Preparation of ω-functionalized eicosane-phosphate building blocks

Article abstract of DOI:10.1016/j.chemphyslip.2004.09.016

New 1,20-substituted eicosanes carrying phosphate headgroups and readily derivatizable thiol, maleimido, and activated carboxylic ester moieties were prepared. The C₂₀-backbone of these molecules was assembled by a halopolycarbon homologation from 1,8-dichlorooctane and 1,6-dibromohexane. 20-Mercapto- and 20-maleimido-icosylphosphates were synthesized via ω-bromo di-t-butyl protected icosylphosphate while 20-phosphonooxy- icosanoic acid N-hydroxysuccinimidoyl ester was prepared via ω-bromo dibenzyl protected icosylphosphate in multistep syntheses. These molecules can serve as model compounds for studying binding and structural organization on different surfaces with potential applications in the fields of biosensors.

Full text of DOI:10.1016/j.chemphyslip.2004.09.016

Chemistry and Physics of Lipids 133 (2005) 103–112
Preparation of ␻-functionalized eicosane-phosphate
building blocks
Istvan Jablonkaia,∗, Peter Oroszlanb
a
Biological Chemistry Group, Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences,
P.O. Box 17, H-1525 Budapest, Hungary
b
Zeptosens AG, CH-4108 Witterswil, Switzerland
Received 5 June 2004; received in revised form 15 September 2004; accepted 20 September 2004
Available online 28 October 2004
Abstract
New 1,20-substituted eicosanes carrying phosphate headgroups and readily derivatizable thiol, maleimido, and activated
carboxylic ester moieties were prepared. The C20-backbone of these molecules was assembled by a halopolycarbon homologation
from 1,8-dichlorooctane and 1,6-dibromohexane. 20-Mercapto- and 20-maleimido-icosylphosphates were synthesized via ␻-
bromo di-t-butyl protected icosylphosphate while 20-phosphonooxy-icosanoic acid N-hydroxysuccinimidoyl ester was prepared
via ␻-bromo dibenzyl protected icosylphosphate in multistep syntheses. These molecules can serve as model compounds for
studying binding and structural organization on different surfaces with potential applications in the fields of biosensors.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Eicosane; Lipid; Bifunctional; Phosphate; Synthesis
1. Introduction
shown that anchoring lipidic thiols to gold (Roberts
et al., 1998) or C18-phosphate to tantalum(V) oxide
surfaces (Brovelli et al., 1999) produces oriented
and well-ordered monolayers via self-assembly.
20-Mercaptoeicosane-1-ol was found to increase
the stability of a lipid monolayer of dioleoyl-l-␣-
phosphatidylcholine (Nakashima et al., 1993). These
layers simulating natural surfaces are suitable to
mimic cell membranes and have potential uses in
adhesion studies and developing biosensors. Thus a
growing interest has stimulated the synthesis of various
phospholipid analogs (Hasegawa et al., 1990; Wang
Naturally occurring phospholipids are important
cell membrane constituents. Due to their amphiphilic
properties they have also gained importance in several
areas of research like drug encapsulation (Walker et al.,
1997) and gene delivery (Behr et al., 1989).
The use of synthetic lipids to create mimics of
cell surfaces has been a focus of research. It has been
∗
Correspondingauthor. Tel.:+3613257554;fax:+3613257554.
E-mail address: jabi@chemres.hu (I. Jablonkai).
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2004.09.016

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I. Jablonkai, P. Oroszlan / Chemistry and Physics of Lipids 133 (2005) 103–112
Fig. 1. Structure of the target ␻-substituted icosylphosphates.
and Hollingsworth, 1999; Ohlsson and Magnusson,
1999).
(1.53 g, 63 mmol) in THF (25 ml) was added slowly
to a stirred, cooled (5–10 ◦C) solution of 1,6-
dibromohexane (17.08 g, 70 mmol) in THF (35 ml)
containing Li2CuCl4 (7 ml, 0.1 M THF solution). The
mixture was stirred for an additional 1 h and 1N HCl
(50 ml) was added. The resulting mixture was di-
luted with water (100 ml) and extracted twice with
dichloromethane (150 ml). The organic layer was dried
over MgSO4 and the solvent was evaporated. The unre-
acted dihalogenated starting materials were distilled off
at 100 ◦C and the residue was purified on silica gel with
light petrolether. The product (9.77 g, 74%) is a white
waxy material (m.p.: 65–68 ◦C, Rf = 0.52 in hexane).
1H NMR (CDCl3), δ (ppm) 1.26 (m, 28H, CH2), 1.48
(m, 4H, CH2CH2CH2Br), 1.86 (m, 4H, CH2CH2Br),
3.41 (t, 4H, CH2Br, J = 6.6 Hz). 13C NMR (CDCl3), δ
(ppm) 26.78, 27.67, 28.91, 29.01, 29.15, 32.01, 32.34,
32.99, 33.42. IR (KBr) cm−1 548, 634, 705, 1198,
1239, 1460, 2840, 2905.
Here we report the preparation of ␻-functionalized
C20 lipidic phosphates (Fig. 1) as model compounds
for studying binding and structural organization on
different surfaces and to increase understanding of
lipid membrane-protein interaction. The introduction
of easily derivatizable groups such as thiol, maleimido,
and N-hydroxysuccinimide-activated carboxylic ester
to the ␻-position of a long alkyl chain phosphate allow
further functionalization of these molecules attached to
surfaces with a variety of biomolecules.
2. Experimental
2.1. Materials and instruments
All chemicals and solvents were purchased from
Sigma-Aldrich Kft. (Budapest, Hungary). TLC alu-
minium sheets (Silica gel 60 F254, 0.2 mm layer thick-
ness) for following the proceeding of the reactions and
silica gel (Silica gel 60, 0.040–0.063 mm) for column
chromatography were purchased from Merck Kft. (Bu-
dapest, Hungary). Melting points were determined by a
2.3. 2-(20-Bromo-icosyloxy)-tetrahydro-pyran (2)
A stirred solution of 1 (10 g, 22.7 mmol) in
dimethylformamide (100 ml) was treated with an-
hydrous sodium acetate (2.8 g, 34.0 mmol) at 80 ◦C
for 1 h. The solvent was removed in vacuo and the
residue was partitioned between water (40 ml) and
dichloromethane (100 ml). The organic phase was
dried over MgSO4 and then evaporated. The silica gel
chromatography of the residue with hexane–ether elu-
ent (9:1) resulted in acetic acid 20-acetoxy-1-bromo-
eicosane (3.6 g, 38%, m.p.: 50–52 ◦C), the unreacted
starting material (2.37 g) and the diacetate (1.0 g, m.p.:
56–58 ◦C). To the monoacetate (2.5 g, 6.0 mmol) dis-
solved in methanol (70 ml) was added concentrated sul-
furic acid (0.2 g, 2 mmol) and the mixture was refluxed
for 1.5 h. The solvent was removed in vacuo and the
¨
Buchi apparatus and are uncorrected. The NMR spec-
tra were recorded with Varian Unity Inova (400 MHz
for 1H and 100 MHz for 13C) and Varian Gemini-2000
(200 MHz for 1H) spectrometer (Varian Inc., Palo Alto,
CA, USA). Infrared spectra were run on Nicolet Avatar
320 FT-IR spectrometer (Thermo Nicolet, Madison,
WI, USA).
2.2. 1,20-Dibromo-eicosane (1)
A Grignard reagent prepared from 1,8-dichloro-
octane (5.5 g, 30 mmol) and magnesium turnings

I. Jablonkai, P. Oroszlan / Chemistry and Physics of Lipids 133 (2005) 103–112
105
residue taken up in dichloromethane (50 ml) and was
2.5. Thioacetic acid S-(20-hydroxy-icosyl)
ester (4)
washed with water (30 ml) and saturated NaHCO3 so-
lution(30 ml). TheorganiclayerwasdriedoverMgSO4
and the solvent was distilled off. The residue 20-
bromo-eicosane-1-ol (2.2 g, m.p.: 55–56 ◦C), a white
waxy material, was used for the THP-ether formation
without purification. To a solution of bromo-eicosanol
(2.2 g, 5.9 mmol) in dry dichloromethane (25 ml) was
added 3,4-dihydro-2H-pyran (1.24 g, 14.7 mmol) and
pyridinium p-toluenesulfonate (0.1 g, 0.4 mmol) and
the mixture was stirred overnight at room tempera-
ture. After dilution with dichloromethane (20 ml) the
solution was washed with water (20 ml) and satu-
rated NaHCO3 solution (20 ml). The organic layer
was dried over MgSO4 and the solvent was evap-
orated. The product was obtained as a white waxy
substance (2.3 g, 84%, m.p.: 38–40 ◦C, Rf = 0.38 in
hexane–ether 95:5) by silica gel chromatography us-
A solution of protected thioacetate 3 (1.6 g,
3.52 mmol) in a mixture of ethanol (15 ml) and
chloroform (15 ml) was stirred with pyridinium p-
toluenesulfonate (0.35 g, 1.4 mmol) at 55 ◦C for 3 h
and at room temperature overnight. The solvent was
then removed in vacuo and the residue was dissolved
in dichloromethane (30 ml) and washed with water
(15 ml) and saturated NaHCO3 solution (15 ml). The
organic layer was dried over MgSO4 then evaporated.
The residue was purified by silica-gel column chro-
matography using a hexane–ether (7:3) eluent. The
product is a white crystalline material (0.91 g, 69%,
m.p.: 55–57 ◦C, Rf = 0.27 in hexane–ether, 7:3). 1H
NMR (CDCl3), δ (ppm) 1.23–1.40 (m, 30H, CH2),
1.22–1.65 (m, 6H, CH2), 2.32 (s, 3H, CH3), 2.86 (t,
2H, CH2S, J = 7.0 Hz), 3.64 (t, 2H, CH2O, J = 6.6 Hz).
13C NMR (CDCl3), δ (ppm) 25.23, 25.72, 28.30, 28.57,
28.64, 28.97, 29.15, 30.07, 30.27, 32.30, 61.78, 62.57,
195.47. IR (KBr) cm−1 640, 719, 730, 958, 1035,
1074, 1123, 1355, 1463, 1473, 1695, 2849, 2918,
3334.
1
ing hexane–ether (97:3) eluent. H NMR (CDCl3), δ
(ppm) 1.25–1.40 (m, 30H, CH2), 1.57–1.94 (m, 12H,
CH2), 3.38(m, 3H, CH2Br, CHHO of alkyl chain),
3.5 (m, 1H, CHHO of THP), 3.70 (m, 1H, OCHH
of alkyl chain), 3.88 (m, 1H, CHHO of THP), 4.58
(m, 1H, OCHO). IR (KBr) cm−1 626, 732, 786,
828, 964, 1014, 1058, 1068, 1142, 1304, 1430, 2920,
2980.
2.6. Thioacetic acid S-(20-phosphonooxy-icosyl)
ester (5)
2.4. Thioacetic acid S-[20-(tetrahydro-pyran-2-
yloxy)-icosyl] ester (3)
In an oven-dried two-necked flask equipped with
a reflux condenser and a dropping funnel was placed
P2O5 (0.1 g, 0.7 mmol) and toluene (5.0 ml) then hex-
amethyldisiloxane (0.69 g, 4.2 mmol) was added under
nitrogen. The mixture was stirred under reflux until
the solution became clear. (ca. 1 h). After cooling, the
alcohol 4 (0.55 g, 1.5 mmol) was added in one por-
tion and the mixture was refluxed for 2 h. Removal
of the solvent in vacuo yielded a viscous oily residue.
Ether (20 ml) was added to the residue then the mix-
ture was triturated with water (20 ml). The solid pre-
cipitate that formed was filtered off. The solid in the
filter was washed successively with 50% ethanol and
ethanol. The product (0.59 g, 86%, m.p.: 75–77 ◦C)
was obtained after drying the precipitate in vacuo. 1H
NMR (CDCl3), δ (ppm) 1.23–1.40 (m, 34H, CH2),
1.55 (m, 2H, CH2), 2.32 (s, 3H, CH3), 2.86 (t, 2H,
CH2S, J = 7.2 Hz), 4.08 (br, 2H, CH2O). IR (KBr)
cm−1 637, 719, 962, 1024(br), 1238, 1472, 1689, 2850,
2917.
A mixture of 2 (3.0 g, 6.5 mmol) and potassium
thioacetate (1.5 g, 13.1 mmol) was stirred in DMF
(100 ml) at 80 ◦C for 1 h. The solvent was removed
in vacuo and the residue was shaken with a mix-
ture of dichloromethane (50 ml) and water (30 ml).
The organic layer was dried over MgSO4 then evap-
orated. The crude product was chromatographed us-
ing hexane–ether (95:5) on silica gel column to ob-
tain the white crystalline thioester 3 (2.7 g, 92%, m.p.:
40–41 ◦C, Rf = 0.20 in hexane–ether, 95:5). H NMR
1
(CDCl3), δ (ppm) 1.25–1.40 (m, 30H, CH2), 1.57–1.94
(m, 12H, CH2), 2.32 (s, 3H, CH3CO), 2.86 (t, 2H,
CH2S, J = 6.9 Hz), 3.38(m, 1H, CHHO of alkyl chain),
3.5 (m, 1H, CHHO of THP), 3.72 (m, 1H, OCHH of
alkyl chain), 3.88 (m, 1H, CHHO of THP), 4.58 (1H,
m, OCHO). IR (KBr) cm−1 629, 717, 816, 870, 906,
964, 1035, 1078, 1122, 1139, 1354, 1472, 1695, 2850,
2918, 3431.

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I. Jablonkai, P. Oroszlan / Chemistry and Physics of Lipids 133 (2005) 103–112
2.7. Phosphoric acid 20-bromo-icosyl ester
2.9. Phosphoric acid di-tert-butyl ester
di-tert-butyl ester (6)
20-mercapto-icosyl ester (8)
To
a
freshly prepared solution of di-tert-
Lithium aluminium hydride (0.8 ml, 3.8 M in ether)
was added dropwise to a stirred solution of thioester
7 (1.70 g, 3.0 mmol) in ether (20 ml) at 0 ◦C. After the
addition was complete, the mixture was stirred for 1 h
at room temperature. The excess of lithium aluminium
hydride was destroyed by the careful addition of water
(2 ml). The precipitated lithium aluminate was filtered
off and the filtrate was dried over MgSO4. The solvent
was removed in vacuo and the crude product was chro-
matographed on silica gel with hexane–ethyl acetate
(9:1). The product (1.37 g, 88%) is a white crystalline
material (m.p.: 56–57 ◦C, Rf = 0.39 in hexane–ethyl ac-
butylphosphate (1.80 g, 8.6 mmol) in acetone
(25 ml) a mixture of methanolic tetramethylammo-
nium hydroxide (4.00 ml 2.2 M methanol solution)
and water (4 ml) was added dropwise at −10 ◦C.
The solution was evaporated and the residue was
dried in vacuo. The white tetramethylammonium
di-tert-butylphosphate (2.4 g) was added to a stirred
solution of 1,20-dibromo-eicosane (3.8 g, 8.6 mmol) in
1,2-dimethoxyethane (20 ml) at 100 ◦C. The mixture
was refluxed overnight. The precipitated salt was
then filtered off and the filtrate was evaporated to
dryness. The residue was purified on silica gel with
hexane–ether (8:2) eluent. The product is a white
waxy material (1.66 g, 34%, m.p.: 29–30 ◦C, Rf = 0.31
in hexane–ethyl acetate, 8:2). 1H NMR (CDCl3),
1
etate, 8:2). H NMR (CDCl3) δ (ppm) 1.23 (m, 32H,
t
CH2), 1.34 (t, 1H, SH),1.46 (m, 18H, Bu), 1.61 (m,
4H, CH2), 2.51 (q, 2H, CH2S, J = 7.1 Hz), 3.93 (q, 2H,
POCH2, J = 6.6 Hz). IR (KBr) cm−1 582, 768, 987,
1033, 1244, 1363, 1381, 1459, 2842, 2913.
t
δ (ppm) 1.28 (m, 32H, CH2), 1.44 (m, 18H, Bu),
1.61 (m, 2H, CH2CH2Br), 1.84 (m, 2H, CH2CH2Br),
3.42 (t, 2H, CH2Br, J = 6.0 Hz), 3.95 (q, 2H, POCH2,
J = 6.4 Hz). 13C NMR (CDCl3), δ (ppm) 25.10, 27.65,
28.23, 28.66, 28.90, 29.00, 29.14, 29.30, 29.38, 29.66,
29.81, 32.33, 33.42, 66.32, 81.41. IR (KBr) cm−1 536,
737, 921, 981, 1044, 1166, 1268, 1360, 1381, 1461,
2847, 2923. MS (APCI) m/z 571.3 (M+H)+, 459.1
(M-2xC(CH3)3+3H)+.
2.10. Phosphoric acid mono-(20-mercapto-icosyl)
ester (9)
A solution of the thiol 8 (0.26 g, 0.5 mmol) in diox-
ane (15 ml) and 4N HCl (3 ml) was stirred for 1 h at
room temperature. The mixture was diluted with water
(15 ml) and the white precipitate formed was filtered
off and washed with 50% ethanol to give the product
1
(0.19 g, 92%, m.p.: 88–90 ◦C). H NMR (DMSO-d6)
2.8. Thioacetic acid S-[20-(di-tert-butoxy-
δ (ppm) 1.27 (m, 32H, CH2), 1.41 (t, 1H, SH), 1.62
(m, 4H, CH2), 2.52 (q, 2H, CH2S, J = 7.0 Hz), 3.95
(q, 2H, POCH2, J = 5.3 Hz). 13C NMR (DMSO-d6) δ
(ppm) 25.26, 26.78, 28.32, 28.66, 28.92, 29.21, 29.35,
29.54, 29.76, 29.90, 30.24, 66.12. IR (KBr) cm−1 715,
1020–1080 (br), 1462, 2844, 2910. Raman, 2572 cm−1
(SH). MS(APCI)m/z412.3(M+H)+, 456.1(M+2Na)+.
phosphoryloxy)-icosyl] ester (7)
A mixture of 6 (0.57 g, 1.0 mmol) and potassium
thioacetate (2.28 g, 2.0 mmol) was stirred in DMF
(20 ml) at 80 ◦C for 3 h. The solvent was removed
in vacuo and the residue was shaken with a mix-
ture of dichloromethane (30 ml) and water (20 ml).
The organic layer was dried over MgSO4 then evap-
orated. The crude product was chromatographed us-
ing hexane–ethyl acetate (85:15) on silica-gel column
to obtain a yellowish thioester 7 (0.4 g, 71%, m.p.:
97–100 ◦C, Rf = 0.27 in hexane–ethyl acetate, 8:2). 1H
NMR (CDCl3) δ (ppm) 1.22 (m, 32H, CH2), 1.44
2.11. Phosphoric acid dibenzyl ester
20-bromo-icosyl ester (10)
To a freshly prepared solution of dibenzylphosphate
(1.00 g, 3.6 mmol) in acetone (10 ml) a mixture of
methanolic tetramethylammonium hydroxide (1.85 ml
20% methanol solution) and water (2 ml) was added
dropwise at −10 ◦C. The solution was evaporated and
the residue was dried in vacuo. The white tetram-
ethylammonium dibenzylphosphate (1.25 g) was
t
(m, 18H, Bu), 1.58 (m, 4H,), 2.31 (s, 3H, CH3),
2.86 (t, 2H, CH2S, J = 7.2 Hz), 3.93 (q, 2H, POCH2,
J = 6.0 Hz). IR (KBr) cm−1 542, 624, 705, 970, 996,
1038, 1056, 1262, 1362, 1382, 1461, 1694, 2845,
2920.

I. Jablonkai, P. Oroszlan / Chemistry and Physics of Lipids 133 (2005) 103–112
107
added to a stirred solution of 1,20-dibromo-eicosane
(1.58 g, 3.6 mmol) in 1,2-dimethoxyethane (20 ml)
at 100 ◦C. The mixture was refluxed overnight then
the precipitated salt was filtered off and the filtrate
was evaporated to dryness. The residue was purified
on silica gel with hexane–ethyl acetate (85:15). The
product is a white waxy material (0.96 g, 41%, m.p.:
solution (20 ml). The organic layer was dried over
MgSO4 and the solvent was distilled off. The residue
was purified by a silica-gel chromatography with
hexane–ethyl acetate (7:3) eluent to give the white crys-
talline product (0.25 g, 84%, m.p.: 60–62 ◦C, Rf = 0.17
in hexane–ethyl acetate, 7:3). 1H NMR (CDCl3) δ
(ppm) 1.25 (m, 32H, CH2), 1.54 (m, 4H, CH2), 3.64
(t, 2H, CH2OH, J = 6.6 Hz), 3.96 (q, 2H, POCH2,
J = 6.6 Hz), 5.01 (CH2OPh), 5.05 (s, 2H, CH2OPh),
7.24–7.36 (m, 10H, arH). 13C NMR (CDCl3) δ (ppm)
26.46, 27.69, 28.44, 29.09, 29.19, 29.68, 32.36, 61.37,
67.59, 68.54, 68.66, 127.36, 128.01, 136.00. IR (KBr)
cm−1 669, 695, 736, 983, 1001, 1069, 1245, 1457,
2850, 2917, 3448.
1
36–38 ◦C, Rf = 0.26 in hexane–ethyl acetate, 8:2). H
NMR (CDCl3) δ (ppm) 1.24 (m, 32H, CH2), 1.60
(m, 4H, CH2), 3.42 (t, 2H, CH2Br, J = 7.0 Hz), 3.98
(q, 2H, POCH2, J = 6.6 Hz), 5.02 (s, 2H, CH2OPh),
5.06 (s, 2H, CH2OPh), 7.32 (m, 10H, arH). 13C
NMR (CDCl3) δ (ppm) 24.86, 27.65, 28.01, 28.24,
28.58, 28.81, 29.15, 29.60, 32.32, 67.59, 68.54, 68.66,
127.36, 128.01, 135.58. IR (KBr) cm−1 498, 695, 723,
1026, 1264, 1448, 1462, 2854, 2924, 3420.
2.14. 20-(Bis-benzyloxy-phosphoryloxy)-
icosanoic acid (13)
2.12. Acetic acid 20-(bis-benzyloxy-phosphoryl-
oxy)-icosyl ester (11)
To the alcohol 12 (0.16 g, 0.28 mmol) dissolved
in acetone (40 ml) and dichloromethane (16 ml) was
added the Jones reagent (0.56 ml) at room temperature.
The solution was stirred for 2.5 h then diluted with
water (55 ml) and the product was extracted by
ether (2 ml × 30 ml). The organic layer was dried
over MgSO4 and the solvent was distilled off. The
residue (0.13 g, 79%, m.p.: 58–61 ◦C, Rf = 0.27 in
hexane–ethyl acetate, 1:1) was sufficiently pure
to use in the next reaction without further purifi-
cation. 1H NMR (CDCl3) δ (ppm) 1.22 (m, 30H,
CH2), 1.62 (m, 4H, CH2), 2.36 (t, 2H, CH2COO,
J = 7.0 Hz), 3.98 (q, 2H, POCH2, J = 6.6 Hz), 5.02
(s, 2H, CH2OPh), 5.06 (s, 2H, CH2OPh), 7.22–7.42
(m, 10H, arH). IR (KBr) cm−1 696, 726, 1001,
1028, 1069, 1174, 1238, 1473, 1727, 2850, 2920,
3430.
A mixture of 5 (0.5 g, 0.78 mmol) and sodium ac-
etate (1.28 g, 1.56 mmol) was stirred in DMF (20 ml) at
80 ◦C for 3 h. The solvent wasremoved in vacuoandthe
residue was shaken with a mixture of dichloromethane
(30 ml) and water (20 ml). The organic layer was
dried over MgSO4 then evaporated. The crude prod-
uct was chromatographed with hexane–ethyl acetate
(8:2) on a silica-gel column to obtain the white crys-
talline product (0.42 g, 87%, m.p.: 29–30 ◦C, Rf = 0.14
in hexane–ethyl acetate, 8:2). 1H NMR (CDCl3) δ
(ppm) 1.25 (m, 32H, CH2), 1.60 (m, 4H, CH2), 2.04
(s, 3H, CH3), 3.90–4.09 (m, 4H, CH2O), 5.01 (s, 2H,
CH2OPh), 5.05 (s, 2H, CH2OPh), 7.22–7.41 (m, 10H,
arH). 13C NMR (CDCl3) δ (ppm) 20.17, 25.86, 27.75,
28.24, 28.59, 29.16, 29.65, 32.32, 67.64, 68.54, 68.66,
69.44, 127.35, 128.01, 135.45, 171.10. IR (KBr) cm−1
504, 602, 697, 736, 872, 1021, 1246, 1273, 1456, 1466,
1738, 2851, 2921, 3444.
2.15. 20-(Bis-benzyloxy-phosphoryloxy)-
icosanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (14)
2.13. Phosphoric acid dibenzyl ester
20-hydroxy-icosyl ester (12)
A mixture of the acid 13 (0.12 g, 0.2 mmol),
N-hydroxysuccinimide (0.23 mg, 0.2 mmol) and di-
cyclohexylcarbodiimide (0.042 g, 0.2 mmol) in dry
dichloromethane (12 ml) was stirred under nitrogen
overnight. After removing the urea byproduct by filtra-
tion the filtrate was evaporated to dryness. The residue
was purified on silica-gel column using hexane–ethyl
acetate (6:4) solvent. The product (0.09 g, 65%, m.p.:
30–32 ◦C, Rf = 0.23 in hexane–ethyl acetate, 6:4) is
To the acetate 11 (0.32 g, 0.52 mmol) dissolved
in methanol (20 ml) was added concentrated sulfu-
ric (0.02 g, 0.2 mmol) and the mixture was refluxed
for 1.5 h. The solvent was removed in vacuo and the
residue taken up in dichloromethane (30 ml) and was
washed with water (20 ml) and saturated NaHCO3

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I. Jablonkai, P. Oroszlan / Chemistry and Physics of Lipids 133 (2005) 103–112
a white waxy material. 1H NMR (CDCl3) δ (ppm)
1.26 (m, 24H, CH2), 1.56 (m, 14H, CH2), 2.61 (t,
2H, CH2CO, J = 7.6 Hz), 2.84 (s, 4H, CH2 succin-
imide), 4.00 (q, 2H, POCH2, J = 7.0 Hz), 5.02 (s,
2H, CH2OPh), 5.07 (s, 2H, CH2OPh), 7.22–7.44
(m, 10H, arH). 13C NMR (CDCl3) δ (ppm) 25.31,
28.46, 28.69, 28.84, 29.09, 29.19, 29.68, 32.36, 67.59,
68.68, 127.36, 128.01, 136.72, 168.90, 169.98. IR
(KBr) cm−1 698, 736, 1002–1078 (br), 1215, 1580,
1628, 1735, 1741, 2851, 2924. MS (APCI) m/z 686.5
(M+H)+.
30.92, 51.55, 66.86, 66.96, 81.50. IR (KBr) cm−1
566, 995, 1036, 1264, 1363, 1391, 1460, 2094, 2842,
2933.
2.18. Phosphoric acid 20-amino-icosyl ester
di-tert-butyl ester (17)
A mixture of 16 (0.4 g, 0.75 mmol) and triph-
enylphosphine was refluxed in toluene (20 ml) for 2 h.
The solvent was removed in vacuo and the residue
dissolved in THF (20 ml) containing water (27 ␮l,
1.5 mmol). The solution was stirred for 20 h then evap-
orated to dryness. The product, a yellow oil (0.3 g, 79%,
Rf = 0.26 in chloroform–methanol–ammonia, 9:1:0.1)
was obtained after silica-gel column chromatogra-
phy with chloroform–methanol–ammonia (95:5:0.5)
eluent. 1H NMR (CDCl3) δ (ppm) 1.22 (m, 32H,
CH2), 1.49 (m, 18H, tBu), 2.22 (br, 2H, NH2),
2.72 (t, 2H, CH2N, J = 7.0 Hz), 3.94 (q, 2H,
POCH2, J = 6.6 Hz). 13C NMR (CDCl3) δ (ppm)
25.68, 26.79, 28.90, 29.25, 29.45, 29.70, 29.78,
29.87, 29.90, 30.08, 30.26, 33.02, 42.45, 66.74,
66.90, 81.34. IR (KBr) cm−1 562, 705, 995 (br),
1040, 1267, 1371, 1394, 1472, 2852, 2920, 2979,
3345.
2.16. 20-Phosphonooxy-icosanoic acid
2,5-dioxo-pyrrolidin-1-yl ester (15)
The hydroxysuccinimide ester 14 (0.08 g,
0.12 mmol) dissolved in ethylacetate (20 ml) was
debenzylated with palladium (10% on carbon) catalyst
(0.02 g) in a hydrogen atmosphere overnight. The cat-
alyst was removed by filtration on Celite. The removal
of the solvent in vacuo provided the white crystalline
product (0.048 g, 84%, m.p.: 105–107 ◦C). H NMR
1
(CDCl3 + CD3OD) δ (ppm) 1.24 (m, 28H, CH2),
1.69 (m, 4H, CH2), 2.64 (t, 2H, CH2CO, J = 7.2 Hz),
2.88 (s, 4H, CH2 succinimide), 3.96 (q, 2H, POCH2,
J = 6.8 Hz). 13C NMR (CDCl3 + CD3OD) δ (ppm)
25.41, 28.96, 29.12, 29.34, 29.44, 29.82, 29.96,
30.18, 30.37, 30.56, 30.70, 66.20, 168.82, 169.35. IR
(KBr) cm−1 653, 744, 964, 1010–1073 (br), 1125,
1210, 1288, 1380, 1467, 1728, 1787, 1818, 2851,
2921.
2.19. 3-[20-(Di-tert-butoxy-phosphoryloxy)-
icosylcarbamoyl]-acrylic acid (18)
A mixture of the amino-phosphate 17 (0.17 g,
0.34 mmol) and maleic anhydride (0.034 g, 0.34 mmol)
was refluxed in dry chloroform (15 ml) for 7 h. The
maleamic acid 18 (0.19 g, 93%, m.p.: 99–102 ◦C,
Rf = 0.11 in chloroform–methanol–ammonia, 9:1:0.1)
obtained after the evaporation of the solvent was suffi-
2.17. Phosphoric acid 20-azido-icosyl ester
di-tert-butyl ester (16)
1
ciently pure to use in the cyclization step. H NMR
A mixture of 6 (0.86 g, 1.5 mmol) and sodium azide
(0.2 g, 3.0 mmol) was stirred in DMF (30 ml) at 80 ◦C
for 6 h. The solvent was removed in vacuo and the
residue was shaken with a mixture of dichloromethane
(40 ml) and water (20 ml). The organic layer was dried
over MgSO4 then evaporated. The crude product was
chromatographed using hexane–ethyl acetate (85:15)
on a silica-gel column to obtain the oily azide 16
(0.6 g, 70%, Rf = 0.16 in hexane–ether, 8:2). 1H NMR
(CDCl3) δ (ppm) 1.22 (m, 32H, CH2), 1.49 (m, 18H,
tBu), 2.77 (t, 2H, CH2N3), 3.94 (q, 2H, POCH2).
13C NMR (CDCl3) δ (ppm) 25.65, 26.74, 28.86,
29.15, 29.33, 29.55, 29.68, 29.87, 29.93, 30.24, 30.36,
(DMSO-d6 + CDCl3) δ (ppm) 1.22 (m, 32H, CH2),
t
1.49 (m, 18H, Bu), 3.21 (t, 2H, CH2N, J = 6.0 Hz),
3.94 (q, 2H, POCH2, J = 6.7 Hz), 6.08 (d, 1H, CH CH,
J = 12.8 Hz), 6.24 (d, 1H, CH CH, J = 12.8 Hz), 8.32
(br, 1H, NH), 9.21 (s, 1H, COOH). 13C NMR (DMSO-
d6 + CDCl3) δ (ppm) 25.86, 26.55, 28.40, 28.86,
29.25, 29.72, 29.77, 29.85, 29.94, 30.18, 30.29, 31.46,
41.07, 66.73, 66.94, 81.50, 131.96, 133.88, 164.99,
165.67. IR (KBr) cm−1 630, 717, 859, 955, 1025,
1071, 1213, 1384, 1472, 1524, 1636, 1722, 2850,
2918, 3280. MS (APCI) m/z 604.6 (M+H)+, 492.4
(M-2xC(CH3)3+3H)+.

I. Jablonkai, P. Oroszlan / Chemistry and Physics of Lipids 133 (2005) 103–112
109
2.20. Phosphoric acid di-tert-butyl ester 20-(2,5-
dioxo-2,5-dihydro-pyrrol-1-yl)-icosyl ester (19)
in the presence of lithium tetrachlorocuprate (II) to
yield alkyl halides with extended chain length. 1,20-
Dibromoeicosane 1 was obtained in good yield (74%)
by this method starting from 1,6-dibromohexane
and 1,8-octanedimagnesium chloride (Scheme 1).
Interestingly, a poor yield (15%) was reported when
1,20-dibromoeicosane was prepared from 1,10-
dibromodecane and 1,5-dibromopentane employing
the same methodology (Naratjan et al., 1987). Since
such bifunctional long chain lipids with phosphate
headgroup and reactive terminal functionalities have
not been reported in the literature, the initial strategy
for obtaining 20-mercapto-icosylphosphate 9 was
based on “protected functionality–non-protected
phosphate headgroup” approach (Scheme 1, route
A). This synthetic route includes the phosphorylation
of the 20-thioacetyl-eicosane-1-ol 4 to a phosphate
monoester prior to the deprotection of the thiol.
Following this approach 1,20-dibromoeicosane 1 was
converted to THP-protected monobromo-eicosanol 2
after acetate displacement of one of the bromides, ester
hydrolysis and THP-ether formation using standard
methods. Subsequent thioester formation followed by
removal of the THP protection provided the eicosanol
4. Phosphorylation was achieved using trimethylsilyl
polyposhphate (Okamoto, 1985) yielding the highly
pure phosphoric acid monoester 5 in excellent yield.
However, the deprotection of this thioester by base-
catalyzed hydrolysis did not offer the desired thiol 9.
Instead, the main product of the hydrolysis was the
disulfide-bonded diphosphate, insoluble in most sol-
vents such as alcohols, ethers, and CHCl3-methanol.
Due to difficulties in handling the disulfide, all efforts
to obtain the thiol-phosphate 9 failed.
In order to overcome solubility and purification
problems that appeared during deprotection of the
thioacetate 5 in the presence of the polar, unprotected
phosphate group, the synthetic strategy was changed
for the “protected functionality–protected phosphate”
approach (Scheme 1, route B). Accordingly, the
protected ␻-functionality was prepared after the
introduction of a protected phosphate group. Cleavage
of the phosphate protection was carried out in the final
step in the presence of an unmasked thiol group. In
order to shorten the synthesis, a rarely used phospho-
rylation method for alkyl halides was employed where
the starting intermediate 1,20-dibromoeicosane (1)
was monophosphorylated with tetramethylammonium
A mixture of the maleamic acid 18 (0.5 g,
0.83 mmol),
1-hydroxybenzotriazole
(0.12 g,
0.83 mmol) and dicyclohexylcarbodiimide (0.17 g,
0.83 mmol) in dry THF (30 ml) was stirred for 20 h
at room temperature. The solvent was removed
in vacuo and the residue was purified on silica
gel with hexane–ethylacetate (9:1) to obtain the
maleimide 19 (0.26 g, 52%, m.p.: 68–70 ◦C, Rf = 0.32
in hexane–ethyl acetate, 8:2) as white crystalline
1
material. H NMR (CDCl3) δ (ppm) 1.25 (m, 32H,
t
CH2), 1.49 (m, 18H, Bu), 1.57 (m, 4H, CH2), 3.50
(t, 2H, CH2N, J = 7.0 Hz), 3.94 (q, 2H, POCH2,
J = 6.7 Hz), 6.68 (s, 2H, CH CH). 13C NMR (CDCl3)
δ (ppm) 26.22, 26.55, 28.00, 28.59, 28.82, 28.95,
29.02, 29.15, 29.94, 30.18, 31.39, 37.42, 51.50, 66.73,
66.94, 81.34, 133.49, 170.33. IR (KBr) cm−1 630,
717, 859, 950, 1025, 1071, 1213, 1368, 1404, 1466,
1524, 1700, 2851, 2919, 3088.
2.21. Phosphoric acid mono-[20-(2,5-dioxo-2,5-
dihydro-pyrrol-1-yl)-icosyl] ester (20)
To a stirred solution of maleimide 19 (0.20 g,
0.34 mmol) in dioxane (20 ml) was added 4N HCl
(5 ml) at room temperature and the mixture was stirred
for 5 h. The cloudy solution was diluted with wa-
ter (25 ml) and the white precipitate formed was fil-
tered off and washed with 50% ethanol to give the
1
product (0.14 g, 90%, m.p.: 119–121 ◦C). H NMR
(DMSO-d6 + CDCl3)δ(ppm)1.25(m, 32H, CH2), 1.57
(m, 4H, CH2), 3.52 (t, 2H, CH2N, J = 7.0 Hz), 3.98
(q, 2H, POCH2, J = 6.8 Hz), 6.71 (s, 2H, CH CH).
13C NMR (DMSO-d6 + CDCl3) δ (ppm) 26.42, 26.65,
28.89, 29.22, 29.25, 29.98, 30.24, 31.41, 37.48, 51.55,
66.83, 66.99, 133.55, 170.30. IR (KBr) cm−1 638, 717,
859, 960–1073 (br), 1124, 1368, 1404, 1466, 1524,
1700, 2851, 2919, 3087.
3. Results
The C20 alkyl chain was assembled from C6 and
C8 units using the halopolycarbon homologation
method (Friedman and Shani, 1974) in which a
Grignard reagent reacts with ␣,␻-dibromoalkanes

110
I. Jablonkai, P. Oroszlan / Chemistry and Physics of Lipids 133 (2005) 103–112
Scheme 1. Preparation of 20-mercapto-icosylphosphate. (a) Li2CuCl4, THF, 5◦, 74%; (b) NaOOCCH3, DMF, 80◦, 37%; (c) H+, MeOH,
99%; (d) DHP, PPTS, rt, 84%; (e) CH3COSK, DMF, 80◦, 92%; (f) PPTS, EtOH-CHCl3, 55◦, 69%; (g) P2O5, (CH3)3SiOSi(CH3)3, 86%; (h)
(CH3)4N+O−P(O)(OtBu)2, DME, 100◦, 34%; (i) CH3COSK, DMF, 80◦, 71%; (j) LiAlH4, THF, 0◦, 88%; (k) 4N HCl, dioxane, rt, 92%.
di-tert-butyl phosphate (Zwierzak and Kluba, 1971)
via nucleophilic substitution. Since the phosphoryla-
tion agent is not available commercially it was prepared
from di-tert-butyl phosphite (Goldwhite and Saunders,
1957) in three steps (Zwierzak and Kluba, 1971). The
thiol group was then introduced as thioacetate by the
treatment of the di-tert-butyl-protected bromoalkyl
phosphate 6 with potassium thioacetate in dimethyl-
formamide. Transformation of the thioester 7 to thiol 8
was accomplished by using lithium aluminium hydride
since a high portion of disulfide byproduct formed
when methanolic KOH was used for the deprotection.
After purification of the thiol-phosphate 8 by chro-
matography, the acid sensitive tert-butyl protective
groups were cleanly removed in dioxane. The product
9 obtained was sufficiently pure after washing the
deprotected material several times with methanol.
Preparation of 20-phosphonooxy-icosanoic acid
N-hydroxysuccinimidoyl ester (15) was carried out
by using a similar approach as described for the thiol
analog (9). However, instead of tert-butyl protection
the synthesis proceeded through benzylated phosphate
intermediates that could be cleaved under neutral con-
ditions without affecting the activated ester function
(Scheme 2). The preparation of tetramethylammonium
dibenzylphosphate was accomplished by a careful
neutralization of tetramethylammonium hydroxide
with the commercially available dibenzylphosphate as

I. Jablonkai, P. Oroszlan / Chemistry and Physics of Lipids 133 (2005) 103–112
111
Scheme 2. Preparation of 20-(N-hydroxysuccinimidyl)-icosylphosphate. (a) (CH3)4N+O−P(O)(OBn)2, DME, 100◦, 41%; (b) NaOOCCH3,
DMF, 80◦, 87%; (c) H+, MeOH, 65◦, 84%; (d) CrO3, H2SO4, rt, 79%; (e) N-hydroxysuccinimide, DCC, rt, 65%; (f) H2, Pd/C, rt, 84%.
described for the tert-butyl analogue (Zwierzak and
Kluba, 1971). Dibenzyl-protected ␻-bromo phosphate
10 was obtained from 1 and tetramethylammonium
dibenzylphosphate in moderate yield (41%). Displace-
ment of the bromine by treatment with sodium acetate
followed by acidic hydrolysis of the acetate 11 afforded
alcohol 12. The activated ester function was generated
by reacting carboxylic acid 13, formed by a Jones oxi-
dation, with N-hydroxysuccinimide in the presence of
dicyclohexylcarbodiimide (DCC). Removal of the ben-
zyl groups from the purified ester 14 by hydrogenolysis
resulted in the desired bifunctional lipid phosphate 15.
For the preparation of icosyl-phosphate carrying the
maleimido group (20), ␻-bromo di-tert-butyl protected
icosylphosphate 6 was treated with sodium azide and
the resulting azide 16 was converted to the correspond-
ing amine 17 by Staudinger reaction (Scheme 3). Syn-
thesis of 17 via a phthalimido derivative afforded a
very low yield (16%) due to the inefficient cleavage of
phthalimide function by hydrazinolysis. The reaction
Scheme 3. Preparation of 20-maleimido-icosylphosphate. (a) NaN3, DMF, 80◦, 70%; (b) PPh3, toluene, reflux then H2O, THF, rt, 79%; (c)
maleic anhydride, CHCl3, reflux, 93%; (d) 1-HOBt, DCC, THF, rt, 52%; (e) 4N HCl, dioxane, rt, 90%.

112
I. Jablonkai, P. Oroszlan / Chemistry and Physics of Lipids 133 (2005) 103–112
of amine 17 with maleic anhydride yielded maleamic
acid derivative 18 which was cyclized to maleimide 19
using DCC as a dehydrating agent in the presence of
1-hydroxybenzotriazole (HOBt) in 52% yield. Carry-
ing out the cyclization without HOBt led exclusively
to isomaleimide formation. Removal of the tert-butyl
groups accomplished by treatment of the maleimide 19
with 4N HCl in dioxane, resulting in the maleimido-
phosphate 20.
In conclusion, new 1,20-substituted eicosanes
carrying phosphate headgroups and readily deriva-
tizable thiol, maleimido, and activated carboxylic
ester moieties were prepared by “protected functiona-
lity–protected phosphate” synthetic approach. In
these ␣,␻-bifunctional lipids the phosphate headgroup
can be tethered to a metal oxide surface while the
other terminal functionality can be derivatized by
binding to various peptides and proteins to create
“biocompatible” surfaces e.g., antibodies used as the
affinity element in biosensing.
Friedman, L., Shani, A., 1974. Halopolycarbonhomologation. J. Am.
Chem. Soc. 96, 7101–7103.
Goldwhite, H., Saunders, B.C., 1957. Esters containing phosphorus.
Part XIV. Some tert-butyl esters and their reactions. J. Chem.
Soc., 2409–2412.
Hasegawa, E., Fukuzumi, M., Nishide, H., Tsuchida, E., 1990. Syn-
thesis of negatively-charged porhinatoiron(II) having a phospho-
serine group and oxygenation in phospholipd bilayer. Chem.
Lett., 123–126.
Nakashima, N., Abe, K., Hirohashi, T., Hamada, K., Kunitake,
M., Manabe, O., 1993. Electrochemistry of cytochrome c at
a lipid Langmiur Blodgett monolayer electrode. Chem. Lett.,
1021–1024.
Naratjan, A., Ferrera, J.D., Youngs, W.D., Sukenik, C.N., 1987. Mi-
cellar control of organic reactions: propellane substrates as stere-
ochemical probes for micellar binding. J. Am. Chem. Soc. 109,
7477–7483.
Ohlsson, J., Magnusson, G., 1999. ␻-Mercapto analogs of natuarally
occurring lipids. Tetrahedron Lett. 40, 2011–2014.
Okamoto, Y., 1985. Synthesis of alkyl dihydrogenphosphate by the
reaction of alcohols and silyl polyphosphate. Bull. Chem. Soc.
Jpn. 58, 3393–3394.
Roberts, C., Chen, C.S., Mrksich, M., Martichonok, V., Ingber, D.E.,
Whitesides, G.M., 1998. Using a mixed self-assembled monolay-
ers presenting RGD and (EG)3OH groups to characterize long-
term attachment of bovine capillary endothelial cell surfaces. J.
Am. Chem. Soc. 120, 6548–6555.
References
Walker, S.A., Kennedy, M.T., Zasadzinski, J.A., 1997. Encapsulation
of bilayer vesicles by self-assembly. Nature 387, 61–64.
Wang, G., Hollingsworth, R.I., 1999. Synthesis and properties of
a bipolar, bisphosphatidyl ethanolamine that forms stable 2-
dimensional self-assembled bilayer systems and liposomes. J.
Org. Chem. 64, 4140–4147.
Zwierzak, A., Kluba, M., 1971. Organophosphorus esters – I. t-Butyl
as protecting group in phosphorylation via nucleophilic substi-
tution. Tetrahedron 27, 3163–3170.
Behr, J.-P., Demeneix, B., Loeffler, J.-P., Perez-Mutul, J., 1989. Effi-
cient gene transfer into mammalian primary endocrine cells with
lipopolyamine-coated DNA. Proc. Natl. Acad. Sci. U.S.A. 86,
6982–6986.
Brovelli, D., Hahner, G., Ruiz, L., Hofer, R., Kraus, G., Waldner,
A., Schloesser, J., Oroszlan, P., Ehrat, M., Spencer, N.D., 1999.
Highly oriented, self-assembled alkanephosphate monolayers on
tantalum(V) oxide surfaces. Langmuir 15, 4324–4327.