Drug-phospholipid conjugates as potential prodrugs: Synthesis, characterization, and degradation by pancreatic phospholipase A2

Article abstract of DOI:10.1016/S0009-3084(00)00167-5

The aim of the present study was the synthesis of phospholipids containing a drug molecule instead of a fatty acid. Valproic acid and ibuprofen served as model compounds. The target molecules were synthesized either starting from sn-glycero-3-phosphocholi

Full text of DOI:10.1016/S0009-3084(00)00167-5

Chemistry and Physics of Lipids
107 (2000) 143–157
www.elsevier.com/locate/chemphyslip
Drug–phospholipid conjugates as potential prodrugs:
synthesis, characterization, and degradation by pancreatic
phospholipase A2
Michael Kurz a, Gerhard K.E. Scriba b,*
a Uni6ersity of Mu¨nster, Department of Pharmaceutical Chemistry, D-48149 Mu¨nster, Germany
b Department of Pharmaceutical Chemistry, Uni6ersity of Jena, School of Pharmacy, Philosophenweg 14, 07743 Jena, Germany
Received 10 April 2000; received in revised form 13 June 2000; accepted 14 June 2000
Abstract
The aim of the present study was the synthesis of phospholipids containing a drug molecule instead of a fatty acid.
Valproic acid and ibuprofen served as model compounds. The target molecules were synthesized either starting from
sn-glycero-3-phosphocholine (1) or using (S)-2-O-benzyl-1-O-tritylglycerol (11) and (R)-2-O-benzyl-1-O-tert-
butyldiphenylsilylglycerol (12), respectively, as key intermediates. With respect to the surface properties and the
aggregation behavior, the drug–phospholipid conjugates resembled natural phosopholipids. Upon incubation with
porcine pancreatic phospholipase A2, only compounds with a fatty acid in the sn-2 position of the glycerol backbone
were degraded. Derivatives with either ibuprofen in the sn-2 position or displaying the unnatural S-configuration
were resistant to enzymatic in vitro hydrolysis. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Drug-phospholipid conjugates; Prodrugs; Phospholipid; Phospholipase A2
substitution of one or more fatty acids of a
triglyceride by a drug molecule in order to confer
1. Introduction
the attached drugs to the metabolic pathways of
Many promising pharmacological agents, dis-
natural lipids. Non-steroidal anti-inflammatory
playing high in vitro efficacy, are less active or
drugs such as aspirin, indomethacin, ibuprofen, or
even inactive after in vivo application, especially
naproxen were covalently bound to diglycerides in
after oral administration. One approach to over-
order to reduce the ulcerogenity of the com-
come this problem is the synthesis of prodrugs.
pounds (Paris et al., 1979; Paris et al. 1980; Paris
Among the prodrugs, triglyceride analogues, also
et al., 1982). The approach has also been utilized
termed pseudoglycerides, have been prepared by
for an improved oral bioavailability of phenytoin
(Scriba et al., 1995). Employing the absorption of
dietary fat, pseudoglycerides have been used for
targeting the lymphatic route in order to avoid
* Corresponding author. Tel.: +49-3641-949830; fax: +49-
3641-949802.
E-mail address: gerhard.scriba@uni-jena.de (G.K.E. Scriba).
1079-3084/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0009-3084(00)00167-5

144
M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157
al., 1994). ‘Pseudophospholipid’ prodrugs bearing
a drug molecule instead of a fatty acid have not so
far been realized. The present study describes the
synthesis of such phospholipid derivatives using
the anticonvulsant valproic acid and the non-
steroidal anti-inflammatory drug ibuprofen as
model compounds. The drugs were primarily at-
tached to the sn-1 position, which is not hydrolyzed
by pancreatic phospholipase A2. The surface activ-
ity of the phospholipid analogues and the degrada-
tion of the lipids by phospholipase A2 were also
investigated.
the first-pass metabolism of
L-DOPA (Garzon-
Aburbeh et al., 1986) and/or target the lymphatic
system for the treatment of lymphatic metastasis
(Garzon-Aburbeh et al., 1983) or filiariasis (De-
verre et al., 1992). Radioionated agents (Weichert
et al., 1995) and compounds containing a nitroxyl
moiety (Gallez et al., 1992) were coupled to glyce-
rides as potential hepatographic agents in nuclear
medicine and nuclear magnetic resonance imaging.
Improvement of the blood–brain barrier penetra-
tion of GABA glycerides has been reported (Jacob
et al., 1990). A related approach has been applied
to L-DOPA, glycine, valproic acid and the
enkephalinase inhibitor thiorphan including amide
isosters of glycerides (Mergen et al., 1991; Lambert
et al., 1993).
2. Results and discussion
The use of phospholipid analogues as drug
carriers has so far been utilized primarily for
cytostatic nucleosides and nucleoside analogues
such as AZT (van Wijk et al., 1992, 1994), acyclovir
(Hostetler et al., 1990, 1993; van Wijk et al., 1992)
or cytidine analogues (Turcotte et al., 1980; Mat-
sushita et al., 1981; Ryu et al., 1982). In these
derivatives, nucleoside monophosphates, diphos-
phates and triphosphates were bound to diacylglyc-
erols, yielding derivatives with increased anti-
neoplastic activity in vitro and in vivo. A phospho-
lipid derivative obtained by coupling of a HIV
protease inhibitor to dipalmitoylphosphatidyl-
ethanolamine has also been described (Hostetler et
2.1. Synthesis
The synthetic sequence starting from sn-glyc-
ero-3-phosphocholine (1), as depicted in Scheme
1, essentially followed the procedure published
by Hermetter et al. (1989) for the synthesis of
unsymmetrical phospholipids, except that the
esterification of the hydroxy group of the trityl
intermediate 2 with palmitic acid in the sn-2
position was carried out in CH2Cl2 using dicyclo-
hexylcarbodiimide
(DCC)
and
4-dimethy-
laminopyridine (DMAP) instead of the acid
imidazolide in dimethylsulphoxide (DMSO) in the
presence of sodium. Despite longer reaction
Scheme 1.

M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157
145
Scheme 2.
times, we found this procedure more suitable,
especially due to the more facile work-up. Both
procedures give comparable yields of compound 3.
We found 77% compared with 74% of the published
procedure. The final product 1-O-valproyl-2-O-
palmitoyl-sn-glycero-3-phosphocholine (4) was ob-
tained in a one-pot reaction by removal of the trityl
group with BF3 in the presence of valproic acid
anhydride according to Hermetter et al. (1989). The
overall yield of this sequence was 40%. However,
attempts to synthesize the corresponding ibuprofen
phospholipid by the same sequence failed.
The second synthetic sequence utilized the key
intermediates 11 and 12 (Peters et al., 1987), which
allowed the preparation of derivatives with the
natural R-configuration of phospholipids as well as
derivatives with the unnatural S-configuration.
Moreover, the corresponding lyso-phospholipids
can be obtained during this sequence. Compounds
11 and 12 were synthesized as outlined in Scheme
2.
D
-Mannitol (5) was converted to the dibenzyli-
dene acetal 6 by stirring in acidified dimethylfor-
mamide (DMF) in the presence of an excess of
benzaldehyde followed by reaction with KOH and
benzyl chloride in DMSO (Johnstone and Rose,
1979) to give 2,5-di-O-benzyl-1,3:4,6-di-O-benzyli-
dene-
tecting groups upon refluxing with diluted HCl in
ethanol gave 2,5-di-O-benzyl- -mannitol (8) with
a yield of 93%. 2-O-Benzyl-1-O-trityl- -mannitol
(9) was obtained by reaction of compound 8 with
trityl chloride in the presence of DMAP as catalyst
and triethylamine as base. Malaprade oxidation of
compound 9 using an excess of 1.5 equivalents of
sodium periodate in THF/water (10:1) resulted in
aldehyde 10, which was reduced with sodium
borohydride to yield (S)-2-O-benzyl-1-O-trityl-
glycerol (11) (Peters et al., 1987). The overall yield
of these three steps starting from compound 8 was
90%. All steps can also be carried out without
extensive isolation of the intermediates. (R)-2-O-
D
-mannitol (7). Removal of the acetal pro-
D
D

146
M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157
benzyl-1-O-tert-butyldiphenylsilylglycerol (12), the
starting material for phospholipids with the unnat-
ural S-configuration, was obtained from com-
pound 11 by protection of the free hydroxy group
as tert-butyldiphenylsilylether (Hanessian and
Lavallee 1975) followed by removal of the trityl
group by refluxing with a 1% solution of pyri-
dinium chloride in ethanol.
pound 11 as outlined in Scheme 3. Upon reaction
with 1.2 equivalents methyldichlorophosphite and
2.5 equivalents of diisopropylethylamine in te-
trahydrofuran (THF) at −78°C, followed by the
addition of 1.5 equivalents of 2-bromoethanol and
oxidation with hydrogen peroxide (Martin et al.,
1994), ((R)-(2-O-benzyl-3-O-tritylglyceryl)-2-bro-
moethyl-methyl phosphate (13) was produced as a
viscous oil that was converted to ((R)-2-O-benzyl-
glyceryl)-2-bromoethyl-methyl phosphate (15) by
The drug–phospholipid conjugates with the nat-
ural R-configuration were synthesized from com-
Scheme 3.

M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157
147
that removal of the silyl ether of compound 14
to give ((S)-2-O-benzylglyceryl)-2-bromoethyl-
methyl phosphate (16) was carried out with tetra-
butylammonium fluoride in THF. The yields of
this sequence were comparable with the R-
configurated series.
2.2. Physicochemical characterization
With respect to the physicochemical properties,
the surface activity and the particle size of aggre-
gates formed in aqueous solutions of compounds
30 and 32 were determined. The surface activity
of the compounds was determined by the Wil-
helmy method. The surface pressure apparent
molecular area curves of compounds 30 and 32
are shown in Fig. 1. Lift off areas of 90 and 110
2
,
A /molecule were found for compounds 30 and
32, respectively. Transition to the solid-condensed
phase occurred continuously. The collapse points
2
,
were 45 and 55 A /molecule, the collapse pressure
2
,
values were 45 and 43 mN/m. Values of 40 A /
mol and 60 and 40 mN/m have been reported for
the standard phospholipids 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-
glycero-3-phosphoglycerol, respectively (Bredlow
et al., 1992). These differences compared with the
ibuprofen-containing pseudophospholipids may
be explained by the smaller size of the lipophilic
part of the molecule due to the substitution of a
palmitic acid residue with ibuprofen.
The aggregation behavior of the ibuprofen
phospholipids 30 and 32 in aqueous solution was
characterized by dynamic laser light scattering.
An average particle size of about 160–180 nm
was determined for a 0.1% aqueous dispersion of
compound 30. This suggests the formation of
spherical particles by compound 30 (Eibl, 1984).
The value was not significantly altered upon in-
creasing the temperature from 20 to 37°C. In
contrast, compound 32 containing ibuprofen in
the sn-2 position displayed a different behavior.
While two maxima at approximately 500 and
1000 nm could be observed when no particle size
was preselected (Fig. 2A), a single maximum at
about 550 nm was recorded when a particle size
of 500 nm (Fig. 2B) or 1000 nm (data not shown)
was preselected. This behavior did not change
Fig. 1. Surface pressure-apparent molecular area isotherms of
compounds 30 and 32 at the air–water interface at 20°C.
refluxing with pyridinium chloride in ethanol. De-
pending on the target molecule the free hydroxy
group was esterified with either ibuprofen,
palmitic acid, or valproic acid to give compounds
17, 19 and 20, respectively. Hydrogenolytic depro-
tection and esterification with the appropriate car-
boxylic acid gave compounds 22, 23 and 25–27,
which were converted to the pseudophospholipids
29, 30, and 32–34 by treatment with trimethy-
lamine. The lyso-phospholipid 28 was obtained by
treatment of compound 21 with triethylamine. All
reactions of the sequence did not affect the stereo-
chemistry of the chiral sn-2 carbon atom. The
chiral purity of all compounds was estimated by
measuring the optical rotation.
The pseudophospholipid 31 with the unnatural
S-configuration was obtained from compound 12
in the same synthetic sequence as described for
the R-configurated compounds (Scheme 3) except

148
M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157
fact that the maximum at 1000 nm is twice the
size of the maximum at 500 nm suggested the
existence of ‘monomers’ and ‘dimers’. The
‘dimers’ should disappear upon dilution. How-
ever, no significant change was observed upon
dilution to 0.01%. As the solution could not be
diluted further due to a strong concomitant de-
crease of the light scattering intensity, the pres-
ence of ‘dimers’ cannot be unequivocally ruled
out. An alternative explanation of the observed
phenomenon might be the existence of hexagonal
structures as the fitting software cannot distin-
guish between light scattering at spherical or
cylindric shape particles.
upon dilution of the solution to 0.01% or upon
increasing the temperature from 20 to 37°C. The
2.3. Enzymatic degradation
The ibuprofen-containing phospholipids 29–32
were incubated in the presence of porcine pan-
creatic phospholipase A2 in order to estimate the
activity of the enzyme towards the drug–phos-
pholipid conjugates. The incubations were ana-
lyzed by high-performance liquid chromatography
(HPLC). Under the conditions applied, only con-
jugate 30, which contained ibuprofen in the sn-1
position and palmitic acid in the sn-2 position
and possesses the natural R-configuration, was
hydrolyzed. No degradation of the other com-
pounds was observed for 96 h. This is in agree-
ment with the fact that pancreatic phospholipase
A2 only cleaves R-configurated phospholipids
and not derivatives with the S-configuration, i.e.
compound 31. Apparently, compounds containing
ibuprofen at the sn-2 position are not substrates
for the enzyme under the conditions applied.
This may be explained by steric hindrance of
the branched 2-methyl-phenylacetic acid moiety
and/or by a different spatial arrangement of
the acid moieties compared with natural phospho-
lipids with linear fatty acids.
Fig. 2. Distribution of particle size of compound 32 deter-
mined by dynamic laser light scattering. (A) No preselected
particle size; (B) preselected particle size, 500 nm.
The time course of the degradation of com-
pound 30 by pancreatic phospholipase A2 at
a starting concentration of 500 mM is shown in
Fig. 3. Between 0 and 3 h, the hydrolysis follows
apparent zero-order kinetics with a half-life of
2.0 h, while the kinetic becomes apparent first
order after 5–6 h with a half-life of 13.1 h.
Fig. 3. Time course of the in vitro degradation of compound
30 by porcine pancreatic phospholipase A2. Each time point
represents the mean of three determinations9S.D.

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149
3. Conclusions
230°C. [h]2D0= −10.0° (c=4.0 in MeOH). 1H-
NMR (MeOD, 200 MHz): l (ppm)=3.14 (s,
11H), 3.55 (m, 2H), 3.95 (m, 2H), 4.04 (m, 1H),
4.22 (m, 2H), 7.17–7.48 (m, 15H). 13C-NMR
(MeOD, 50 MHz): l (ppm)=54.58, 60.31, 66.07,
67.29, 68.82, 71.28, 87.85, 128.13, 128.81, 129.85,
145.32. MS (chemical ionization (CI), ammonia):
m/z=500 ([M+H]+).
The target ‘pseudophospholipids’ containing ei-
ther valproic acid or ibuprofen instead of a fatty
acid were synthesized. The surface properties and
aggregation behavior resembled the properties of
natural phospholipids. Upon incubation with
pancreatic phospholipase A2, only the com-
pounds containing palmitic acid in the sn-2 posi-
tion of the glycerol backbone were degraded,
while the compounds containing either ibuprofen
in the sn-2 position or displaying the unnatural
S-configuration were resistant to enzymatic hy-
drolysis. The pharmacological activity of the
compounds is currently under investigation.
4.1.2. 1-O-Trityl-2-O-palmitoyl-sn-glycero-3-
phosphocholine (3)
Compound 3 was prepared from 8.0 g com-
pound 2 (16 mmol), 5.0 g palmitic acid (20
mmol), 5.0 g DCC (24 mmol) and 1 g DMAP
(8.2 mmol) in 80 ml CH2Cl2. Filtration, evapora-
tion of the solvent and recrystallization from
methanol:water (4:1, v/v) followed by recrystal-
lization from acetone, yielded 5.7 g (77%). [h]2D0=
4. Experimental
1
−7.4° (c=4.0 in MeOH). H-NMR (CDCl3, 200
3
4.1. Synthesis
MHz): l (ppm)=0.88 (t, 3H, J=5.8 Hz), 1.25
3
(s, 24H), 1.60 (m, 2H), 2.34 (t, 2H, J=7.5 Hz),
sn-Glycero-3-phosphocholine was a gift from
Nattermann Phospholipid GmbH (Cologne, Ger-
many). All other chemicals were obtained from
commercial suppliers and were used without fur-
ther purification unless noted otherwise. THF
and diethyl ether were distilled from sodium and
benzophenone ketyl, chloroform was distilled
from phosphorous pentoxide, and the amines and
DMF were distilled from calcium hydride. Reac-
tions involving air- or moisture-sensitive reagents
were carried out under nitrogen. Column chro-
matography was performed using Merck silica gel
60 (70–230 mesh). Melting points are uncor-
rected. Nuclear magnetic resonance (NMR) spec-
tra were obtained on either a Varian Gemini 200
or a Varian Unityplus 600. Mass spectra were
obtained on a Varian MAT 44S. The optical
rotation, [h]2D0, was recorded using a Perkin-Elmer
341 instrument. Elemental analyses were within
90.4% of the theoretical values.
3.14 (s, 11H), 3.65 (m, 2H), 3.95 (m, 2H), 4.13
(m, 2H), 5.24 (m, 1H), 7.16–7.42 (m, 15H). 13C-
NMR (CDCl3, 50 MHz): l (ppm)=14.15, 22.71,
25.05, 29.30, 29.73, 31.94, 34.61, 54.23, 59.14,
63.05, 64.20, 66.21, 72.44, 86.46, 127.09, 127.84,
128.90, 143.70, 173.42. MS (CI, ammonia): m/
z=738 (M+).
4.1.3. 1-O-Valproyl-2-O-palmitoyl-sn-glycero-
3-phosphocholine (4)
Compound 4 was prepared from compound 3
(3.0 g, 4 mmol), valproic acid anhydride (2.2 g,
8 mmol) and 16 mmol BF3·Et2O according to the
procedure by Hermetter et al. (1989). Yield: 1.85
g (77%). [h]2D0= +6.4° (c=4.0 in CHCl3). 1H-
NMR (CDCl3, 200 MHz): l (ppm)=0.88 (t, 9H,
3J=7.6 Hz), 1.26 (s, 28H),1.50 (m, 6H), 2.29
3
(t, 2H, J=7.6 Hz), 2.34 (m, 1H), 3.38 (s, 9H),
3.80 (m, 2H), 3.89 (m, 2H), 4.30 (m, 2H), 4.41
(m, 2H), 5.18 (m, 1H). 13C-NMR (CDCl3, 50
MHz):
4.1.1. 1-O-Trityl-sn-glycero-3-phosphocholine (2)
Compound 2 was synthesized from 10.3 g sn-
glycero-3-phosphocholine (1) (40 mmol), 5.5 g
ZnCl2 (40 mmol) and 12.2 g trityl chloride (44
mmol) according to Hermetter et al. (1989).
Yield: 14.3 g (72%); melting point (m.p.), 228–
l (ppm)=14.12, 20.57, 22.71, 24.99, 29.27, 29.75,
31.97, 34.39, 34.49, 45.14, 54.44, 59.45, 62.92,
63.55, 66.55, 70.76, 173.11, 176.21. MS (CI,
ammonia): m/z=623 ([M+H]+). C32H64NO8P·1
.2H2O (643.45): Calc.: C, 59.73; H, 10.40; N,
2.18; found: C, 59.44; H, 10.33; N, 2.26.

150
M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157
2
3
4.1.4. 2,5-Di-O-benzyl-1,3:4,6-di-O-benzylidene-
Hz, 2H), 3.27 (dd, J=10.0 Hz, J=5.2 Hz, 2H),
2
3
D
-mannitol (7)
3.37 (dd, J=10.0 Hz, J=4.2 Hz, 2H), 3.74 (m,
2H), 3.97 (dd, 3J=5.7, 6.5 Hz, 2H), 4.53 (d,
2J=11.3 Hz, 2H), 4.70 (d,2J=11.3 Hz, 2H),
7.18–7.46 (m, 40H). 13C-NMR (CDCl3, 50 MHz):
Compound 7 was synthesized according to
Johnstone and Rose (1979) from 45 g 1,3:4,6-di-
O-benzylidene- -mannitol (6) (Baggett and Strib-
D
blehill, 1977), 60 g KOH (1 mol) and 60 ml benzyl
chloride (0.5 mol). Yield: 61 g (90%); m.p., 108–
110°C. [h]2D0= −37.5° (c=1.3 in CHCl3). 1H-
NMR (CDCl3, 200 MHz): l (ppm)=3.76 (t,
2J=3J=9.8 Hz, 2H), 3.90–4.11 (m, 4H), 4.42
l
(ppm)=63.83, 69.90, 73.31, 79.89, 87.13,
127.08, 127.73, 127.84, 127.91, 128.01, 128.06,
128.43, 128.46, 128.79, 138.34, 144.02. MS (CI,
ammonia): m/z=863 ([M+NH3]+).
3
(dd,2J=10.0 Hz, J=4.4 Hz, 2H), 4.64 (s, 4H),
4.1.7. (R)-2-O-Benzyl-3-O-tritylglycerol aldehyde
(10)
5.46 (s, 2H), 7.16–7.60 (m, 20H). 13C-NMR
(CDCl3, 50 MHz): l (ppm)=67.00, 69.70, 72.72,
77.58, 101.13, 126.30, 127.97, 128.02, 128.51,
128.86, 137.85, 138.08. MS (EI): m/z=538 (M+).
To 83 g of compound 9 (100 mmol) dissolved in
400 ml THF, 32 g sodium periodate (150 mmol)
in 100 ml water were added and stirred for 2 h.
Then, 400 ml ethyl acetate and 300 ml water are
added, and the aqueous phase was extracted with
ethyl acetate. The combined organic phases were
washed with brine and evaporated. Compound 10
was obtained as a colourless oil. Yield: 78.7 g
(95%). [h]2D0= +2.5° (c=1.1 in CHCl3). 1H-
4.1.5. 2,5-Di-O-benzyl- -mannitol (8)
D
Compound 7 (54 g, 0.1 mol) was refluxed for 4
h in 400 ml ethanol containing 40 ml of 0.5 M
HCl. After neutralization with NaHCO3, the sol-
vents were removed in vacuo. The residue was
dissolved in hot ethyl acetate and washed once
with warm water. Upon removal of the ethyl
acetate, the residue was suspended in a mixture of
diethyl ether (200 ml) and xylene (200 ml), and
stirred for 0.5 h. The white solid was filtered,
washed with petrol ether and dried. Yield: 33.7 g
(93%); m.p., 123–125°C. [h]2D0= −8.1° (c=1.0
2
NMR (CDCl3, 200 MHz): (ppm)=3.41 (dd, J=
3
2
10.1 Hz, J=5.3 Hz, 1H), 3.46 (dd, J=10.1 Hz,
3J=4.4 Hz, 1H), 3.93 (dd, J=4.4, 5.3 Hz, 1H),
3
4.68 (s, 2H), 7.19–7.46 (m, 20H), 9.72 (s, 1H).
13C-NMR (CDCl3, 50 MHz): l (ppm)=63.15,
72.57, 82.85, 87.10, 127.17, 127.26, 127.84, 127.89,
127.96, 128.02, 128.53, 128.71, 128.76, 138.42,
143.53, 201.74. MS (CI, ammonia): m/z=440
([M+NH3]+).
1
EtOH). H-NMR (MeOD, 200 MHz): l (ppm)=
2
3
3.61 (m, 2H), 3.75 (dd, J=11.8 Hz, J=4.5 Hz,
2H), 3.93 (dd, 2J=11.8 Hz, 3J=3.6 Hz, 2H), 3.98
(d, J=7.9 Hz, 2H), 4.58 (d, J=11.6 Hz, 1H),
3
2
4.1.8. (S)-2-O-Benzyl-1-O-tritylglycerol (11)
2
4.73 (d, J=11.6 Hz, 1H), 7.20–7.50 (m, 10H).
Compound 10 (78.5 g, 186 mmol) was dissolved
in 300 ml MeOH, 8 g sodium borohydride (200
mmol) added and the mixture was stirred for 4 h
at room temperature. The solvent was removed in
vacuo, the residue was dissolved in diethyl ether
and washed with water. The residue obtained
upon evaporation of the ether was purified by
column chromatography (cyclohexane/ethyl ace-
tate 10:1). Yield: 76.7 g (92%); m.p., 80–83°C.
[h]2D0= −26.3° (1.2% in CHCl3). 1H-NMR
(CDCl3, 200 MHz): l (ppm)=2.01 (s, 1H), 3.24
(dd, 2J=11.0 Hz, 3J=5.8 Hz, 1H), 3.31 (dd,
2J=11.0 Hz, 3J=4.8 Hz, 1H), 3.50–3.80 (m,
13C-NMR (MeOD, 50 MHz): l (ppm)=62.28,
70.41, 73.54, 81.42, 128.55, 128.97, 129.25, 140.01.
MS (CI, ammonia): m/z=379 ([M+NH3]+).
4.1.6. 2,5-Di-O-benzyl-1,6-di-O-trityl-D-mannitol
(9)
Compound 8 (36.2 g, 0.1 mol), 1 g DMAP and
35 ml triethylamine (250 mmol) were suspended in
250 ml CH2Cl2, 61.3 g tritylchloride (220 mmol)
were added and the mixture was stirred for 6 h at
room temperature. The organic phase was washed
with water and evaporated. The residue was re-
crystallized from 2-propanol. Yield: 83 g (98%);
m.p., 64–66 °C. [h]2D0= −22.1°. 1H-NMR
(CDCl3, 200 MHz): l (ppm)=2.84 (d, 3J=5.7
2
2
3H), 4.53 (d, J=11.7 Hz, 1H), 4.68 (d, J=11.7
Hz, 1H), 7.15–7.55 (m, 20H). 13C-NMR (CDCl3,

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151
phase was washed with water. Column chro-
matography (cyclohexane/ethyl acetate, 3:1)
yielded a colourless oil. Yield: 30.5 g (78%).
[h]2D0= −5.5° (c=0.8 in CHCl3). 1H-NMR
(CDCl3, 200 MHz): l (ppm)=3.26 (d, 3J=5.1
Hz, 2H), 3.42 (dt, 3J=6.2 Hz, 4JH,P=0.8 Hz,
50 MHz): l (ppm)=63.24, 63.65, 72.31, 78.84,
87.09, 127.16, 127.32, 127.84, 127.87, 127.94,
128.00, 128.03, 128.54, 128.78, 138.43, 143.97.
4.1.9. (R)-2-O-Benzyl-1-O-tert-
butyldiphenylsilylglycerol (12)
2
Twenty-eight grams of tert-butyldiphenylsilyl
chloride (100 mmol) were added to 40 g com-
pound 11 (94 mmol) and 15 g imidazole (220
mmol) in 125 ml DMF. The mixture was stirred
at room temperature overnight, poured into 400
ml water and extracted with CH2Cl2. The product
obtained upon evaporation of the solvent was
refluxed without further purification in 150 ml
ethanol containing 1.25% of pyridinium chloride
for 2 h. Upon addition of 1.1 equivalents of
NaHCO3 in water, the solvents were removed in
vacuo. The residue was dissolved in CH2Cl2 and
washed with brine. The organic phase was evapo-
rated in vacuo. Column chromatography (cyclo-
hexane/ethyl acetate, 10:1) yielded 33.4 g (84.5%)
of a colourless oil. [h]2D0= +23.7° (c=4.7 in
2H), 3.64–3.78 (m, JH,P=11.3 Hz, 4H), 4.15–
4.30 (m, 4H), 4.62 (s, 2H), 7.19–7.47 (m, 20H).
13C-NMR (CDCl3, 50 MHz): l (ppm)=29.86,
54.88, 63.14, 67.17, 68.14, 72.77, 77.38, 86.96,
126.27, 126.87, 127.12, 127.58, 127.74, 127.80,
127.87, 128.18, 128.29, 128.40, 128.73, 138.17,
143.83.
4.1.11. ((S)-2-O-Benzyl-3-O-tert-butyldiphenyl-
silylglyceryl)-2-bromoethyl-methylphosphate (14)
Compound 12 (26.3 g, 62.5 mmol) was reacted
as described for compound 13. Yield: 28.0 g
(72%). [h]2D0= +8.4° (c=1.3 in CHCl3). 1H-
NMR (CDCl3, 200 MHz): l (ppm)=1.06 (s, 9H),
3
4
3.44 (dt, J=6.2 Hz, JH,P=0.7 Hz, 2H), 3.71–
3
3.77 (m, JH,P=11.2 Hz, 6H), 4.18–4.30 (m, 4H),
1
CHCl3). H-NMR (CDCl3, 200 MHz): l (ppm)=
4.58 (s, 2H), 7.24–7.69 (m, 15H). 13C-NMR
(CDCl3, 50 MHz): l (ppm)=19.26, 26.89, 29.42,
54.49, 62.66, 66.73, 67.25, 72.24, 78.16, 127.73,
127.80, 128.38, 129.84, 133.21, 133.31, 135.62,
135.66, 138.17. MS (EI): m/z=623 (M+).
1.06 (s, 9H), 2.07 (brs, 1H), 3.55–3.90 (m, 5H),
2
2
4.51 (d, J=11.7 Hz, 1H), 4.63 (d, J=11.7 Hz,
1H), 7.10–7.75 (m, 15H). 13C-NMR (CDCl3, 50
MHz): l (ppm)=19.31, 26.97, 62.96, 63.74, 72.26,
79.79, 127.82, 127.85, 128.11, 128.18, 128.53,
128.82, 129.89, 130.27, 133.33, 133.44, 135.70,
135.73, 138.50. MS (CI, ammonia): m/z=437
([M+NH3]+).
4.1.12. ((R)-2-O-Benzyl-glyceryl)-2-bromoethyl-
methyl phosphate (15)
Compound 12 (27.1 g, 43.3 mmol) was refluxed
with 1.2 g pyridinium chloride (10 mmol) in 200
ml EtOH for 2 h. Upon addition of 1.1 equiva-
lents of NaHCO3 in water, the solvents were
removed in vacuo. The residue was dissolved in
CH2Cl2 and washed with brine. Evaporation of
the organic solvent yielded 14.5 g (87%) of a
colourless oil. [h]2D0= +7.0° (c=0.9 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=2.72 (s,
1H), 3.50 (2× dt, 3J=6.1 Hz, 4JH,P=0.8 Hz,
4.1.10. ((R)-2-O-Benzyl-3-O-tritylglyceryl)-
2-bromoethyl-methyl phosphate (13)
Ten grams of methyldichlorophosphite (75
mmol) and 30 ml diisopropylethylamine (175
mmol) were dissolved in 200 ml THF under nitro-
gen and cooled to −78°C. Then, 26.5 g com-
pound 11 (62.5 mmol) dissolved in 50 ml THF
were added slowly over a period of 1 h. After
stirring for an additional hour, 7.1 ml 2-bro-
moethanol (100 mmol) were added dropwise. The
reaction mixture was allowed to warm to room
temperature and stirred for 2 h prior to evapora-
tion of the solvents. The residue was dissolved in
CH2Cl2 and treated for 2 h with 20 ml hydrogen
peroxide solution (30%, 200 mmol) The organic
3
2H), 3.64–3.74 (m, 3H), 3.79 (2× d, JH,P=11.3
Hz, 3H), 4.16–4.36 (m, 4H), 4.62 (d, 2J=11.7 Hz,
1H), 4.71 (d, 2J=11.7 Hz, 1H), 7.25–7.40 (m,
5H). 13C-NMR (CDCl3, 50 MHz): l (ppm)=
29.46, 54.70, 61.09, 66.40, 66.95, 72.22, 77.73,
127.86, 127.98, 128.54, 137.88. MS (EI): m/z=
383 (M+).

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3
4.1.13. ((S)-2-O-Benzyl-glyceryl)-2-bromoethyl-
methyl phosphate (16)
d, JH,P=11.3 Hz, 3H), 3.83 (m, 1H), 4.06–4.35
(m, 6H), 4.64 (s, 2H), 7.30–7.40 (m, 5H). 13C-
NMR (CDCl3, 50 MHz): l (ppm)=14.14, 22.72,
24.93, 29.18, 29.31, 29.44, 29.64, 29.72, 31.95,
34.18, 54.68, 62.33, 66.60, 66.87, 72.27, 75.18,
127.89, 128.17, 128.48, 137.66, 173.47. MS (EI):
m/z=621 (M+).
Compound 14 (9.9 g, 19 mmol) was dissolved
in 32 ml 1 M tetrabutylammonium fluoride in
THF and stirred for 1 h. Then, 200 ml ethyl
acetate were added and the solution was washed
with brine. Column chromatography (diethyl
ether/acetone, 5:1) gave compound 18 as a colour-
less oil. Yield: 5.5 g (90%). [h]2D0= −6.9° (c=1.0
in CHCl3). The other spectroscopic data were
identical to compound 15.
4.1.17. (3-O-[2-Propyl pentanoyl]-(R)-2-O-
benzylglyceryl)-2-bromoethyl-methyl phosphate
(20)
Yield: 92%. [h]D20= +0.6° (c=1.3 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.87 (t,
3J=7.3 Hz, 6H), 1.16–1.68 (m, 8H), 2.39 (m,
4.1.14. Esterification of the sn-1-position —
general procedure
3
Compound 15 or 16 (3.8 g, 10 mmol) was
dissolved in 50 ml CH2Cl2 and 12 mmol car-
boxylic acid, 3.1 g DCC (15 mmol) and 250 mg
DMAP (2 mmol) were added and stirred for 2 h.
After filtration, 50 ml CH2Cl2 were added and the
organic phase was washed with 0.1 M HCl, satu-
rated NaHCO3 and brine. The solvent was evapo-
rated and the residue purified by column
chromatography (cyclohexane/ethyl acetate 5:1–
2:1). Compound 18 was not extensively character-
ized but used directly for the next synthetic step.
1H), 3.47 (m, 2H), 3.77 (2× d, JH,P=11.3 Hz,
3H), 3.83 (m, 1H), 4.05–4.38 (m, 6H), 4.65 (s,
2H), 7.25–7.45 (m, 5H). 13C-NMR (CDCl3, 50
MHz): l (ppm)=13.96, 20.63, 29.39, 34.57, 45.24,
54.60, 62.14, 66.70, 66.86, 72.24, 75.44, 127.87,
128.25, 128.75, 137.72, 176.01. MS (EI): m/z=
509 (M+).
4.1.18. Deprotection and esterification of the
sn-2-position — general procedure
Compounds 17–20 (6 mmol) were dissolved in
50 ml CH2Cl2 and 750 mg Pd/C (10% m/m) were
added. The suspension was stirred for 2 h under
an atmosphere of hydrogen (1.05 bar). The reac-
tion mixture was filtered and removed under re-
duced pressure to isolate compound 21. In all
other cases, 7.5 mmol respective carboxylic acid,
1.85 g DCC (9 mmol) and 250 mg DMAP (2
mmol) were directly added and the solution was
stirred for 2 h. Work-up as described for com-
pounds 17–20 yielded compounds 22–27 as
colourless, viscous oils. Compound 24 was not
extensively characterized but used directly for the
next synthetic step.
4.1.15. (3-O-[2-(4-Isobutylphenyl propanoyl)]-
(R)-2-O-benzylglyceryl)-2-bromoethyl-methyl
phosphate (17)
Yield: 91%. [h]D20= +1.1° (c=1.0 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.88 (d,
3
3J=6.7 Hz, 6H), 1.49 (2× d, J=7.2 Hz, 3H),
3
1.82 (m, 1H), 2.43 (d, J=7.1 Hz, 2H), 3.45 (m,
3
2H), 3.64–3.84 (m, 2H), 3.74 (2× d, JH,P=11.3
Hz, 3H), 3.96–4.36 (m, 6H), 4.54 (2× s, 2H),
7.05–7.40 (m, 9H). 13C-NMR (CDCl3, 50 MHz):
l (ppm)=18.41, 18.45, 22.40, 29.40, 30.15, 45.09,
45.13, 54.55, 62.90, 63.02, 66.54, 66.83, 72.25,
72.35, 75.21, 75.41, 127.18, 127.23, 127.83, 128.42,
129.40, 137.50+137.53, 137.75, 140.61, 174.33.
MS (EI): m/z=571 (M+).
4.1.19. (3-O-[2-(4-Isobutylphenyl propanoyl)]-(R)-
2-glyceryl)-2-bromoethyl-methyl phosphate (21)
Yield: 90%. 1H-NMR (CDCl3, 200 MHz): l
3
3
4.1.16. (3-O-Palmitoyl-(R)-2-O-benzylglyceryl)-
2-romoethyl-methyl phosphate (19)
(ppm)=0.89 (d, J=6.6 Hz, 6H), 1.50 (d, J=
3
7.2 Hz, 3H), 1.84 (m, 1H), 2.45 (d, J=7.0 Hz,
3
Yield: 88%. [h]2D0= +0.9° (c=1.3 in
CHCl3).1H-NMR (CDCl3, 200 MHz): l (ppm)=
2H), 2.62 (s, 1H), 3.52 (t, J=6.1 Hz, 2H), 3.73
(q, 3J=7.2 Hz, 2H), 3.79 (d, 3JH,P=11.3 Hz, 3H),
3.94–4.08 (m, 4H), 4.16 (d, 3J=4.5 Hz, 2H),
3
0.87 (t, J=6.5 Hz, 3H), 1.29 (s, 24H), 1.60 (m,
3
3
3
2H), 2.31 (t, J=7.6 Hz), 3.49 (m, 2H), 3.74 (2×
4.31 (dd, J=6.1 Hz, J=14.1 Hz, 2H), 7.09 (d,

M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157
153
3
3
3J=8.1 Hz, 2H), 7.19 (d, J=8.1 Hz, 2H). 13C-
(d+t, J=6.6 Hz, 9H), 1.25 (s, 24H), 1.49 (2×
3
NMR (CDCl3, 50 MHz): l (ppm)=18.41, 22.44,
29.42 (d, 3JC,P=8.7 Hz), 30.21, 45.01, 45.09, 54.85
(d, 2JC,P=7.3 Hz), 64.62+64.73, 67.12+67.16,
d, J=7.1 Hz, 3H), 1.54 (m, 2H), 1.84 (m, 1H),
2.15+2.28 (2× t, 3J=7.8 Hz, 2H), 2.43 (d,
3J=7.1 Hz, 2H), 3.44+3.51 (2× dt, 3J=7.1 Hz,
3
2
68.68+68.73 (d, JC,P=5.5 Hz), 68.89 (d, JC,P
=
4JH,P=0.8 Hz, 2H), 3.66 (m, 1H), 3.77 (d, 3JH,P
=
5.5 Hz), 127.18, 129.49, 137.53, 140.82, 174.69.
11.3 Hz, 3H), 4.02–4.40 (m, 6H), 5.21 (m, 1H),
3
3
7.10 (d, J=7.6 Hz, 2H), 7.20 (d, J=7.6 Hz,
2H). 13C-NMR (CDCl3, 50 MHz): l (ppm)=
14.16, 18.43, 22.42, 22.73, 24.77, 24.87, 29.17,
29.31, 29.46, 29.73, 30.21, 31.95, 33.88, 34.05,
44.84, 44.98, 45.08, 54.70, 61.52, 61.43, 65.45,
65.83, 66.90, 67.10, 69.71, 69.78, 127.18, 128.18,
137.21, 137.26, 140.70, 173.16, 173.82. MS (CI,
ammonia): m/z=739 ([M+NH3]+).
4.1.20. ((R)-2,3-Di-O-[2-(4-isobutylphenyl
propanoyl)]-glyceryl)-2-bromoethyl-methyl
phosphate (22)
Yield: 77%. [h]D20= +0.5° (c=1.3 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.89 (d,
3J=6.5 Hz, 12H), 1.36–1.53 (m, 6H), 1.84 (m,
2H), 2.44 (d, 3J=7.1 Hz, 4H), 3.38–3.55 (m, 2H),
3.57–3.67 (4× q, 3J=4.4 Hz, 2H), 3.70–3.78
3
(4× d, JH,P=11.3 Hz, 3H), 3.98–4.42 (m, 6H),
4.1.23. (3-O-Palmitoyl-(R)-2-O-[2-propyl
5.17 (m, 1H), 7.05–7.25 (m, 8H). 13C-NMR
(CDCl3, 50 MHz): l (ppm)=18.16, 18.25, 18.34,
18.45, 22.22, 22.36, 29.29, 30.12, 44.84, 44.95,
45.06, 54.46, 54.57, 61.65, 61.80, 65.22, 65.47,
66.80, 66.87, 69.68, 69.73, 127.12, 129.33, 137.22,
137.30, 140.59, 173.56, 173.62, 173.89, 173.98. MS
(CI, ammonia): m/z=689 ([M+NH3]+).
pentanoyl])-2-bromoethyl-methyl phosphate (26)
Yield: 76%. [h]D20= +2.3° (c=1.3 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.85 (m,
9H), 1.10–.32 (m, 28H), 1.38 (m, 2H), 1.44–.63
3
(m, 4H), 2.25 (t, J=7.5 Hz, 2H), 2.35 (m, 1H),
3
3
3.48 (t, J=6.1 Hz, 2H), 3.75 (2× d, JH,P=11.2
Hz, 3H), 4.05–.34 (m, 6H), 5.22 (m, 1H). 13C-
NMR (CDCl3, 50 MHz): l (ppm)=13.88, 14.01,
20.45, 20.49, 22.62, 24.78, 29.08, 29.13, 29.19,
29.34, 29.53, 29.61, 31.86, 33.95, 34.45, 34.50,
45.06, 45.16, 54.58, 61.69, 65.65, 65.71, 66.91,
69.17, 173.00, 175.33. MS (CI, ammonia): m/z=
676 ([M+NH3]+).
4.1.21. (3-O-[2-(4-Isobutylphenyl propanoyl)]-
(R)-2-O-palmitoylglyceryl)-2-bromoethyl-methyl
phosphate (23)
Yield: 75%. [h]D20= +0.3° (c=1.4 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.85
3
(d+t, J=6.4 Hz, 9H), 1.26 (s, 24H), 1.49 (2×
3
d, J=7.2 Hz, 3H), 1.57 (m, 2H), 1.82 (m, 1H),
4.1.24. ((R)-2,3-Di-O-[2-propyl pentanoyl]-
glyceryl)-2-bromoethyl-methyl phosphate (27)
Yield: 79%. [h]D20= +1.1° (c=1.3 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.80 (t,
3J=7.1 Hz, 12H), 1.08–.62 (m, 16H), 2.21–.38
2.23 (t, 3J=7.4 Hz, 2H), 2.44 (d, 3J=7.1 Hz,
3
2H), 3.52 (t, J=6.2 Hz, 2H), 3.58–3.66 (m, 1H),
3
3.77 (d, JH,P=11.3 Hz, 3H), 3.98–4.40 (m, 6H),
3
5.20 (m, 1H), 7.09 (d, J=8.1 Hz, 2H), 7.19 (d,
3J=8.1 Hz, 2H). 13C-NMR (CDCl3, 50 MHz): l
(ppm)=14.12, 18.33, 22.42, 22.72, 24.88, 29.16,
29.32, 29.46, 29.73, 30.19, 31.97, 34.09, 45.05,
45.11, 54.44, 61.80, 61.92, 65.61, 66.96, 69.26,
69.40, 127.20, 129.40, 137.38, 140.73, 172.72,
174.11. MS (CI, ammonia): m/z=739 ([M+
NH3]+).
(m, 2H), 3.45 (t, 3J=6.1 Hz, 2H), 3.71 (d, 3JH,P
=
11.3 Hz, 3H), 3.99–.35 (m, 6H), 5.17 (m, 1H).
13C-NMR (CDCl3, 50 MHz): l (ppm)=13.81,
20.42, 29.16, 34.32, 34.36, 44.96, 45.06, 54.43,
61.47, 65.54, 65.60, 66.84, 69.25, 175.17, 175.57.
MS (CI, ammonia): m/z=564 ([M+NH3]+).
4.1.25. Synthesis of the choline moiety — general
procedure
Compound 21 (4 mmol) or compounds 22–27
in 10 ml toluene and 20 ml freshly condensed
(−50°C) trimethylamine were heated for 2 days
at 50°C in an autoclave. After cooling to −20°C,
4.1.22. ((R)-2-O-[2-(4-Isobutylphenyl propanoyl)]-
3-O-palmitoylglyceryl)-2-bromoethyl-methyl
phosphate (25)
Yield: 73%. [h]D20= +2.7° (c=1.1 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.87

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M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157
the solution was poured into a mixture of 200 ml
methanol, 100 ml of 1 M HCl and 100 ml water,
and extracted with CH2Cl2. The organic phase
was evaporated. Column chromatography
(methanol) gave the products as waxy solids.
3J=7.1 Hz, 2H), 3.27 (s, 9H), 3.60–3.95 (m, 5H),
3.99–4.47 (m, 4H), 5.28 (m, 1H), 7.04 (d, J=7.9
3
Hz, 2H), 7.14 (d, 3J=7.9 Hz, 2H). 13C-NMR
(CDCl3, 50 MHz): l (ppm)=14.10, 18.56, 22.42,
22.71, 24.95, 29.26, 29.39, 29.62, 29.75, 30.19,
31.95, 34.28, 45.04, 45.09, 54.35, 59.33, 63.28,
63.49, 66.39, 70.54, 127.21, 129.33, 137.50, 137.57,
140.55, 173.04, 174.47. MS (CI, ammonia): m/z=
684 (M+). C37H66NO8P·H2O (701.91): calc.; C,
63.31; H, 9.76; N, 2.00; found: C, 63.15; H, 10.15;
N, 1.99.
4.1.26. 1-O-[2-(4-Isobutylphenyl propanoyl)]-2-
lyso-sn-glycero-3-phosphocholine (28)
Yield: 71%. [h]D20= +3.3° (c=1.4 in CHCl3).
1H-NMR (CDCl3, 600 MHz): l (ppm)=0.87 (d,
3
3J=6.6 Hz, 3H), 1.43 (d, J=7.0 Hz, 3H), 1.81
3
3
(sept., J=6.7 Hz, 1H), 2.41 (s, J=7.1 Hz, 2H),
3.22 (s, 9H), 3.54 (m, 0.5 H), 3.65–3.72 (m, 3H),
3.78 (m, 0.5H), 3.89 (m, 2H), 3.97+4.15 (2× m,
4.1.29. 3-O-[2-(4-Isobutylphenyl propanoyl)]-2-O-
palmitoyl-sn-glycero-1-phosphocholine (31)
3
2H), 4.25 (m, 2H), 7.06 (d, J=7.5 Hz, 2H), 7.16
Yield: 73%. [h]D20= −7.9° (c=1.2 in CHCl3).
1H-NMR (CDCl3, 600 MHz): l (ppm)=0.88
(d, 3J=7.5 Hz, 2H). 13C-NMR (CDCl3, 150
MHz): l (ppm)=18.80, 18.97, 22.34, 30.12, 44.83,
44.93, 53.97, 59.31, 65.51, 65.68, 65.88, 66.94,
68.66, 127.20, 129.27, 137.55, 137.70, 140.45,
3
(d+t, J=8.5 Hz, 9H), 1.26 (s, 24H), 1.45 (d,
3
3J=6.9 Hz, 3H), 1.52 (m, 2H), 1.83 (sept., J=
3
6.6 Hz, 1H), 2.20 (m, 2H), 2.42 (s, J=6.9 Hz,
174.75.
MS
(CI,
ammonia):
m/z=416
2H), 3.29 (s, 9H), 3.63–3.71 (m, 1H), 3.74 (m,
2H), 3.84–3.95 (m, 2H), 4.13 (m, 1H), 4.26 (m,
([MꢀC2H6]+). C21H36NO7P·1.2 H2O (467.11):
calc.: C, 54.00; H, 8.29; N, 3.05; found: C, 53.77;
H, 8.49; N, 3.05.
2
2H), 4.36+4.42 (2× d, J=11.2 Hz, 1H), 5.17
3
3
(m, 1H), 7.06 (d, J=7.5 Hz, 2H), 7.15 (d, J=
7.5 Hz, 2H). 13C-NMR (CDCl3, 150 MHz): l
(ppm)=14.02, 18.32, 18.42, 22.29, 22.59, 24.78,
25.19, 29.11, 29.27, 29.30, 29.39, 29.50, 29.53,
29.57, 29.63, 30.08, 31.83, 34.09, 34.11, 44.85,
44.93, 54.20, 59.19, 59. 23, 63.21, 63.25, 63.25,
63.35, 66.14, 66.18, 70.22, 70.28, 70.33, 127.10,
127.11, 129.18, 129.24, 137.31, 137.38, 140.43,
172.92, 172.99, 174.36. MS (CI, ammonia): m/z=
684 (M+). C37H66NO8P·H2O (701.91): calc.: C,
63.31; H, 9.76; N, 2.00; found: C, 62.99; H, 10.27;
N, 2.40.
4.1.27. 1,2-Di-O-[2-(4-Isobutylphenyl propanoyl)]-
sn-glycero-3-phosphocholine (29)
Yield: 78%. [h]D20= +8.0° (c=1.0 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.86 (d,
3J=6.7 Hz, 12H), 1.27–1.43 (m, 6H), 1.80 (sept.,
3J=6.7 Hz, 2H), 2.40 (d, J=7.1 Hz, 4H), 3.19
3
3
(s, 9H), 3.53–3.65 (2× q, J=7.0 Hz, 2H), 3.63
(m, 2H), 3.74–3.95 (m, 2H), 3.95–4.30 (m, 3H),
4.30–4.50 (m, 1H), 5.04–5.24 (m, 1H), 6.98 (m,
8H). 13C-NMR (CDCl3, 50 MHz): l (ppm)=
18.27, 18.38, 18.53, 18.71, 22.40, 30.13, 44.78,
44.87, 44.95, 54.24, 59.34, 63.12, 63.36, 66.13,
70.94, 127.21, 127.31, 129.29, 137.46, 137.57,
140.52, 174.01, 174.35. MS (CI, ammonia): m/z=
634 (M+). C34H52NO8P·1.5 H2O (660.77): calc.:
C, 61.63; H, 8.40; N, 2.11; found: C, 61.39; H,
8.49; N, 1.92.
4.1.30. 1-O-Palmitoyl-2-O-[2-(4-isobutylphenyl
propanoyl)]-sn-glycero-3-phosphocholine (32)
Yield: 79%. [h]D20= +9.1° (c=1.0 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.88
3
(d+t, J=6.6 Hz, 9H), 1.20 (s, 24H), 1.34–1.60
(m, 5H), 1.80 (m, 1H), 2.12 (m, 2H), 2.40 (d,
3J=7.2 Hz, 2H), 3.25+3.29 (2× s, 9H),
3.60–3.98 (m, 5H), 4.01–4.44 (m, 4H), 5.27 (m,
4.1.28. 1-O-[2-(4-Isobutylphenyl propanoyl)]-2-
O-palmitoyl-sn-glycero-3-phosphocholine (30)
Yield: 77%. [h]D20= +8.3° (c=1.0 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.85 (m,
9H), 1.24 (s, 24H), 1.46 (d, J=6.8 Hz, 3H), 1.50
(m, 2H), 1.81 (m, 1H), 2.13–2.23 (m, 2H), 2.41 (d,
3
3
1H), 7.04 (d, J=8.0 Hz, 2H), 7.14 (d, J=8.0
Hz, 2H). 13C-NMR (CDCl3, 50 MHz):
l
(ppm)=14.08, 18.49, 18.83, 22.40, 22.73, 24.78,
24.89, 29.21, 29.35, 29.57, 29.72, 30.17, 31.94),
33.94, 34.14, 44.98, 45.24, 45.07, 54.34, 59.31,
3

M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157
155
62.94, 63.40, 66.30, 71.05,127.18, 127.32, 129.25,
129.31, 137.50, 137.62, 140.44, 140.55, 173.35,
173.44, 174.09, 174.16. MS (CI, ammonia): m/z=
684 (M+). C37H66NO8P·2.5 H2O (728.94): calc.: C,
60.97; H, 9.82; N, 1.92; found: C, 60.99; H, 9.99;
N, 1.85.
monolayers were continuously compressed by the
2
,
motor-driven Teflon barriers at 20 A /molecule/
min. Every experiment was performed three times.
4.2.2. Laser light scattering
The size of the aggregates was determined by
dynamic laser light scattering (scattering angle,
90°) using a ZetaMasterS Autosizer 4700 (Mal-
vern Instruments, operating with a He–Ne laser at
633 nm) and fitting software. The size of the
aggregates is expressed as the mean diameter of
three determinations. The solutions were prepared
by dissolving 100 mg phospholipid in 100 ml
bidistilled water and sonicating for 2 h at 40°C.
4.1.31. 1-O-Palmitoyl-2-O-[2-propyl
pentanoyl]-sn-glycero-3-phosphocholine (33)
Yield: 74%. [h]D20= +0.7° (c=1.0 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.85 (t,
3J=7.1 Hz, 9H), 1.22 (m, 28H), 1.28–1.64 (m,
6H), 2.18–2.40 (m, 3H), 3.33 (s, 9H), 3.68–3.84
(m, 2H), 3.90–4.10 (m, 3H), 4.20–4.35 (m, 3H),
5.17 (m, 1H). 13C-NMR (CDCl3, 50 MHz): l
(ppm)=14.03, 20.54, 22.65, 24.87, 24.97, 29.15,
29.22, 29.28, 29.31, 29.43, 29.51, 29.57, 29.66,
31.90, 34.12, 34.16, 34.45, 45.06, 45.20, 54.26,
59.40, 63.23, 64.96, 66.20, 69.00, 175.75, 176.42.
MS (CI, ammonia): m/z=621 (M+). C32H64NO8-
P·2.5 H2O (666.87): calc.: C, 57.33; H, 10.43; N,
2.09; found: C, 56.99; H, 10.41; N, 2.26.
4.3. Enzymatic studies
4.3.1. HPLC analysis
The HPLC system consisted of a Shimadzu LC
6A solvent delivery module, a Shimadzu SPD 6A
UV detector operated at 220 nm, and a Shimadzu
C-R6A integrator. The chromatographic separa-
tion was obtained on a LiChrospher Si 60 column
(250×4.6 mm2, 5 mm) (Merck, Germany). The
mobile phase consisted of acetronitrile/methanol/
phosphoric acid (70:30:0.5, v/v/v), and the flow
rate was 1.5 ml/min. Phospholipid and lyso-phos-
pholipid concentrations were calculated from cali-
bration curves obtained by analysis of the pure
compounds under identical chromatographic
conditions.
4.1.32. 1,2-Di-O-[2-propyl
pentanoyl]-sn-glycero-3-phosphocholine (34)
Yield: 75%. [h]D20= +6.2° (c=1.0 in CHCl3).
1H-NMR (CDCl3, 200 MHz): l (ppm)=0.83 (t,
3J=7.0 Hz, 12H), 1.10–1.62 (m, 16H), 2.29 (m,
2H), 3.36 (s, 9H), 3.80 (m, 2H), 3.85–4.15 (m, 2H),
4.28 (m, 3H), 4.46 (m, 1H), 5.15 (m, 1H). 13C-
NMR (CDCl3, 50 MHz): l (ppm)=14.00, 20.53,
34.33, 34.44, 45.09, 45.19, 54.41, 59.32, 63.16,
66.46, 70.65, 175.69, 176.20. MS (CI, ammonia):
m/z=510 (M+). C24H48NO8P·0.5 H2O (518.61):
calc.: C, 55.20; H, 9.53; N, 2.68; found: C, 55.01;
H, 9.91; N, 2.60.
4.3.2. Kinetics
Enzymatic hydrolysis was studied at 3790.5°C
in 100 mM tris(hydroxymethyl)aminomethane
(TRIS) buffer, pH 8.0, containing 10 mM CaCl2
and 100 U porcine pancreatic phospholipase A2
4.2. Physicochemical measurements
(assayed by Sigma using soybean
L
-a-phos-
4.2.1. Surface tension
phatidylcholine as substrate). Two micromoles of
the compounds were incubated in 4 ml buffer and
the mixtures were vigorously stirred during the
kinetic run. Aliquots were sampled at selected time
intervals and extracted with methanol/chloroform/
2 N HCl (2:2:2, v/v/v) according to Bligh and
Dyer (1959). The organic phase was dried under a
stream of nitrogen. The residue was dissolved in
n-hexane/2-propanol (3:1, v/v) and analyzed by
HPLC.
The surface tension of the compounds was de-
termined using a computer-assisted Nima Type
622 Wilhelmy film balance. The Teflon trough
(200×600 mm2) was rinsed with ethanol, distilled
water and bidistilled water before use. The mono-
layers were formed by spreading 50 ml chloroform
solution containing 100 nmol phospholipids onto
the surface of the water. After a period of 10 min,
in order for the organic solvent to evaporate, the

156
M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157
Hostetler, K.Y., Stuhmiller, L.M., Lenting, H.B.M., van den
Acknowledgements
Bosch, H., Richman, D.D., 1990. Synthesis and antiretrovi-
ral activity of phospholipid analogs of azidothymidine
and other antiviral nucleosides. J. Biol. Chem. 265, 6112–
6117.
The gift of sn-glycero-3-phosphocholine by
Nattermann Phospholipid GmbH and the finan-
cial support by the Fonds der Chemischen Indus-
trie are gratefully acknowledged. We also thank
Dr. Claus Ru¨diger for assistance with the film
balance measurements and Dr. Jo¨rg Breitkreutz
for assistance with laser light scattering
experiments.
Hostetler, K.Y., Parker, S., Sridhar, C.N., Martin, M.J., Li,
J.-L., Stuhmiller, L.M., van Wijk, G.M.T., van den Bosch,
H., Gardner, M.F., Aldern, K.A., Richman, D.D., 1993.
Acyclovir diphosphate dimyristoylglycerol: a phospholipid
prodrug with activity against acyclovir-resistant herpes sim-
plex virus. Proc. Natl. Acad. Sci. USA 90, 11835–11839.
Hostetler, K.Y., Richman, D.D., Forssen, E.A., Selk, L.,
Basava, R., Gardner, M.F., Parker, S., Basava, C., 1994.
Phospholipid prodrug inhibitors of the HIV protease. An-
tiviral activity and pharmacokinetics in rats. Biochem.
Pharmacol. 48, 1399–1404.
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