
(0.2987 g, 2.5 mmol) until pH of solution reached 8, followed by the
active ester p-nitrophenyl N-Cbz-D-phenylalanine (0.2652 g,
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To a suspension of 1-palmitoyl-2-hydroxy-sn-glycero-3-phos-
phocholine 6 (0.3704 g, 0.7 mmol) in 25 mL of CHCl3 was added
N-BOC-gly (0.5305 g, 3 mmol), followed by DCC (0.6204 g, 3 mmol),
DMAP (0.3704 g, 3 mmol) and 1 g of glass beads. The reaction was
sonicated for 1 h at 25 ꢀC. Next, to the mixture were added 8 mL of
Dowex-H+ and stirred for 10 min. The resin was filtered and
washed with 30 mL of CHCl3:MeOH (1:1). The combined solution
was evaporated under reduced pressure to one third of volume and
then was promoted the chromatographic purification on silica gel
using as eluent. and then was loaded on a silica gel column, eluted
first with CHCl3/MeOH (7:3), folloed by CHCl3/MeOH/H2O
(65:25:4). The fractions corresponding to the product were
combined, evaporated, re-dissolved in benzene and freeze-dried
to give a white solid 14 (0.4325 g, 0.66 mmol, 94.5%). IR (Nujol):
3364 br m, 1746 vs, 1714 vs, 1253 m, 1168 m cmꢁ1. 1H NMR (CDCl3,
0.63 mmol) at room temperature. After 24 h more active ester
(0.1802 g, 0.43 mmol) was added. After 48 h stirring at room
temperature, to the mixture was added 15 mL Dowex-H+ and it was
stirred for 10 min. The suspension was filtered and the resin was
washed with 30 mL CHCl3/MeOH (1:1). The solvents collected were
evaporated under reduced pressure to one third of the volume and
loaded on a silica gel column, eluted first with CHCl3/MeOH (3:1),
followed by CHCl3/MeOH/H2O (65:25:4). The fractions correspond-
ing to the product were combined, evaporated, re-dissolved in
benzene and freeze-dried to give a white solid 13 (0.3815 g,
0.39 mmol, 81.3%). IR (Nujol): 3292 w, 1728 s,1693 m,1643 vs,1540
200 MHz) d 0.85 (br t, 3H),1.23 (br s, 24H),1.40 (s, 9H),1.52 (m, 2H),
2.26 (t, 2H, J = 6.7 Hz), 3.26 (br s, 9H), 3.75–4.01 (m, 6H), 4.10–4.18
(m, 2H), 4.25 (m, 2H), 5.22 (m, 1H), 6.21 (m, 1H). 13C NMR (CDCl3,
50 MHz)
d 14.01, 22.58, 24.71, 28.33, 29.08, 29.22, 29.25, 29.44,
29.56, 29.60, 31.82, 33.89, 42.31, 54.16, 59.36, 62.43, 63.62, 65.98,
71.50, 79.39, 156.04, 170.36, 173.47. Rf (CHCl3/MeOH/H2O 65:25:4)
m,1301wcmꢁ1.1HNMR(CDCl3, 200 MHz)
d
0.85(brt, 3H),1.25(brs,
0.38. Anal. Cald for C31H61N2O10
P H2O C, 55.50; H, 9.47;
ꢂ
24H),1.52 (m, 2H), 2.23 (t, 2H, J = 6.7 Hz), 2.73 (m, 2H), 3.05 (m, 2H),
3.17 (br s, 9H), 3.71 (m, 2H), 4.05–4.20 (m, 4H), 4.35(m, 2H), 4.62 (m,
2H), 4.81–5.02 (m, 4H), 5.25 (m, 1H), 6.03 (m, 1H), 6.90–7.26 (m,
N, 4.18, found: C, 55.50; H, 9.49; N, 4.04. FAB-MS MH+
25 ꢀ
C
C31H61N2O10PH Calcd: 653.4137, found: 653.4165. [
a
]
+8.80
D
(c 1.00, CHCl3/MeOH 4:1).
15H), 8.01 (m, 1H), 8.32 (m, 1H). 13C NMR (CDCl3, 50 MHz)
d 14.10,
427
22.65, 24.74, 29.12, 29.33, 29.51, 29.63, 29.68, 31.89, 33.86, 37.69,
38.43, 41.32, 54.21, 54.70, 55.89, 59.85, 62.08, 64.43, 65.93, 66.58,
71.52, 126.66, 126.83, 127.65, 128.02, 128.33,128.48, 129.33, 129.46,
136.47, 137.05, 155.97, 169.54, 171.60, 172.27, 173.55. Rf (CHCl3/
4.8. Enzymatic hydrolysis of the phospholipids
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In a typical experiment prodrug 10 (4.7 mg, 5.8
m
mol) was
added to a mixture containing dipalmitoyl phosphatidylcholine
(DPPC, 17.9 mg, 23.4 mol), in 4.1 mL Tris buffer (0.05 M, pH 8.50),
MeOH/H2O 65:25:4) 0.55. Anal. Cald for C52H77N4O12
P
ꢂ
4H2O C,
m
59.30; H, 8.13; N, 5.32; found: C, 59.75; H, 7.78; N, 5.51. FAB-MS MH+
with 0.1 mL Triton X-100 and CaCl2 (7.2 mg, 0.049 mmol) The
mixture was vortexed, for 5 min, followed by incubation of the
resulting dispersion at 40 ꢀC for 10 min in a constant-temperature
water-bath. To the optically clear dispersion that resulted was
25
C52H77N4O12PH Calcd: 981.5348, found: 981.5375. [
(c 0.97, CHCl3/MeOH 4:1).
a
]
ꢁ6.57
D
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375
4.6. 1-Palmitoyl-2-(FMOC-phe-gly)-sn-glycero-3-phosphocholine
(110)
added bee-venom phospholipase A2 (40 mg in 200 mL buffer) to
initiate the reaction. The reaction mixture was kept at 40 ꢀC,
and formation of the products was analyzed by thin layer
chromatography (CHCl3/MeOH/H2O, 65:25:4). The compounds
were visualized by iodine adsorption, molybdic acid spray and
ninhydrin spray. TLC analysis showed complete hydrolysis of the
phospholipids (DPPC and the synthetic phospholipid prodrug 10)
by PLA2 within 90 min, leading to the formation of lysophospha-
tidylcholine 6, and the oligopeptide 4. PLA2 catalyzed hydrolysis of
DPPC under the same conditions in absence of compound 10 was
completed in 10 min.
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To a suspension of 1-palmitoyl-2-hydroxy-sn-glycero-3-phos-
phocholine 6 (0.5002 g, 1 mmol) in 25 mL of CHCl3 were added
FMOC-phe-gly-OH (0.5393 g, 1.2 mmol), DCC (0.2498 g, 1.2 mmol),
DMAP (0.1479 g, 1.2 mmol) and 1 g of glass beads. The reaction was
sonicated for 48 h at 25 ꢀC, the mixture was then filtered to remove
DCC-urea and glass beads. The solvent was evaporated to one third
of the volume and then loaded on a silica gel column for
chromatography. A stepwise gradient of CHCl3/MeOH (5:1 and 5:2)
was applied to elute DMAP and some impurities, followed by
CHCl3/MeOH/H2O (65:25:4). The fractions corresponding to the
product were combined, evaporated, re-dissolved in benzene and
freeze-dried to give 110 as a white solid (0.5352 g, 0.58 mmol, 58%).
In a somewhat similar experimental setup, prodrug 13 (3.4 mg,
0.5 mmol) was added to a mixture containing DPPC (15 mg, 1.5 m
mol), in 4.1 mL Tris buffer (0.05 M, pH 8.50), with 0.1 mL
Triton X-100 and 50 mM CaCl2. The mixture was vortexed, for
5 min, kept at 40 ꢀC for 10 min in a constant-temperature
water-bath. To the resulting dispersion was added bee-venom
IR (Nujol): 3297 br m,1728 vs,1693 s,1654 vs,1536 m,1252 wcmꢁ1
1H NMR (CDCl3, 200 MHz)
0.85 (br t, 3H), 1.25 (br s, 24H),1.50 (m,
.
d
390
391
392
393
394
395
2H), 2.20 (t, 2H, J = 6.7 Hz), 2.95 (m, 2H), 3.17 (br s, 9H), 3.67 (br s,
2H), 3.95–4.30 (br m, 10H), 4.44 (m, 2H), 5.36 (m, 1H), 6.15 (m, 1H),
7.21–7.47 (m, 11H), 7.72 (d, 2H, J = 7.4 Hz), 8.66 (m, 1H). 13C NMR
phospholipase A2 (16 mg in 80 mL 0.05 M Tris buffer, pH 8.5) to
initiate the reaction. The reaction mixture was kept at 40 ꢀC, and
formation of the products was analyzed by thin layer chromatog-
raphy (CHCl3/MeOH/H2O, 65:25:4). The compounds were
visualized by UV-absorption, iodine adsorption, and molybdic
acid spray. TLC analysis showed complete hydrolysis of the DPPC
(CDCl3, 50 MHz)
d 14.28, 22.85, 24.92, 29.31, 29.52, 29.71, 29.83,
29.87, 32.08, 34.04, 38.94, 41.47, 47.13, 54.34, 55.90, 59.63, 62.44,
64.18, 66.28, 67.07, 71.96, 120.08, 125.27, 125.47, 126.94, 127.26,
Please cite this article in press as: Rosseto, R., Hajdu, J., Peptidophospholipids: Synthesis, phospholipase A2 catalyzed hydrolysis, and