Synthesis and intermembrane transfer of pyrene-labelled liponucleotides: Ceramide phosphothymidines

Article abstract of DOI:10.1016/S0009-3084(99)00006-7

Phospholipid conjugates of 3'-azido-3'-deoxythymidine (AZT) show activity against human immunodeficiency virus (HIV) in vitro. Here we report on the synthesis and characterization of two pyrene containing conjugates: 2-N-(4-(pyren-1-yl)butanoyl)ceramide 5'-phosphothymidine (Pbs-Cer-P-T) (XII) and 2-N-(10-(pyren-1-yl)decanoyl)ceramide 5'-phosphothymidine (Pds-Cer-P-T) (XIII). These fluorescent labelled conjugates served as model compounds to study incorporation of sphingoliponucleotides into membranes. The complex compounds were prepared by condensation of 3'-acetylthymidine and labelled ceramides using the phosphite triester coupling procedure. UV absorption, fluorimetry as well as ¹H-, ³¹P-, ¹³C-NMR analyses were used for structure confirmation of the synthesized substances. When incorporated into small unilamellar 1-palmitoyl-2-oleoyl-glycerophosphatidylcholine (POPC) vesicles and incubated with unlabelled acceptor POPC vesicles, the compounds (XII) and (XIII) exhibited spontaneous transfer. Kinetic data suggest that transfer from donor to acceptor vesicles occurred via the intervening aqueous phase. The non-specific lipid transfer protein from bovine liver stimulated the transfer of Pds-Cer-P-T between phospholipid vesicles in a concentration dependent manner. Copyright (C) 1999 Elsevier Science Ireland Ltd.

Full text of DOI:10.1016/S0009-3084(99)00006-7

Chemistry and Physics of Lipids
99 (1999) 73–86
Synthesis and intermembrane transfer of pyrene-labelled
liponucleotides: ceramide phosphothymidines
Olga V. Oskolkova a,b, Vitaly I. Shvets a, Albin Hermetter b, Fritz Paltauf b,*
a Department of Biotechnology, Moscow Lomonoso6 State Academy of Fine Chemical Technology, Prospect Vernadskogo 86,
117571 Moscow, Russia
b Department of Biochemistry and Food Chemistry, Technical Uni6ersity Graz, Petersgasse 12/II, 8010 Graz, Austria
Received 12 September 1998; accepted 11 January 1999
Abstract
Phospholipid conjugates of 3%-azido-3%-deoxythymidine (AZT) show activity against human immunodeficiency virus
(HIV) in vitro. Here we report on the synthesis and characterization of two pyrene containing conjugates:
2-N-(4-(pyren-1-yl)butanoyl)ceramide 5%-phosphothymidine (Pbs–Cer–P–T) (XII) and 2-N-(10-(pyren-1-
yl)decanoyl)ceramide 5%-phosphothymidine (Pds–Cer–P–T) (XIII). These fluorescent labelled conjugates served as
model compounds to study incorporation of sphingoliponucleotides into membranes. The complex compounds were
prepared by condensation of 3%-acetylthymidine and labelled ceramides using the phosphite triester coupling
1
procedure. UV absorption, fluorimetry as well as H-, 31P-, 13C-NMR analyses were used for structure confirmation
of the synthesized substances. When incorporated into small unilamellar 1-palmitoyl-2-oleoyl-glycerophosphatidyl-
choline (POPC) vesicles and incubated with unlabelled acceptor POPC vesicles, the compounds (XII) and (XIII)
exhibited spontaneous transfer. Kinetic data suggest that transfer from donor to acceptor vesicles occurred via the
intervening aqueous phase. The non-specific lipid transfer protein from bovine liver stimulated the transfer of
Pds–Cer–P–T between phospholipid vesicles in a concentration dependent manner. © 1999 Elsevier Science Ireland
Ltd. All rights reserved.
Keywords: Nucleoside-phospholipid conjugates; Sphingophospholipids; Phosphite triester approach; Fluorescence
spectroscopy; Spontaneous transfer; Protein mediated transfer
1. Introduction
chains have become important and frequently in-
dispensable tools in studies of the structure and
dynamics of membranes. Since such probes retain
head groups and resemble the molecular shape of
native membrane lipids, they largely mimic the
behaviour of their natural prototypes in biological
membranes. These probes are applied in investiga-
Fluorescently labelled phospholipids carrying a
fluorophore group in one of the hydrocarbon
* Corresponding author. Tel.: +43-316-873-6450; fax: +
43-316-873-6952.
E-mail address: f548palt@mbox.tu-graz.ac.at (F. Paltauf)
0009-3084/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0009-3084(99)00006-7

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O.V. Oskolko6a et al. / Chemistry and Physics of Lipids 99 (1999) 73–86
tions dealing with biophysical aspects of mem-
branes, including lateral organization and mobil-
ity (Galla and Hartmann, 1980; Bergelson et al.,
1985; Molotkovsky et al., 1991; Bredlow et al.,
1992; Barenholz et al., 1996), and also in studies
of intermembrane and transmembrane transfer of
phospholipids (for reviews see Pownall and Smith,
1973; Pagano and Sleight, 1985; Nichols, 1993)
and phospholipid metabolism (Pagano and Long-
muir, 1983; Kok and Hoekstra, 1993; Gatt et al.,
1996).
ers different from pyrene had to be described by a
sum of two exponential functions. The fast com-
ponent was assigned to lipid transfer via soluble
monomers and the slow component to transbi-
layer motion (Arvinte and Hildenbrand, 1984; Bai
and Pagano, 1997). Pyrene lipid probes, while
more bulky as compared to antrylvinyl and 7-ni-
tro-2-1,3-benzoxadiazol-4-yl (NBD) fluorophores
(Bredlow et al., 1992), were of considerable use
for studies of kinetics and mechanism of the
transfer because the spectroscopic properties of
pyrene are concentration-dependent (Foerster,
1969; Pownall and Smith, 1973; Galla and Hart-
mann, 1980) and allow facile analysis of lipid
exchange without separation of donor and accep-
tor particles (Galla et al., 1979; Roseman and
Thompson, 1980; Correa-Freire et al., 1982;
Massey et al., 1982a,b; Pownall et al., 1982;
Frank et al., 1983; Pownall et al., 1983; Via et al.,
1985; Homan and Pownall, 1988; Jones and
Thompson, 1989, 1990; van Wijk et al., 1992).
This makes pyrene-labelled lipid probes ‘lipid-spe-
cific’ in the sense that the structure and conforma-
tion of their polar head groups are identical or
very close to those of corresponding natural lipids
(Correa-Freire et al., 1982; Frank et al., 1983).
Formerly, we have synthesized rac-erythro-2-
N-stearoyl-sphinganine 1-phospho thymidine (Os-
kolkova et al., 1996) via phosphite triester and
H-phosphonate approaches and applied these
methodologies for synthesis of ceramide phospho-
3%-azidothymidine and ceramide phospho-2%,3%-
didehydro-2%,3%-dideoxythymidine (Oskolkova et
al., 1997). In this report we present the chemical
synthesis and kinetic data for the rates of the
intermembrane transfer of fluorescent nucleoside
phospholipid conjugates- racemic ceramidephos-
phoryl thymidines containing 4-(1-pyrenyl)bu-
tanoic or 10-(1-pyrenyl)decanoic acids attached to
the amine group of sphinganine. The synthesis
directed towards both target compounds com-
prises two main stages: the acylation of a free
amino group of racemic erythro-3-benzoyl sphin-
ganine to give ceramide fluorescently labelled with
pyrene fatty acids, and subsequent phosphityla-
tion and condensation with the free hydroxy
group at the 5%-end of protected thymidine using a
phosphite triester approach followed by a depro-
The lateral diffusion and intermembrane trans-
fer of phospholipids have been the subject of
numerous studies. Many of the physical and
chemical determinants that govern these processes
have been elucidated. In general, these studies
have shown that the lateral diffusion of mem-
brane lipids is strongly dependent on the fluidity
and composition of the host membrane, but rela-
tively independent of the chemical nature of the
diffusing species. In contrast, intermembrane dif-
fusion rates are strongly dependent on the compo-
sition and length (i.e. hydrophobicity) of the
phospholipid acyl chains. A number of mecha-
nisms have been proposed for transport of lipids
among various membranes or plasma lipo-
proteins. These include (a) transport by specific
carriers that are usually proteins, (b) fusion occur-
ring at the point of contact between the donor
and acceptor surfaces, and (c) passive transport
via the surrounding aqueous phase. Phospholipid
molecules spontaneously migrate between differ-
ent artificial membranes and/or membranous par-
ticles (Roseman and Thompson, 1980; McLean
and Philips, 1981; Massey et al., 1982a,b; Nichols
and Pagano, 1982; McLean and Philips, 1984;
Petrie and Jonas, 1984). The experimental criteria
for this mechanism given by Charlton et al. (1976)
are that the process is first order and independent
of donor to acceptor concentration and of the
identity of the acceptor. However, it has been
demonstrated that phospholipid transfer between
bilayer vesicles at higher vesicle concentrations is
characterized not only by a first-order desorption
rate (spontaneous migration) but also by a sec-
ond-order process (collision) dependent on vesicle
concentration (Jones and Thompson, 1989, 1990).
The transfer kinetics of lipids labelled with mark-

O.V. Oskolko6a et al. / Chemistry and Physics of Lipids 99 (1999) 73–86
75
tection procedure to give target rac-erythro-2-N-
(4-(pyren-1-yl)butanoyl)sphinganine-1-O-phosph-
oryl-5%-thymidine (XII) and rac-erythro-2-N-(10-
(pyren - 1 - yl)decanoyl)sphinganine - 1 - O - phosph-
oryl-5%-thymidine (XIII), respectively. Hydropho-
bicity of the latter compound closely resembles
oxalyl
chloride
(Aldrich),
sebacic
acid
monomethyl ester (Aldrich) were used as such.
The solution of 1H-tetrazol (3.5%) in acetonitrile
was obtained from Aldrich. TLC was carried out
on silica gel 60 F254 aluminum sheets (0.2 mm,
Merck) using the following developing solvents:
petroleum ether/diethyl ether, 4:1, v/v (system 1);
chloroform/methanol/acetone, 10:0.5:0.5, v/v/v
(system 2); petroleum ether/ethyl acetate/TEA,
10:4:0.5, v/v/v (system 3); chloroform/methanol,
10:1, v/v (system 4); chloroform/methanol, 10:3,
v/v (system 5); chloroform/methanol/water,
65:25:4, v/v/v (system 6). Short column chro-
matography was performed on Kieselgel 60 (230–
400 mesh, Merck). Compounds with an aromatic
moiety were visualized under UV-light (254 and/
or 360 nm). Phosphorus containing material was
detected using phosphomolybdic acid (Dittmer
and Lester, 1964). All organic substances were
detected by charring at 120°C after spraying with
50% sulfuric acid. Melting points were determined
on a Bu¨chi 510 apparatus (Switzerland) in open
capillary tubes.
that
of
2-N-palmitoyl-ceramide
phospho-
thymidine. Liponucleotides (XII) and (XIII) were
incorporated into POPC vesicles prepared by the
ethanol-injection method (Batzri and Korn, 1973).
Inclusion of the pyrene fluorophore enabled us to
monitor continuously time-dependent changes in
the concentration of labelled lipids in vesicle bi-
layers. The partially purified non-specific lipid
transfer protein (ns-PLTP) from bovine liver stim-
ulated the transfer of Pds–Cer–P–T between
vesicles in a concentration dependent manner. In
contrast, ns-PLTP had no effect on the transfer
rate of the short-chain Pbs–Cer–P–T.
2. Experimental procedures
2.1. Materials and general methods
2.2. Chemical syntheses
Pyrene butanoic acid pentafluorophenyl ester
was a gift from K. Balakin (Shemyakin and
Ovchinnikov Institute of Bioorganic Chemistry,
2.2.1. 10-(1-Pyrenyl)decanoic acid
Sebacic acid monomethyl ester chloride was
obtained by mixing of sebacic acid monomethyl
ester with oxalyl chloride. The mixture was kept
overnight, triple evaporated with water-free THF
and then used without isolation. Pyrenedecanoic
acid was prepared with modifications according to
Galla et al. (1979) by a Friedel–Crafts reaction of
pyrene and sebacic acid monomethyl ester chlo-
ride in presence of aluminum chloride in
dichloromethane. After work up, pyrenoyl-
nonanoic acid methyl ester was purified by
column chromatography eluting with petroleum
ether/ diethyl ether (6:1 by vol.). The yield was
48.9%, Rf 0.21 (system 1). Pyrenedecanoic acid
was obtained from pyrenoylnonanoic acid methyl
ester by a Kishner–Wolf reaction with hydrazine
hydrate and potassium hydroxide in ethylene gly-
col. Purification was achieved on silica gel using a
gradient of methanol (0–1%) in chloroform. A
single spot was observed on TLC (Rf 0.31, system
2). Pyrenedecanoic acid was obtained with 89.0%
Moscow).
sulfate was synthesized according to Shapiro
(1969). -erythro-3-O-benzoyl-sphinganine was
D,L-erythro-3-O-benzoyl-sphinganine
D,L
obtained from its sulfate by shaking with a satu-
rated sodium bicarbonate solution. The non-spe-
cific lipid transfer protein was isolated essentially
as described by Bloj and Zilversmit (1983) and
kindly provided by Dr. A. Fell (Technical Univer-
sity Graz).
Dichloromethane was distilled from phospho-
rus pentoxide and used freshly. Water-free
pyridine was prepared by refluxing with calcium
hydride for 12 h and distilled. Methanol was
refluxed with magnesium methoxide and then dis-
tilled. Triethylamine (TEA) was purified by reflux-
ing with ninhydrine and subsequent distillation.
3%-Acetylthymidine (Sigma) was dried by evapora-
tion with absolute acetonitrile and then dried in
vacuo. Aluminum chloride, sodium azide, Tris–
HCl, EDTA (all from Merck), pyrene (Sigma),

76
O.V. Oskolko6a et al. / Chemistry and Physics of Lipids 99 (1999) 73–86
yield; m.p. 104–108.5°C (99°C (Galla and Hart-
mann, 1981), 110°C (Galla et al., 1979)).
J 3.70 Hz, J 8.17 Hz, 3-CHꢀSph), 6.30 (d, 1H,
NH), 7.40–8.35 (m, 14H, Ar).
2.2.5. 3-O-Benzoyl-2-N-((1-pyrenyl)decanoyl)-
2.2.2. 10-(1-Pyrenyl)decanoic acid p-nitrophenyl
ester
D,L
-erythro-sphinganine (III)
A mixture of 111.0 mg (0.274 mmol) 3-benzoyl
The activated ester was obtained from pyren-
edecanoic acid using a standard procedure by
reacting the respective acid with p-nitrophenol in
presence of dicyclohexylcarbobiimide in dichloro-
methane. Column chromatography on silica gel
gave the pure product with 94.0% yield. Rf 0.55
sphinganine (I) and 178.0 mg (0.361 mmol)
pyrenedecanoic acid p-nitrophenyl ester in 3 ml
pyridine and 50.0 ml (0.361 mmol) TEA was kept
at room temperature for 5 h. The solvent was
removed under reduced pressure, the residue was
applied to a silica gel column and eluted with
chloroform/petroleum ether (8:2, v/v), then with
chloroform and with 1% methanol in chloroform
to give 114.8 mg (55.1%) of ceramide (III). Rf 0.74
(system 2), m.p. 105–105.5°C (petroleum ether/di-
1
(system 1), 0.82 (system 2), m.p. 99–103°C. H-
NMR (CDCl3): 1.10–1.65 (m, 10H, 5CH2), 1.65–
2.05 (m, 4H, 3-CH2, 9-CH2), 2.58 (t, 2H, J2,3 7.42
Hz, 2-CH2), 3.35 (t, 2H, J9,10 7.69 Hz, 10-CH2),
7.83–8.31 (m, 13H, Ar).
1
ethyl ether, 2:1). H-NMR (CDCl3): 0.85 (t, 3H,
CH3ꢀSph), 0.97–2.10 (m, 40H, CH2ꢀSph, Pds),
2.20 (t, 2H, J2,3 7.50 Hz, 2-CH2ꢀPds), 3.28–3.61
(m, 4H, 1-CH2ꢀSph, 10-CH2ꢀPds), 4.16 (m, 1H,
2-CHꢀSph), 5.08 (dt, 1H, J 3.94 Hz, J 7.95 Hz,
3-CHꢀSph), 6.47 (d, 1H, J2,NH 8.52 Hz, NH),
7.40–7.63 (m, 5H, Bz), 7.80–8.30 (m, 9H, Ar).
An alternative procedure was carried out start-
ing from 3-benzoyl sphinganine (121.8 mg, 0.300
mmol) (I) and 178.0 mg (0.361 mmol) pyrenede-
canoic acid pentafluorophenyl ester. The purifica-
tion was performed as described above giving
49.0% ceramide (III).
2.2.3. 10-(1-Pyrenyl)decanoic acid
pentafluorophenyl ester
Pentafluorophenyl ester of pyrenedecanoic acid
was obtained as described above with 91.9% yield;
Rf 0.94 (system 1), m.p. 80–81°C. 1H-NMR
(CDCl3): 1.15–1.65 (m, 10H, 5CH2), 1.65–2.00
(m, 4H, 3-CH2, 9-CH2), 2.64 (t, 2H, J2,3 7.37 Hz,
2-CH2), 3.35 (t, 2H, J9,10 7.79 Hz, 10-CH2), 7.82–
8.35 (m, 9H, Ar).
2.2.4. 3-O-Benzoyl-2-N-(4-(1-pyrenyl)butanoyl)-
D
,L
-erythro-sphinganine (II)
A mixture of racemic 3-benzoyl-sphinganine (I)
2.2.6. 3-O-Benzoyl-2-N-(4-(1-pyrenyl)butanoyl)-
(496.8 mg, 1.227 mmol) and pyrenebutanoic acid
pentafluorophenyl ester (669.5 mg, 1.473 mmol) in
17 ml pyridine was kept at room temperature for
3.5 h, then concentrated in vacuo, the residue was
subsequently co-evaporated with toluene (3×5
ml) to remove the rest of pyridine. The resulting
oil was applied to a silica gel column and eluted
with chloroform to give the crystalline product
(II). Yield 375.6 mg (45.0%). Rf 0.61 (system 2),
m.p. 91–94°C. 1H-NMR (CDCl3): 0.88 (t, 3H,
CH3-sphinganine (Sph)), 1.1–1.45 (m, 26H,
CH2ꢀSph), 1.55–2.00 (m, 4H, 4-CH2ꢀSph, 3-
CH2ꢀPbs), 2.26 (dt, 2H, Ja,b=J2-Pbs,3-Pbs 7.92 Hz,
2-CH2ꢀPbs), 2.36 (dt, 2H, Ja,b=J3-Pbs,4-Pbs 6.19
Hz, 3-CH2ꢀPbs), 3.10 (bt, 1H, ꢀOH), 3.41 (t, 2H,
J3-Pbs,4-Pbs 7.33 Hz, 4-CH2ꢀPbs), 3.66 (m, 2H, 1-
CH2ꢀSph), 4.21 (m, 1H, 2-CHꢀSph), 5.07 (dt, 1H,
D,L-erythro-sphinganine-1-O-(N,N-diisopropyla-
mino)-2-cyanoethylphosphine (IV)
To a solution of dried (by coevaporation with a
dichloromethane-acetonitrile mixture (1:1 v/v,
3×3 ml)) 2-N-pyrenebutanoyl-ceramide (II)
(100.0 mg, 0.148 mmol) and diisopropylammo-
nium tetrazolide (17.0 mg, 0.099 mmol) in 1 ml
dichloromethane, bis(N,N-diisopropylamino)-2-
cyanoethylphosphine (0.1 ml, 0.315 mmol) was
added. After completion of the reaction, methanol
(0.1 ml) was added. After 15 min, the mixture was
concentrated in vacuo, diluted with a 10% TEA
solution in ethyl acetate (50 ml), washed with 10%
sodium bicarbonate (2×10 ml) and water (2×10
ml). The organic layer was dried over sodium
sulfate and the product was purified by flash
chromatography on silica gel (elution with

O.V. Oskolko6a et al. / Chemistry and Physics of Lipids 99 (1999) 73–86
77
petroleum ether/ethyl acetate/TEA, 10:4:0.5, v/v/
v) to give 116.0 mg (89.2%) of oily phospho-
ramidite (X). Rf 0.28 and 0.33 (diastereoisomers
on phosphorus) (system 3). 31P-NMR (CDCl3):
148.18 and 148.54 ppm.
3H, CH3ꢀthymidine (Thd)), 1.87 (s, 3H,
CH3ꢀCO), 2.08 (m, 4H, 2-CH2ꢀPbs, 2%-
CH2ꢀThd), 2.21 (m, 2H, 3-CH2ꢀPbs), 2.73 (q,
6H, 3CH2ꢀTEA), 3.26 (t, 2H, 4-CH2ꢀPbs),
3.75–3.93 (m, 3H, 1-CH2ꢀSph, 4%-HꢀThd), the
region 3.93–4.18 ppm was overlapped with a
bright hydroxyl signal from methanol, 4.30 (m,
1H, 2-CHꢀSph), 5.09, 5.18 (2 m, 2H, 3-CHꢀSph,
3%-CHꢀThd), 6.11 (2t, 1H, J1%,2% 7.5 Hz, 1%-
CHꢀThd), 7.39–7.49 (m, 3H, Ar), 7.63–8.20 (m,
12H, Ar, 6-CHꢀThd).
2.2.7. 3-O-Benzoyl-2-N-(10-(1-pyrenyl)decanoyl)-
D,L-erythro-sphinganine-1-O-(N,N-diisopropyla-
mino)-2-cyanoethylphosphine (V)
The synthesis and isolation of amidophosphite
(V) was performed under identical conditions as
above, starting from the corresponding 3-ben-
zoyl ceramide (III) (100.0 mg, 0.132 mmol).
Yield 127.1 mg (75.9%) (oil). Rf 0.31 and 0.37
(diastereoisomers on phosphorus) (system 3).
31P-NMR (CDCl3): l 148.05 and 148.79 ppm.
2.2.9. 3-O-Benzoyl-2-N-(10-(1-pyrenyl)decanoyl)-
D,L-erythro-sphinganine-1-O-phospho-5%-(3%-O-
acetyl)thymidine, triethylammonium salt (XI)
The synthesis of protected ceramide phospho-
thymidine (XI) was carried out identically as de-
scribed above, starting from phosphoramidite
(V) (114.0 mg, 0.119 mmol) and 3%-
acetylthymidine (36.8 mg, 0.129 mmol). The
product was purified by column chromatography
on silica gel, elution with chloroform/methanol/
TEA (100:1:1, v/v/v). Yield 78.1 mg (54.5%), oil.
Rf 0.23 (system 4), 0.52 (system 5), 0.66 (system
6). 1H-NMR (CDCl3): 0.88 (2t, 3H, CH3ꢀSph),
1.00–1.50 (m, 45H, CH2ꢀSph, CH2ꢀPds,
3CH3ꢀTEA), 1.52–1.92 (m, 6H, 4-CH2ꢀSph, 3-
CH2ꢀPds, 9-CH2ꢀPds), 1.97 (s, 3H, CH3ꢀThd),
2.04 (s, 3H, CH3ꢀCO), 2.12–2.35 (m, 4H, 2-
2.2.8. 3-O-Benzoyl-2-N-(4-(1-pyrenyl)butanoyl)-
D,L-erythro-sphinganine-1-O-phospho-5%-(3%-O-
acetyl)thymidine, triethylammonium salt (X)
Amidophosphite (IV) (200.0 mg, 0.228 mmol)
and 3%-acetylthymidine (70.6 mg, 0.248 mmol)
were dried by coevaporation with water-free
dichloromethane (4×10 ml), dissolved in 4 ml
dichloromethane, and a solution of 3.5% 1H-te-
trazole in acetonitrile (0.64 ml, 22.4 mg, 0.320
mmol) was added. After 30 min at room tem-
perature, the TLC analysis (system 3) showed
the disappearance of the starting phosphoramid-
CH2ꢀPds,
2%-CH2ꢀThd),
2.99
(q,
6H,
ite (IV) and the formation of
a
new
3CH2ꢀTEA), 3.32 (2t, 2H, J 1.83 Hz, J 7.71 Hz,
10-CH2ꢀPds), 3.84 (t, 2H, J1,2 5.66 Hz, 1-
CH2ꢀSph), 3.93–4.20 (m, 3H, 5%-CH2ꢀThd, 4%-
CHꢀThd), 4.41 (m, 1H, 2-CHꢀSph), 5.30 (m,
2H, 3%-CHꢀThd, 3-CHꢀSph), 6.35 (2t, 1H, J1%,2%
8.13 Hz, 1%-CHꢀThd), 7.30–8.42 (m, 17H, Ar,
NHꢀSph, NHꢀThd, 6-CHꢀThd).
diastereomeric product of lower mobility. A 5.5
M solution of tert-butyl hydroperoxide (0.247
ml, 1.359 mmol) was added to the reaction mix-
ture. After 40 min when the oxidation was com-
plete, the solvents were evaporated and the
reaction mixture was dissolved in 1ml of toluene
and 1 ml triethylamine, kept at room tempera-
ture for 4 h, evaporated in vacuo, and co-evap-
orated with toluene (2×2 ml). The product was
purified by column chromatography on silica gel
2.2.10. 2-N-(4-(1-pyrenyl)butanoyl)-
sphinganine-1-O-phospho-5%-thymidine (XII)
To 3-benzoyl-pyrenebutanoyl phosphoce-
D,L-erythro-
by eluting isocratically with
a chloroform/
methanol/TEA mixture (100:5:1, v/v/v), afford-
ing homogenous (X) as oil (206.0 mg, 61.9%).
Rf 0.12 (system 4), 0.37 (system 5), 0.63 (system
6). H-NMR (CDCl3ꢀCD3OD, 3:1): 0.72 (t, 3H,
CH3ꢀSph), 0.90–1.50 (m, 35H, CH2ꢀSph,
3CH3ꢀTEA), 1.59 (m, 2H, 4-CH2ꢀSph), 1.70 (s,
ramide 3%-acethylthymidine (X) (261.8 mg, 0.233
mmol) in diethyl ether/methanol (1:1 by vol., 6
ml), a 0.2N solution of sodium methoxide (6
ml) was added. The mixture was kept at room
temperature for 1 h, then evaporated in vacuo.
The residue was chromatographed on silica gel,
1

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O.V. Oskolko6a et al. / Chemistry and Physics of Lipids 99 (1999) 73–86
elution with chloroform–methanol from 10:1 to
10:4 (by vol.). Yield of product (XII) was 184.0
mg (90.1%). Rf 0.51 (system 6). 1H-NMR
(CDCl3ꢀCD3OD, 3:2): 0.85 (t, 3H, CH3ꢀSph),
1.08–1.40 (m, 26H, CH2ꢀSph), 1.52 (m, 2H, 4-
CH2ꢀSph), 1.88 (2s, 3H, CH3ꢀThd), 2.18 (m,
2H, 3-CH2ꢀPbs), 2.38 (t, 2H, J 6.64 Hz, 2-
CH2ꢀPbs), 3.36 (t, 2H, J 7.51 Hz, 4-CH2ꢀPbs),
3.64 (m, 2H, 2%-CH2ꢀThd), 3.87–4.02 (m, 5H,
1-CH2ꢀSph, 5%-CH2ꢀThd, 4%-CHꢀThd), 4.25 (m,
1H, 2-CHꢀSph), 4.44 (m, 1H, 3%-CHꢀThd), the
signal of 3-CHꢀSph overlapped with a bright
signal of water, 6.24 (t, 1H, 1%-CHꢀThd), 7.64
(d, 1H, J 8.01 Hz, 6-CHꢀThd), 7.82–8.35 (m,
9H, Ar). 13C-NMR (CDCl3ꢀCH3OHꢀH2O,
65:25:4): 12.02 (CH3ꢀThd), 14.04 (CH3ꢀSph),
22.71 (17-CH2ꢀSph), 25.74 (5-CH2ꢀSph), 27.88,
29.40, 29.70, 31.95, 32.96 (CH2ꢀSph), 33.76 (16-
CH2ꢀSph), 36.16 (3-CH2ꢀPbs), 39.44 (2-
(17-CH2ꢀSph), 26.20 (5-CH2ꢀSph), 26.50, 28.16,
29.80, 29.97, 30.15, 30.29, 31.63, 32.36, 32.44,
34.05, 36.93, 39.94, 40.00 (CH2-Sph, CH2ꢀPds),
54.62 (2-CHꢀSph), 61.81 (10-CH2ꢀPds), 65.20
(1-CH2ꢀSph, 5%-CH2ꢀThd, 2%-CH2ꢀThd), 70.19,
70.31 (d, 3-CHꢀSph, JCꢀP 9.28 Hz), 70.97, 71.23
(2s, 3%-CHꢀThd), 85.30, 85.36 (2s, 1%-CHꢀThd),
86.04, 86.17 (d, 4%-CHꢀThd, JCꢀP 9.28 Hz),
123.95, 125.06, 125.22, 126.22, 126.90, 127.54,
127.73, 127.99 (CHꢀAr), 137.17 (6-CHꢀThd).
31P-NMR (CDCl3ꢀCH3OH, 2:1): 1.52 and 1.60
ppm.
2.3. Spectroscopic analyses
2.3.1. NMR measurements
1H-NMR spectra were measured in deuterated
solvents at 199.97 MHz, using a Varian Gemini
200 impulse spectrometer. Chemical shifts (l)
are given in ppm relative to tetramethylsilane as
a standard. 31P- and 13C-NMR spectra were
recorded on a Bruker MSL 300 spectrometer.
31P-NMR spectra were measured proton-noise
decoupled at 121.49 MHz; chemical shifts (l,
ppm) are reported relative to 85% orthophos-
phoric acid as external standard. The 13C-NMR
spectra were recorded at 75.47 MHz using
DEPT sequence; chemical shifts are given in
ppm (l) related to deuterated solvents.
CH2ꢀPbs),
54.22
(2-CHꢀSph),
57.52
(4-CH2ꢀPbs), 64.98 (1-CH2ꢀSph, 5%-CH2ꢀThd, 2%-
CH2ꢀThd), 70.00, 70.10 (ds, 3-CHꢀSph, JCꢀP
7.47 Hz), 70.48, 70.70 (2s, 3%-CHꢀThd), 85.05
(1%-CHꢀThd), 85.42, 85.52 (d, 4%-CHꢀThd, JCꢀP
7.25 Hz), 123.32, 124.80, 124.86, 124.95, 125.90,
126.73, 127.28, 127.41, 127.49 (CHꢀAr), 136.71
(6-CHꢀThd). 31P-NMR (CDCl3ꢀCH3OHꢀH2O,
3:2:1): 1.17 and 1.26 ppm.
2.2.11. 2-N-(10-(1-pyrenyl)decanoyl)-D,L-erythro-
sphinganine-1-O-phospho-5%-thymidine (XIII)
2.3.2. Absorbance and fluorescence measurements
A molar extinction coefficient of 42,000 M−1
cm−1 (at 342 nm) in ethanol was used to deter-
mine the pyrene lipid concentration (measure-
ments were performed on a Hitachi U-3210
spectrophotometer). Fluorimetry was registrated
on a Shimadzu RF-5301PC spectrofluorophoto-
meter equipped with a stirring device and ther-
mostated cuvette holder. Fluorescence spectra
were recorded at excitation wavelength of 342
nm with 3 nm band-passes for both excitation
and emission. For transfer experiments, excita-
tion and emission wavelengths were 342 and 378
nm, respectively with corresponding slit widths
of 1.5 and 5.0 nm. The small excitation band-
pass ensured minimal photodecomposition of
the pyrene probes.
This compound was prepared as described for
(XII) starting from N-pyrenedecanoyl phospho-
ceramide 3%-acethylthymidine (XI) (78.1 mg,
0.065 mmol). Yield of (XIII) 55.8 mg (89.9%).
Rf 0.55 (system 6). 1H-NMR (CDCl3ꢀCD3OD,
3:1): 0.71 (t, 3H, CH3ꢀSph), 0.95–1.58 (m, 40H,
CH2ꢀSph, CH2ꢀPds), 1.76 (s, 3H, CH3ꢀThd),
1.95–2.30 (m, 4H, 9-CH2ꢀPds, 2-CH2ꢀPds), the
signals of 10-CH2ꢀPbs (3.12–3.34) and of 3%-
CHꢀThd (3.95–4.22) overlapped with the signals
of methanol, 3.45, 3.60, 3.85 (3m, 8H, 2%-
CH2ꢀThd, 1-CH2ꢀSph, 5%-CH2ꢀThd, 4%-CHꢀThd,
2-CHꢀSph), 4.29 (m, 1H, 3-CHꢀSph), 6.11 (m,
1H, 1%-CHꢀThd), 7.37 (s, 1H, 6-CHꢀThd), 7.68–
8.18 (m, 9H, Ar). 13C-NMR (CDCl3ꢀCD3OD,
3:1): 12.48 (CH3ꢀThd), 14.41 (CH3ꢀSph), 23.11

O.V. Oskolko6a et al. / Chemistry and Physics of Lipids 99 (1999) 73–86
79
2.4. Donor 6esicle preparation
from the donor vesicles was observed, a 10- or
40-fold excess of unlabelled acceptor vesicles
(1.0 ml, 25 or 100 mM respectively) was added
to a stirred solution of the donor vesicles (1.0
ml). The spontaneous transfer of labelled ce-
ramide phosphothymidines from donor to accep-
tor vesicles was monitored continuously by
measuring the pyrene monomer fluorescence in-
crease at 378 nm (excitation at 342 nm) as a
function of time up to 480 s. Protein-mediated
transfer was measured upon addition of ns-
PLTP after a stable signal of spontaneous trans-
fer was achieved (see legends to the figures). For
the transfer rate calculation, the linear range
during first seconds after addition of acceptor
vesicles or protein was covered.
Single-walled phosphatidylcholine vesicles were
prepared by ethanol injection (Batzri and Korn,
1973). Final ethanol concentrations were ad-
justed to 1% by vol. A mixture of POPC/Pbs–
Cer–P–T (85:15 mole%) or Pds–Cer–P–T
(85:15 mole%) was evaporated to dryness under
a gentle stream of argon. The dried lipids were
redissolved in ethanol at a final concentration of
250 mM of total lipids. For measurements of
excimer to monomer fluorescence intensity at
different concentrations of pyrene phospholipids,
aliquots of above-described solutions were di-
luted with a 250 mM solution of POPC to give
two sets of stock solutions with different molar
fractions of pyrene probes. Aqueous lipid dis-
persions were prepared by injecting a 42 ml
ethanolic phospholipid solution through
a
3. Results and discussion
Hamilton syringe into 4158 ml of an intensively
stirred buffer solution. During injection, the
temperature was kept at 37°C. The buffer solu-
tion used throughout this work was 10 mM
Tris–HCl, 1 mM EDTA, and 0.02% sodium
azide adjusted to pH 7.40. For one measure-
ment of intermembrane transfer, 1 ml of vesicle
solution (9 mole% of the pyrene-labelled conju-
gates) was used. Dynamic light scattering analy-
sis (Orthaber and Glatter, 1998) of obtained
liposomes (Pbs- and Pds-phospholipids, 9 mole%
for both, 37°C) revealed a size distribution of
vesicles of average diameter about 60 nm.
3.1. Chemical syntheses
The synthetic routes leading to the nucle-
otide–lipid conjugates are outlined in Fig. 1.
The starting compound for the preparation of
pyrene-labelled ceramide phosphorylthymidines
(XII) and (XIII) was racemic 3-O-benzoyl-sphin-
ganine (I) with both free amino- and hydroxyl-
groups. The most crucial step in the synthesis of
lipids carrying fluorescently labelled fatty acids
is the choice of the selective method for acyla-
tion of the amino-group. For the synthesis of
ceramides acylation could be carried out using
an acid anhydride (Singh and Schmidt, 1989),
acid-halides (Shapiro, 1969; Vunnam and Radin,
1979; Dokolina et al., 1983; Zimmerman et al.,
1988), mixed carbodiimide (Hammarstroem,
1971), N-hydroxy succinimidates (Julina et al.,
1986) or mixed anhydrides formed by ethylchlo-
roformate and a fatty acid (Acquotti et al.,
1986). Alternative methods, using coupling
reagents such as dicyclohexylcarbodimide (Co-
hen et al., 1984; Kiso et al., 1986; Molotkovsky
et al., 1991) or diethylphosphoryl cyanide in the
presence of triethylamine (Anand et al., 1996),
have also been applied. The procedures involv-
ing these acylation or activation agents have
2.5. Acceptor 6esicle preparation
Acceptor vesicles consisting of POPC were
prepared by the ethanol injection method (final
lipid concentration 25 or 100 mM) in analogy to
the procedure outlined above. In transfer experi-
ments, the concentration of the acceptor sample
was either ten or 40 times greater than the
donor vesicle concentration. This protocol mini-
mized the contribution of reverse transfer to the
signal.
2.6. Transfer assays
After a stable fluorescence signal originating

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O.V. Oskolko6a et al. / Chemistry and Physics of Lipids 99 (1999) 73–86
several disadvantages including the requirement
for a large molar excess of fatty acid, low yield,
long reaction time, and undesirable side reaction
such as O-acylation. In our case, this methodol-
ogy must be neither too complicated nor include
the use of acyl chlorides or anhydrides because of
the possible degradation of pyrene fatty acids
during preparations of their derivatives. On the
other hand, the capacity of p-nitro- and pen-
tafluorophenyl esters of carboxylic acids to acy-
late under certain conditions the amino group of
various types of compounds—for example, ser-
ine, threonine, and hydroxylamine (Lapidot et al.,
1967); sphingosine-1-phosphocholine (Ahmad et
al., 1985); sphingoid bases or their derivatives
(Tkaczuk and Thornton, 1981; Dokolina et al.,
1983; Bruzik, 1988; Ohashi et al., 1989; Shibuya et
al., 1992) led us to utilize these reagents in the
acylation of 3-O-benzoyl-sphinganine (I). Thus,
we performed this procedure with activated esters
of pyrene fatty acids in water-free pyridine. The
efficacy of the acylation of the amino group with
p-nitrophenyl ester or with pentafluorophenyl es-
ter of pyrenedecanoic acid, or with pen-
tafluorophenyl ester of pyrenebutanoic acid was
comparable and did not exceed 50% yields. This
points to equivalent efficiency of p-nitrophenyl
ester and pentafluorophenyl ester during deriva-
tization. The use of other solvents or addition of
small amounts of triethylamine did not signifi-
cantly improve the yields. However, acylation of
the amino group of sphingolipids with p-nitro-
phenyl oleate (Tkaczuk and Thornton, 1981), p-
nitrophenyl esters of myristic or lauric acids
(Groenberg et al., 1991), p-nitrophenyl stearate
(Kann et al., 1991), or p-nitrophenyl palmitate
(Ohashi et al., 1989; Shibuya et al., 1992) have
been shown to be more effective. The low yields
Fig. 1. Synthrtic routes of pyrene labelled ceramide phosphothymidines (XII) and (XIII).

O.V. Oskolko6a et al. / Chemistry and Physics of Lipids 99 (1999) 73–86
81
of acylation observed with pyrene-labelled acid
esters could be explained by the presence of the
bulky pyrene group at the terminus of the fatty
acids.
NMR spectra were recorded, which were in
agreement with the structures of the synthesized
ceramide phosphothymidines.
To create the phosphodiester structure, we
chose the mild phosphitylation procedure, which
had previously been used for the successful syn-
thesis of ceramide phosphothymidine and its an-
tiviral analogs (Oskolkova et al., 1996, 1997) as
well as for glycerophospholipid synthesis (Mo-
rillo et al., 1996) and other substance classes
(Beaucage and Iyer, 1992, 1993a,b). The corre-
sponding pyrene-labelled ceramides (II, III) were
converted with good yields (89.2 and 75.9%, re-
spectively) using the bifunctional reagent,
3.2. Spectral properties
The absorption (342 nm) and emission (378,
398 nm) wavelengths found in the UV- and
fluorescence spectra in dilute solutions in
ethanol of the synthesized substances (XII) and
(XIII) were typical for pyrene-labelled phospho-
lipids described earlier (Pownall et al., 1982; van
Wijk et al., 1992) and revealed only monomer
fluorescence.
The two compounds were used to study their
membranotropic features as components of
phospholipid vesicles. Both conjugates (XII) and
(XIII) were readily incorporated into donor vesi-
cles prepared by ethanol-injection. At low con-
centrations of pyrene ceramide phospho-
thymidines in single bilayer POPC vesicles, the
fluorescence spectra were similar to those
recorded in ethanol. At higher concentrations
(above 0.5 mole%) of the labelled lipids, an ex-
cimer fluorescence band centered at 478 nm ap-
peared. This indicates that ceramide phospho-
thymidines (XII) and (XIII) maintain the char-
acteristic features of pyrene groups displaying a
different emission spectrum depending on the
degree of self-interactions. Particularly, the
prevalence of the two emission peaks at 398 and
378 nm, observed in organic solvent solution, is
typical of non-interacting pyrene molecules,
while the preponderance of the wide peak at 478
nm, observed at higher concentrations, indicates
interactions within the hydrophobic tails carry-
ing the pyrene group. The intensity of the ex-
cimer fluorescence at 478 nm (E) relative to the
monomer fluorescence at 398 nm (M) of each
pyrenyl derivative increased with its microscopic
concentration in POPC vesicles. The relationship
between the E/M ratio and the molar% concen-
tration of Pyr–Cer–P–T in vesicles was linear
up to about 4 mole% for both short and long-
chain pyrene derivatives (Fig. 2), which is in
agreement with published data (Massey et al.,
1982b; Somerharju et al., 1985; Ollman et al.,
1987). However, pyrene-labelled phospholipids
bis(N,N-diisopropylamino)-2-cyanoethyl
phos-
phine in the presence of diisopropylammonium
tetrazolide as catalyst. The 31P-NMR spectra of
purified phosphoramidites (IV) and (V) were in-
dicative of the presence of two diastereoisomers
each (148.18 and 148.54 ppm for (IV), and
148.05 and 148.79 ppm for (V)). 1H-tetrazole
catalyzed the condensation of phosphoramidites
(IV) and (V) with the free hydroxyl group of
3%-O-acetylthymidine. The resulting phosphite
triesters (VI) and (VII) were oxidized in situ
with tert-butyl hydroperoxide to give the phos-
phate triesters (VIII) and (IX), respectively.
These compounds were partly deblocked without
isolation by the action of triethylamine to form
phosphodiesters (X) and (XI) with good yields.
The structure of the two latter compounds was
verified by 1H-NMR spectroscopy data. The
proton signals of both, lipid and nucleoside
moieties, were fully present in the spectra. Fi-
nally, removal of benzoyl- and acetyl-protective
groups by the action of sodium methylate in
methanol furnished with 90.1 and 89.9% yields
the target ceramide phosphothymidines (XII)
and (XIII), respectively. The 31P-NMR data of
(XII) and (XIII) exhibit two signals (1.17 and
1.26 ppm for (XII); 1.52 and 1.60 ppm for
(XIII)), which most likely belong to
D- and L-
diastereoisomers at the sphinganine residue.
Analogous pairs of signals have been found in
31P-NMR spectra of unlabelled ceramide phos-
phothymidines (Oskolkova et al., 1996, 1997).
For phosphates (XII) and (XIII), 1H- and 13C-

82
O.V. Oskolko6a et al. / Chemistry and Physics of Lipids 99 (1999) 73–86
tion that the attachment of a paraffinic chain to
pyrene reduces its ability to form excimers, which
may be related to lower mobility and/or steric
restriction, which decreases the ability to give
excimers.
3.3. Spontaneous transfer studies
As a measure of the capacity of Pyr–Cer–P–T
(XII) and (XIII) to exchange between membranes,
we determined their transfer from donor unil-
amellar vesicles to a 10- and 40-fold excess of
acceptor vesicles. Fig. 3 shows the spontaneous
transfer of pyrenebutanoyl- and pyrenedecanoyl
ceramide phosphothymidines (lines I and II, re-
spectively) at 37°C. Compared to Pds–Cer–P–T
(XIII), the spontaneous transfer of Pbs–Cer–P–T
(XII) is approximately 26-fold faster, with esti-
mated transfer rates of 14.89 pmol/min for (XII)
and 0.57 pmol/min for (XIII) at the donor to
acceptor ratio 1:10. Therefore, the short-chain
lipid conjugate (XII) was less stably integrated in
a POPC bilayer compared to its long-chain coun-
terpart (XIII). The intermembrane transport (Fig.
3) could be described as a first order kinetic
process. Furthermore, the transfer rate for both
lipids was independent of acceptor concentration.
These lines of evidence support the notion that
the lipid transfer occurs via rate-limiting desorp-
tion of the pyrene probe from the donor surface
into the surrounding aqueous phase, followed by
a rapid diffusion-controlled association of pyrene
lipid with the acceptor (Galla et al., 1979; Rose-
man and Thompson, 1980; McLean and Philips,
1981; Correa-Freire et al., 1982; Massey et al.,
1982a,b; Nichols and Pagano, 1982; Pownall et
al., 1982, Frank et al., 1983; Pownall et al., 1983;
Arvinte and Hildenbrand, 1984; McLean and
Philips, 1984; Via et al., 1985; Homan and Pow-
nall, 1988). Thus, our data conform to the criteria
for lipid transfer occurring via the aqueous phase.
Fig. 2. Dependence of the excimer (478 nm) to monomer (398
nm) intensity ratio on the Pyr-Cer-P-T (XII, n=3) (ꢀ) and
(XIII, n=9) (ꢁ) concentration in POPC vesicles at 37°C.
Each data point is an average value out of three determina-
tions of the E/M fluorescence intensity (SD9 55%). For
details see Section 2.4.
don’t always show the linear E/M dependence
upon their concentration, thus supporting evi-
dence for regular distribution of labelled phos-
pholipids in vesicles (Viani et al., 1988; Tang and
Chong, 1992; Chong et al., 1994) due to the
immiscibility of pyrene lipids in phospholipid bi-
layers (for example, pyrenedodecanoyl sphin-
gomyelin in stearoyl sphingomyelin or in POPC
(Barenholz et al., 1996)). On the other hand,
pyrene-labelled phosphatidylcholine in dimyris-
toylphosphatidylcholine membranes is an example
of a system with nearly ideal mixing (Hresko et
al., 1986).
The Fig. 2 shows the higher tendency of the
long-chain derivative (XIII) to form excimer
structures. This is apparent from the higher E/M
intensities compared to E/M ratio observed with
the short chain compound (XII) at the same
concentrations and temperature. Within a phos-
pholipid bilayer, pyrene residues of long chain
acyl rests are located close to the boundary be-
tween two membrane leaflets and are therefore
more mobile and the probability of excimer for-
mation is increased. In contrast, with the short-
chain compound (XII) the pyrene label
experiences lower mobility and is thus less likely
to form excimers.
Another
liponucleotide
analogue,
3%-de-
oxythymidine diphosphate diglyceride, has been
shown to be transported by the same mechanism
(van Wijk et al., 1992). The rate of intermembrane
transfer is determined by several factors: (1) insol-
ubility of the bulky polar headgroup in the hydro-
Our data are in agreement with published re-
sults (Barenholz et al., 1996) supporting the no-

O.V. Oskolko6a et al. / Chemistry and Physics of Lipids 99 (1999) 73–86
83
acids which were used for sphingomyelin labeling.
The transfer half-times increase with increasing
chain length of the amide-linked fatty acid in
sphingomyelins. A similar acyl chain length de-
pendence has been noted for a variety of phos-
pholipids labelled with the NBD fluorophore
(Nichols and Pagano, 1982) as well as for pyrene-
labelled glycerophospholipids, cerebrosides and
other amphiphiles (Massey et al., 1982a; Pownall
et al., 1983; Via et al., 1985). Studies performed
by Homan and Pownall (1988) have shown that
an increase of the sn-1-acyl chain length by two
methylene groups reduced the rate of spontaneous
transfer approximately 8-fold.
Fig. 3. Spontaneous intermembrane transfer of fluorescently
labelled ceramide phosphothymidines (XII) and (XIII). Donor
vesicle lipids (10 ml, total lipid concentration 250 mM, 9 mole%
of the labeled lipids) were injected into buffer (990 ml) (arrow
A). After 3 min of intensive stirring, a 10-fold excess of
acceptor vesicles (1 ml, 25 nmol) was added (arrow B). Traces:
I, Pbs-Cer-P-T (XII); II, Pds-Cer-P-T (XIII). The temperature
was held constant at 37°C.
3.4. Protein-mediated transfer
The effect of an enriched non-specific lipid
transfer protein preparation on the rate of trans-
fer of Pbs- (XII) and Pds–Cer–P–T (XIII) was
tested using a 10-fold excess of acceptor. The
ns-PLTP from bovine liver was shown to acceler-
ate transfer of the ceramide phosphothymidine
bearing the pyrenedecanoic acid moiety (XIII)
(Fig. 4). In contrast, the protein did not affect the
phobic membrane interior results in a slow flip–
flop rate in lipid model systems; (2) bulkiness and
charge of the polar headgroup facilitate phospho-
lipid transmembrane transport; (3) the length of
fatty acid chains is responsible for the solubility of
lipids in an aqueous phase. The latter factor is a
major determinant as it affects the first stage of
spontaneous transport, namely dissociation of
lipids from donor vesicles (McLean and Philips,
1981; Ferrell et al., 1985). Manipulations of the
acyl chain lengths greatly affects the t1/2 values for
spontaneous transfer.
Pownall et al. (1982) have reported data on
spontaneous
transfer
of
N-(9-(1-pyrenyl)-
nonanoyl)sphingomyelin between single-bilayer
vesicles composed of DMPC, beef brain sphin-
gomyelin, or 1-palmitoyl-2-palmitoleoylphospha-
tidylcholine. It was found that the transfer of
bovine brain sphingomyelin was about an order
of magnitude slower below the phase transition of
this lipid than above it. Where comparable, how-
ever, respective half-times were about an order of
magnitude smaller than those described by others
(Frank et al., 1983). The origin of this difference
is due to the different lengths of pyrenyl fatty
Fig. 4. Protein-mediated transfer of Pds-Cer-P-T (XIII) cata-
lyzed by non-specific phospholipid transfer protein from
bovine liver. The arrow A shows addition of the donor vesicles
(1 ml, total lipid content 2.5 nmol, 9 mole% of the labelled
lipid in POPC). At arrow B, acceptor vesicles (1 ml, 25 nmol)
were added and the spontaneous transfer was monitored. At
arrow C, lipid-transfer protein (20 ml, 1 mg protein/ml) was
added. After addition of Triton X-100 (10 ml, 20%) (arrow D)
the cesicles were completely disintegrated.

84
O.V. Oskolko6a et al. / Chemistry and Physics of Lipids 99 (1999) 73–86
Fig. 5. Binding of Pds-Cer-P-T by ns-PLTP. After a stable
signal from donor vesicles (1 ml, 2.5 nmol total lipid content,
9 mole% of label) (arrow A) was obtained, ns-PLTP was
added (20 ml, 3 mg protein/ml) (arrow B), followed by 1 ml (25
nmol) acceptor vesicles (arrow C). At arrow D, Triton X-100
(10 ml, 20%) was added.
Fig. 6. Dependence of protein mediated transport of Pds-Cer-
P-T (XIII) from donor to acceptor vesicles on the protein
content. For details see Section 2.
8
senschaftlichen Forschung in Osterreich’ (SFB-
project F 102 to F.P. and F 107 to A.H.). C.
Illaszewicz is gratefully acknowledged for record-
ing of NMR spectra. The authors wish to acknowl-
edge D. Orthaber for light scattering measurements
of vesicles.
transfer rate of the short-chain Pbs–Cer–P–T
(data not shown), most likely due to the consider-
ably higher rate of spontaneous transfer of the
short-chain labelled lipid (XII). Upon addition of
ns-PLTP to the donor vesicle dispersion containing
the liponucleotide (XIII), the monomer fluores-
cence intensity increases instantaneously (Fig. 5).
Upon addition of the acceptor vesicles, a further
gradual increase occurs. This experiment demon-
strates that upon mixing ns-PLTP and Pds–Cer–
P–T the lipid is rapidly bound by the protein and
then transferred to the acceptor vesicles. ns-PLTP
from bovine liver stimulated the transfer of Pds–
Cer–P–T between vesicles in a concentration de-
pendent manner (Fig. 6). It was previously
demonstrated that this protein was capable of
stimulating the transfer of the CDP-DG analogue
3%-deoxythymidine diphosphate diglyceride be-
tween vesicles (van Wijk et al., 1992). Our data
demonstrate that ns-PLTP also catalyzes the inter-
membrane transfer of ceramide phosphothymidine.
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Acknowledgements
This investigation was carried out with financial
support by the ‘Fonds zur Fo¨rderung der wis-

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