Small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) studies of amide phospholipids

Article abstract of DOI:10.1016/j.chemphyslip.2004.09.013

Varying chemically the structure of phospholipids in the region between hydrophobic and hydrophilic segments is expected to have a strong influence on the interaction with water and the phase behavior. This is studied in this work with the motivation to investigate these lipids as potential inhibitors of phospholipase A₂. Thus the amide phospholipids l-ether-amide-PC (1-O-hexadecyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine), l-ester-amide-PC (1-palmitoyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3- phosphocholine) and l-ether-amide-PE (1-O-hexadecyl-2-N-palmitoyl-2-deoxy-sn- glycero-3-phosphoethanolamine) have been synthesized and characterized. The phase behavior and thermal transitions in buffer dispersions are examined by a combination of high-sensitivity differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS) experiments between 10 and 80°C at pH 8.9. The onset temperatures determined from DSC measurements agree well with the starting temperatures of changes in the repeat distance obtained by SAXS measurements. The phases observed are lamellar both below and above the main phase transition. The phase transition temperatures and enthalpies depend strongly on the substitutions in sn-1 position and head group structure. The lamellar repeat distance in gel and liquid-crystalline phases increases with increasing temperature for l-ester-amide-PC and l-ether-amide-PC, whereas the temperature dependence is opposite for the l-ether-amide-PE. The observed behavior is discussed and compared with that of DPPC and DPPE, indicating the strong dependence of hydration and phase behavior on head group structure.

Full text of DOI:10.1016/j.chemphyslip.2004.09.013

Chemistry and Physics of Lipids 133 (2005) 79–88
Small angle X-ray scattering (SAXS) and differential scanning
calorimetry (DSC) studies of amide phospholipids
Xiuhong Zhaia,b, Markus Bartelc, Gerald Brezesinskib,∗, Bernd Rattayc,
b
a,1
¨
Helmuth Mohwald , Junbai Li
a
International Joint Lab, Key Lab of Colloid and Interface Science, The Center for Molecular Science, Institute of Chemistry,
Chinese Academy of Sciences, Zhong Guan Cun, Beijing 100080, China
b
Max Planck Institute of Colloids and Interfaces, Am Mu¨hlenberg 1, D-14476 Golm/Potsdam, Germany
c
Institute of Pharmaceutical Chemistry, Martin-Luther-University, D-06120 Halle/S., Germany
Received 4 June 2004; received in revised form 6 September 2004; accepted 8 September 2004
Available online 11 November 2004
Abstract
Varying chemically the structure of phospholipids in the region between hydrophobic and hydrophilic segments is ex-
pected to have a strong influence on the interaction with water and the phase behavior. This is studied in this work with
the motivation to investigate these lipids as potential inhibitors of phospholipase A2. Thus the amide phospholipids l-ether-
amide-PC (1-O-hexadecyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine), l-ester-amide-PC (1-palmitoyl-2-
N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine) and l-ether-amide-PE (1-O-hexadecyl-2-N-palmitoyl-2-deoxy-
sn-glycero-3-phosphoethanolamine) have been synthesized and characterized. The phase behavior and thermal transitions in
buffer dispersions are examined by a combination of high-sensitivity differential scanning calorimetry (DSC) and small angle
X-ray scattering (SAXS) experiments between 10 and 80 ◦C at pH 8.9. The onset temperatures determined from DSC measure-
ments agree well with the starting temperatures of changes in the repeat distance obtained by SAXS measurements. The phases
observed are lamellar both below and above the main phase transition. The phase transition temperatures and enthalpies depend
strongly on the substitutions in sn-1 position and head group structure. The lamellar repeat distance in gel and liquid-crystalline
phases increases with increasing temperature for l-ester-amide-PC and l-ether-amide-PC, whereas the temperature dependence
is opposite for the l-ether-amide-PE. The observed behavior is discussed and compared with that of DPPC and DPPE, indicating
the strong dependence of hydration and phase behavior on head group structure.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Amide phospholipids; Small angle X-ray scattering (SAXS); Differential scanning calorimetry (DSC); Phase transition; Lamellar
phases
∗
Corresponding author. Tel.: +49 331 567 9234; fax: +49 331 567 9202.
E-mailaddresses:brezesinski@mpikg-golm.mpg.de (G. Brezesinski), jbli@iccas.ac.cn (J. Li).
Tel.: +86 10 82614087; fax: +86 10 82612484.
1
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2004.09.013

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X. Zhai et al. / Chemistry and Physics of Lipids 133 (2005) 79–88
1. Introduction
of amide phospholipids in monolayers at the air/water
interface (Zhai et al., 2003). For the investigation of
the thermodynamic properties of amide phospholipids
bilayers, differential scanning calorimetry (DSC) has
become a standard method. DSC focuses on the deter-
mination of temperatures and enthalpies of transitions
between gel and liquid-crystalline phases of lipid bi-
layers (Lee, 1977; Nagle, 1980; Nagle and Wilkinson,
1978). Small angle X-ray scattering (SAXS) is the most
important method to investigate structural and dynam-
ical properties of these phases.
In the present work we described the phase
behavior of aqueous dispersions of newly synthe-
sized amide phospholipids (l-ester-amide-PC, l-ether-
amide-PC and l-ether-amide-PE) investigated by DSC
and SAXS.
Phospholipase A2 (PLA2) is a calcium dependent
enzyme, which stereospecifically hydrolyzes the fatty
acid ester bond at the sn-2-position of naturally oc-
curring phospholipids (Janda and Bottone, 1981; Gelb
et al., 1992; Verger and de Haas, 1973; Dahmen-
Levison et al., 1998). Because mainly unsaturated fatty
acids are esterified at this position (e.g. arachidonic
acid) the reaction is the basis for the biosynthesis
of physiologically and pathophysiologically important
mediators as prostaglandines, leukotrienes and throm-
boxanes. Therefore, the investigation of the mechanism
of the enzymatic reaction and the search for opportuni-
ties to influence this process is of great interest for the
therapy of inflammation and allergic diseases.
Amide phospholipids bind to secretory PLA2 but
they cannot be hydrolyzed by the enzyme. This is
due to the special binding mode of amide phospho-
lipids to PLA2, which is characterized by a hydrogen
bond of the lipid amide-hydrogen with the imidazole-
nitrogen of the PLA2-His 48 that is involved in the cat-
alytic triad of the enzyme reaction (Slaich et al., 1992;
Plesniak et al., 1993). Amide phospholipids are also
known as competitive inhibitors of the secretory PLA2
when they have an ester-, thioester-, ether- or a sim-
ple alkyl chain at the sn-1 position of the glycerol
backbone or at the respective position of non-glycero-
phospholipids (Bonsen et al., 1972; Davidson et al.,
1986; de Haas et al., 1990; Yu et al., 1990).
Phospholipids display a rich polymorphism de-
pending on the molecular structures and on thermo-
dynamic variables and provide valuable model sys-
tems for biological membranes. Apart from the well-
known lamellar structures, non-lamellar cubic and
hexagonal phases have been detected (Brandenburg
et al., 1993), which may play a role as transient or
local intermediates in a number of biological pro-
cesses (Seddon, 1990; Seddon and Templer, 1993;
Rapp et al., 1995). The phase behavior of aqueous
lipid dispersions and two-dimensional model systems
(monolayers at the air/water interface) of membrane
lipids, particularly phosphatidylcholines (PCs) and
phosphatidylethanolamines (PEs), which are the major
components of biological membranes, has been stud-
ied by an enormous number of physicochemical tech-
niques (Small, 1986). In our previous study we have in-
vestigated the phase behavior and morphology changes
2. Materials and methods
2.1. Materials
The sn-2 amide phospholipid analogs l-ether-
amide-PC and l-ether-amide-PE were synthesized
according to procedures described by Bartel et al.
(2000) and have been characterized by NMR. The l-
ester-amide-PC (1-palmitoyl-2-N-palmitoyl-2-amino-
2-deoxy-sn-glycero-3-phosphocholine) was synthe-
sized as follows: l-serine-methylester and choline
tosylate were purchased from Aldrich. Phosphorous
trichloride and pivaloyl chloride were ACROS prod-
ucts. Trityl chloride (Aldrich) was recrystallized be-
fore use from hexane under addition of thionylchloride.
(S)-N-palmitoyl-serine-methylester 1 was synthesized
in the same way as described by Chandrakumar and
Hajdu (1981). Scheme 1 presents the different steps of
the synthesis.
2.1.1. (S)-N-palmitoyl-3-O-trityl-serine
methylester (2)
To a solution of N-palmitoylserine methylester 1
(5 g; 14 mmol) in 180 ml dry chloroform triethylamine
(2 g; 2.75 ml; 19.6 mmol) was added and a solution
of tritylchlorid (5.5 g; 19.6 mmol) in 35 ml dry chlo-
roform was slowly dropped into the mixture which
turns red during the addition. After stirring for 12 h
at r.t. the progress of the reaction was checked by TLC
(ether/hexane 1:1, Rf ca. 0.3).

X. Zhai et al. / Chemistry and Physics of Lipids 133 (2005) 79–88
81
Scheme 1. The synthesis of the l-ester-amide-PC.
The solution was washed with ice water until the pH
was neutral. Drying of the organic layer with Na2SO4
and evaporation in vacuo gave 7.24 g of a crystalline
yellow/brown crude product. Two-fold crystallization
from ethanol and drying in vacuo at 78 ◦C gave 2.70 g
2 (55.4%).
ganic phases were washed with 15 ml water, dried
with Na2SO4 and evaporated in vacuum. The oily
crude product was purified by chromatography over
a silica gel column with CHCl3/ether 9:1 as eluent.
DC:Rf (CHCl3/ether 8:2) = 0.23
EA (MW = 571.84)
DC:Rf (ether/hexane 1:1) = 0.21
EA (MW = 599.85)
Calc.
C (79.82) H (9.34) N (2.45)
C (78.90) H (9.25) N (2.32)
Found
Calc.
C (78.09)
C (77.50)
H (8.91)
H (9.02)
N (2.34)
N (2.19)
ES-MS: m/z
594.4 (M + Na+)
Found
1H NMR: δ = 1.9 (3H, H3C–CH2–), δ = 1.3
(24H, H3C–(CH2)12–CH2–), δ = 1.6 (2H,
H3C–(CH2)12–CH2–), δ = 2.2 (2H, –H2C–CONH–),
δ = 3.3 (1H, –H2C–OTr), δ = 3.6 (1H, –H2C–OTr),
δ = 3.8 (3H, –OCH3), δ = 4.7 (1H, –CH–NH–CO–),
δ = 6.25 (1H, NH), δ = 7.2–7.4 (15H, OTr).
1H NMR: δ = 1.9 (3H, H3C–CH2–), δ = 1.3
(24H, H3C–(CH2)12–CH2–), δ = 1.6 (2H,
H3C–(CH2)12–CH2–), δ = 2.15 (2H, –H2C–CONH–),
δ = 2.7(1H, OH), δ = 3.3(2H, –H2C–OTr), δ = 3.6–3.85
(2H, –H2C–OH), δ = 4.1 (1H, –CH–NH–CO–), δ = 5.9
(1H, NH), δ = 7.2–7.4 (15H, OTr).
2.1.2. 2-N-palmitoyl-3-O-trityl-2-deoxy-2-amino-
sn-glycerol (3)
2.1.3. 1,2-Dipalmitoyl-3-O-trityl-2-
deoxy-2-amino-sn-glycerol (4)
Compound 2 (500 mg; 0.83 mmol) was dissolved
in 9 ml of abs. ether. LiBH4 (30 mg; 1.25 mmol) was
added and the mixture was refluxed for 20 min fol-
lowed by stirring at r.t. for further 40 min (TLC-control
ether/hexane 1:1). Then the mixture was cooled in
an ice bath and 1 N HCl was added in drops un-
til the mixture had a pH of 8–9. The mixture was
washed with 15 ml water and the water layer was
extracted with 2 × 15 ml CH2Cl2. The collected or-
Compound 3 (280 mg; 0.49 mmol) was dissolved
in 20 ml chloroform and 4-dimethylaminopyridin
(DMAP; 120 mg; 0.98 mmol) was added. Then palmi-
toyl chloride (160 mg; 0.98 mmol) was slowly dropped
into the solution. The mixture was stirred for 48 h at r.t.
and the progress of the reaction was checked by TLC
(petroleumether/diethylether 5:2). In the case of an in-
complete conversion another 40 mg DMAP and 50 mg
palmitoyl chloride were added and stirring was contin-

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X. Zhai et al. / Chemistry and Physics of Lipids 133 (2005) 79–88
ued for further 12 h. The mixture was then washed with
3 × 20 ml water and dried with Na2SO4. After evapo-
ration of the solvent the residue was suspended in water
and filtered off by suction. The remaining solid crude
product was used without further purification.
–H2C–OPO3), δ = 4.2 (3H, –H2C–O–CO–C15H33,
–CH–NH–CO–), δ = 4.3 (2H, –CH2–CH2–N(CH3)3),
δ = 6.8 (1H, NH).
l-Dipalmitoylphosphatidylcholine (l-DPPC) was
purchased from Sigma and was used without further
purification. Thecorrespondingchemicalstructuresare
shown in Fig. 1.
ES-MS: m/z 832.6 (M + Na+)
The samples for DSC and SAXS measurements
were prepared in buffer solutions (pH 8.9 containing
150 mM NaCl, 5 mM CaCl2 and 10 mM Tris). Chloro-
form of >99% purity was purchased from ACROS.
DC:Rf (petroleum ether/ether 5:2) = 0.15
2.1.4. 1,2-Dipalmitoyl-2-deoxy-2-amino-sn-
glycerol (5)
2.2. Differential scanning calorimetry (DSC)
Compound 4 (ca. 0.49 mmol) was dissolved in
25 ml dry CHCl3. After addition of 1 ml BF3 (10% in
CH3OH) at 0 ◦C the mixture was stirred for 20 min
(TLC-control CHCl3/ether 8:2). The solution was
transferred into a separation funnel and was washed
with 2 × 25 ml water. Drying of the organic layer with
Na2SO4 and evaporation of the solvent gave the crude
product which was purified by column chromatogra-
phy on silica gel with CHCl3/ether 9:1.
DSC measurements were performed using the
Micro-Calorimetry System (MCS DSC, MicroCal Inc.,
Northampton, MA, USA) as described elsewhere
(Johnson et al., 1992; Branchu et al., 1999). The ex-
periments were carried out over a temperature range
from 10 to 80 ◦C with a scan rate of 1 K/min. For sam-
ple preparation, all phospholipids were dried in vac-
uum for 2 h at room temperature before weighing. The
phospholipid samples were mixed with the buffer so-
lution (1 mg/ml buffer solution). The phospholipid dis-
persions were formed during heating to 60 ◦C and vor-
texing. After this procedure, the samples were degassed
for 20 min. The dispersion was injected into the sample
cell, and buffer solution was injected into the reference
cell. The transitions were reversible as observed by res-
canning the samples after allowing them to cool down
to the starting temperature.
ES-MS: m/z 568.5 (M + H+)
DC:Rf (CHCl3/ether 8:2) = 0.36
EA
Calc.
C (74.02)
C (74.40)
H (12.25)
H (12.36)
N (2.47)
N (2.39)
Found
1H NMR: δ = 1.9 (6H, 2H3C–CH2–), δ = 1.3
(48H, 2H3C–(CH2)12–CH2–), δ = 1.6 (4H,
2H3C–(CH2)12–CH2–), δ = 2.2 (2H, –H2C–CONH–),
δ = 2.35 (2H, –H2C–COO–), δ = 2.7 (1H, –OH), δ = 3.6
(2H, –H2C–OH), δ = 4.2 (3H, –H2C–O–CO–C15H31,
–CH–NH–CO–), δ = 5.9 (1H, NH).
2.3. Small angle X-ray scattering (SAXS)
Time-resolved SAXS experiments were performed
at the soft condensed matter beamline A2 at HASY-
LAB, DESY, Hamburg, Germany, atafixedwavelength
of 0.15 nm. The diffracted intensity was recorded by a
linear delay-line detector during heating and cooling
scans at a rate of 1 K/min. The data acquisition system
has been described elsewhere (Rapp et al., 1995; Lag-
gner et al., 1991; Bringezu and Brezesinski, 2001). To
minimize the X-ray dose on the sample, a fast shutter
was kept closed when no data were acquired. In the
experiments an alternating sequence of 10 s exposure
time and 50 s waiting time was applied. The recipro-
cal spacings s = 1/d were calibrated with the diffrac-
tion pattern of dry rat-tail collagen with a long spac-
2.1.5. 1,2-Dipalmitoyl-2-deoxy-2-amino-sn-
glycero-3-phosphocholine (I)
The preparation of the final compound I – starting
from 5 – was carried out in the same way as described
previously (Bartel et al., 2000).
1H NMR: δ = 1.9 (6H, 2H3C–CH2–), δ = 1.3
(48H,
2H3C–(CH2)12–CH2–),
δ = 1.6
(4H,
2H3C–(CH2)12–CH2–), δ = 2.2 (2H, –H2C–CONH–),
δ = 2.35 (2H, –H2C–COO–), δ = 3.3 (9H, –N(CH3)3),
δ = 3.75 (2H, –CH2–N(CH3)3), δ = 3.9 (2H,

X. Zhai et al. / Chemistry and Physics of Lipids 133 (2005) 79–88
83
Fig. 1. Chemical structures of (a) l-dipalmitoylphosphatidylcholine (l-DPPC), (b) 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3-
phosphocholine (l-ester-amide-PC), (c) 1-O-hexadecyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine (l-ether-amide-PC), (d)
1-O-hexadecyl-2-palmitoylamino-2-deoxy-sn-glycero-3-phosphoethanolamine (l-ether-amide-PE).
ing of 65 nm (SAXS). Samples were prepared as de-
scribed above (15–20 wt.% of lipid in buffer) and trans-
ferred into glass capillaries (1 mm diameter) (Hilgen-
berg, Malsfeld, Germany). The flame-sealed capillar-
ies were placed in the temperature controlled sample
holder.
35 ◦C) to a lamellar liquid-crystalline phase L␣ with a
spacing of 7.5 nm (at 40 ◦C) between 35 and 40 ◦C. In
the gel phase, the lamellar repeat distance d increases
linearly from 7.8 to 8.3 nm on increasing temperature
(dd/dT = 0.03 nm/K) (Fig. 3). This increase in the gel
phase is much larger than that of DPPC. The temper-
ature dependence of d in the L␣ phase is smaller by
a factor of 2 (dd/dT = 0.014 nm/K). The corresponding
DSC curve is also presented in Fig. 3 as an insert (heat
flux versus temperature). The transition peak does not
show any indication of a pre-transition. The onset of the
transition is at 39 ◦C in good agreement with the SAXS
data. Both the transition temperature and transition en-
thalpy of l-ester-amide-PC are smaller compared with
the main phase transition of DPPC in the same buffer
solution (see Table 1). During cooling, the structure
changes from L␣ back to the gel phase without any
hysteresis.
3. Results
3.1. Amide lipids with a PC head group
The l-ester-amide-PC dispersion in 80% buffer so-
lution (w/w) forms different structures depending on
temperature. A sequence of SAXS patterns around the
main phase transition taken during heating and cooling
scans is presented in Fig. 2a and b. The first and sec-
ond order reflections of a lamellar phase (s1:s2 = 1:2)
can be seen. The full width at half maximum (FWHM)
of the Bragg peaks is much larger in the gel phase
compared with the liquid-crystalline phase indicating
a much better interbilayer ordering above the melt-
ing transition. The SAXS patterns show a transition
from a lamellar gel phase with a spacing of 8.3 nm (at
In comparison with DPPC, the d value in the gel
phase of l-ester-amide-PC is much larger than that of
DPPC (the d value of DPPC is 6.37 nm) (Rappolt et al.,
2000). Additionally, the d values of DPPC do not show
such a large jump at the phase transition. DPPC ex-
hibits a pre-transition at 36.4 ◦C and a main phase tran-

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X. Zhai et al. / Chemistry and Physics of Lipids 133 (2005) 79–88
Table 1
DSC data (main transition temperatures (Tm) and enthalpies (ꢀH))
obtained in buffer dispersions with a concentration of 1 mg/ml
ꢀH (kJ/mol)
Tm (◦C)
DPPC
34.8
26.1
70.3
75.5
42.2
39.7
47.1
61.5
Ester-amide-PC
Ether-amide-PC
Ether-amide-PE
sition at 42.1 ◦C in the buffer solution. The main phase
transition of l-ester-amide-PC occurs at a lower tem-
perature compared with DPPC. The smaller enthalpy
of the chain melting transition indicates that there must
be a different packing (changed interactions between
hydrophobic as well as hydrophilic parts) compared
with DPPC.
Compared with l-ester-amide-PC, l-ether-amide-
PC exhibits an ether bond at the sn-1 position of the
glycerol backbone. The replacement of ester by ether
bonds leads in the case of PCs to a small increase in
the main transition temperature and to a change of the
gel phase structure (Laggner et al., 1987; Maruyama
et al., 1996). The SAXS patterns of l-ether-amide-PC
around the main phase transition taken during heating
and cooling are presented in Fig. 4a and b, respectively.
On heating, the coexistence of two phases with differ-
ent d-values can be seen between 47 and 48 ◦C. Again,
the FWHM of the Bragg peaks is much larger in the gel
phase compared with the liquid-crystalline phase. The
structures observed have not such strong temperature
dependence as in the case of l-ester-amide-PC. The d
spacing increases slightly with increasing temperature
in the gel phase (0.007 nm/K). In this case, the d value
is similar to the one observed for DPPC. Assuming a
comparable hydration of the PC head group, the ob-
served d value could be an indication of tilted chains.
The transition into the L␣ phase is connected with a
drastic increase of the bilayer repeat distance from 6.1
to 7.0 nm, in contrast to the corresponding ester com-
pound. There must be much more water between the
bilayers after the chain melting. The transition occurs
between 47 and 48 ◦C. In the L␣ phase, the d value in-
creases linearly with increasing temperature. The tem-
perature dependence is similarly weak (0.007 nm/K)
(Fig. 5). The cooling process is connected with a pro-
nounced hysteresis in the phase transition (Fig. 5). The
L␣ phase transforms into the lamellar gel phase only
below 40 ◦C. In the metastable region between 47 and
Fig. 2. Small angle X-ray diffraction patterns of l-ester-amide-PC
taken in a temperature scan with 1 ◦C/min: (a) during heating process
at temperatures of 30, 35, 40 and 45 ◦C, (b) during the cooling process
at temperatures of 40, 35 and 30 ◦C.
Fig. 3. Long spacings d of the lamellar gel and lamellar liquid-
crystalline phases (L␣) obtained for l-ester-amide-PC, measured on
heating (᭹) and cooling (ꢀ), as a function of temperature. The cor-
responding DSC curve is shown as a function of temperature.

X. Zhai et al. / Chemistry and Physics of Lipids 133 (2005) 79–88
85
Fig. 5. Long spacings d of the lamellar gel and lamellar liquid-
crystalline phases (L␣) obtained for l-ether-amide-PC, measured on
heating (᭹) and cooling (ꢀ), as a function of temperature. There is
a large hysteresis between heating and cooling. The corresponding
DSC curve is shown as a function of temperature.
The spacing observed for l-ether-amide-PC in the
gel phase is similar to the values for DPPC, but much
smaller than for the l-ester-amide-PC. This indicates
that the bond at the sn-1 position plays an important
role for the packing properties. Obviously, the struc-
tures of the two amide phospholipids are different due
to a different hydration behavior. The amide group in
sn-2 position should affect the intermolecular hydrogen
bonds. This might explain the different repeat distances
of l-ester-amide-PC and DPPC. Due to the different
substitutions in sn-1 position changed molecular inter-
actions could lead to different chain packing structures.
Compared with DPPC and l-ester-amide-PC, the phase
transition temperature of l-ether-amide-PC is therefore
much higher.
Fig. 4. Small angle X-ray diffraction patterns of l-ether-amide-PC
taken in a temperature scan with 1 ◦C/min: (a) during heating process
at temperatures of 45, 46, 47, 48 and 49 ◦C, (b) during the cooling
process at temperatures of 41, 40, 39, 38 and 37 ◦C.
40 ◦C, the d values increase from 6.8 to 7.2 nm. First,
the d value decreases with decreasing temperature and
below 45 ◦C the temperature dependence is opposite
to the observed one in the thermodynamically stable
regions. After reaching the highest value of 7.2 nm, the
system returns back to the gel phase. The d values of
the gel phase reached after cooling are slightly larger
than in the first heating cycle, which could be con-
nected with a slightly changed hydration behavior. The
DSC measurement confirms the main phase transition
temperature of 47.1 ◦C (DSC curve is also presented in
Fig. 5) on heating. Interestingly, the enthalpy value is
very large for a gel-L␣ transition. In contrast to the cor-
responding ester compound, the transition peak shows
a pronounced shoulder on the low-temperature side in-
dicating the existence of a kind of pre-transition, which
could not be seen in the SAXS data.
3.2. Amide lipid with a PE head group
Theheadgroupofl-ether-amide-PEissmallercom-
pared with a PC head group. A sequence of X-ray
diffraction patterns recorded during heating and cool-
ing scans is shown in Fig. 6a and b, respectively. As typ-
ical for PEs, only the first order reflection can be seen.
The d value decreases slightly with increasing temper-
ature both in the gel phase (−0.0043 nm/K) and in the
L␣ phase (−0.0115 nm/K). The repeat distance in the
gel phase amounts to approximately 5.7 nm. The tran-
sition into the liquid-crystalline L␣ phase is connected
with a pronounced decrease to d = 5.4 nm (Fig. 7). The

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X. Zhai et al. / Chemistry and Physics of Lipids 133 (2005) 79–88
Fig. 7. Long spacings d of the lamellar gel and lamellar liquid-
crystalline phases (L␣) obtained for l-ether-amide-PE, measured on
heating (᭹) and cooling (ꢀ), as a function of temperature. The cor-
responding DSC curve is shown as a function of temperature.
ture without any indication of a pre-transition. In com-
parison with l-ether-amide-PC, the transition temper-
ature of the corresponding PE is much higher due to
the changed head group structure.
Taking into account the small PE head group, the
chain packing structure should be much tighter. As-
suming a non-tilted arrangement of the chains in the
bilayers, one comes to the conclusion that there must
be much less water between the bilayers. Solvent
molecules play an important role in structure forma-
tion, phase stability, and intermembrane interactions
of lipid membranes (Kinoshita et al., 1997, 2001).
Fig. 6. Small angle X-ray diffraction patterns of l-ether-amide-PC
taken in a temperature scan with 1 ◦C/min: (a) during heating process
at temperatures of 60, 61, 62, 63, 64 and 65 ◦C, (b) during the cooling
process at temperatures of 65, 64, 63, 62, 61 and 60 ◦C.
phase transition occurs at ca. 61 ◦C similar to DPPE
indicating that the head group structure has a large in-
fluence on the chain melting temperature. The cooling
withascanrateof1 K/mindoesnotleadtoaremarkable
hysteresis, however the transition is broadened over a
temperature range between 64 and 58 ◦C. The d value
showed a gradual increase instead of a jump in a nar-
row region. In order to illustrate the phase transition
more clearly, we present a DSC scan of l-ether-amide-
PE under the same conditions in Fig. 7. The DSC peak
shows a clear pre-transition at 57.3 ◦C and the main
phase transition at 61.4 ◦C. The reason of the observed
pre-transition remains unclear because the X-ray ex-
periments did not show an indication of a change in
the phase structure. DSC measurements performed in
an aqueous solution instead of the used buffer show
only one sharp transition peak at the same tempera-
4. Discussion
The synthesis of compound I was carried out
by a combination of synthesis steps optimized for
the preparation of sn-1,2-diacyl-2-deoxy-2-amino-
glycerophospholipids. After starting from l-serine
methylester-hydrochloride, which was acylated by
fatty acid chloride in chloroform, the remaining hy-
droxy group was protected by tritylation. This way was
more convenient than the protection by the THP group
(ChandrakumarandHajdu, 1981). Forthefollowingre-
duction of the estergroup the use of LiBH4/Ether was
the best choice. In contrast to NaBH4 (Dijkman et al.,
1990) it does not influence the consisting amide group.
The acylation of the resulting hydroxy group by fatty
acyl chloride/4-DMAP and subsequent removal of the
trityl moiety by BF3/methanol gave the sn-1,2-diacyl-

X. Zhai et al. / Chemistry and Physics of Lipids 133 (2005) 79–88
87
2-deoxy-2-amino-sn-glycerol 5. The phosphorylation
of this compound to the final product I was carried out
as already described and discussed in a previous report
(Bartel et al., 2000).
the strong effect of the ethanolamine head group on the
phase behavior.
The DSC results for the amide phospholipids show
that the thermodynamical parameters of the main tran-
sition are different compared with DPPC or DPPE.
This shows the strong influence of the amide group
in sn-2 position. Substitutions in sn-1 position lead in
general to the same changes as observed for DPPC
and DPPE. The replacement of ester by ether bonds
(DPPC–DHPC) increases the main phase transition
temperature (Laggner et al., 1987; Maruyama et al.,
1996). The same behavior can be observed for the in-
vestigated amide phospholipids. However, the effect on
the transition temperature is slightly bigger and on the
transition enthalpy unexpectedly large (see Table 1).
The methylation of the head group going from DPPE
to DPPC reduces the transition temperature drastically.
The same has been observed for the amide phospho-
lipids. Comparing the d-values below and above the
main transition shows that in the case of DPPC the d-
value in the gel phase increases slightly with increasing
temperature, but the temperature dependence is much
smaller(dd/dT = 0.003 nm/K)thanthatobservedforthe
ester-amide-PC. In the case of DPPC, the d-value in the
L␣ phase is larger than in the gel phase. For the ester-
amide-PC the opposite effect was observed. The reason
for the larger d-value in the L␣ phase of DPPC was the
drastically increased water layer between the bilayers.
5. Conclusions
The amide phospholipids, l-ester-amide-PC, l-
ether-amide-PC and l-ether-amide-PE, synthesized
and characterized here can be in future work tested
as substances that modify the interactions between the
secretory PLA2 and supramolecular structures of phos-
pholipids (e.g. bilayers, monolayers). All lipids inves-
tigated exhibit lamellar phases. The l-ester-amide-PC
has a much larger repeat distance than DPPC most
probably due to a much larger water layer thickness,
whereas l-ether-amide-PC shows very similar d-values
both in the gel and liquid-crystalline phases in compar-
ison with DPPC. The amide group in sn-2 position can
form intermolecular hydrogen bonds. The influence of
these bonds on the phase behavior depends on the sub-
stitutions (ester or ether) in the sn-1 position. The dif-
ferent substitutions in sn-1 position change obviously
the molecular interactions and lead to different chain
packingstructures. WhenthePCheadgroupisreplaced
by the smaller PE head group, the chain melting tem-
perature of l-ether-amide-PE is much higher than that
of amide-PCs. The d-values are very similar to those
of DPPE showing that the head group has a very strong
effect on molecular interactions.
ꢁ
Assuming a L␤ phase for the ester-amide-PC the water
layer would have a thickness of 3.5 nm compared with
1.8 nm for DPPC (Marsh and Phil, 1990, CRC press).
Even in the case of a L␤ phase the water layer thickness
amounts to 2.9 nm. This shows that the amide group
leads to a strong increase in the water layer thickness
(hydration behavior). Assuming the same lipid layer
thickness in the L␣ phase, the water layer thickness
between the bilayers is drastically increased: 3.2 nm
for the ester-amide-PC instead of 2.4 nm for DPPC.
The behavior of the ether-amide-PC is more similar to
DPPC. At least the d-values in the gel phase are very
similar and the melting transition leads to an increase.
The d-values in the L␣ phase are slightly larger and
have an opposite temperature dependence. In the case
of the ether-amide-PE, the d-values are very similar
to those of DPPE. In both cases the melting transition
leads to a decrease in the d-values. This demonstrates
Acknowledgements
Authors wish to acknowledge the Chinese Academy
of Sciences as well as the collaboration project of the
German Max Planck Society. We thank HASYLAB at
DESY, Hamburg, for beam time and support and Dr.
S.S. Funari for his valuable help at the soft condensed
matter beamline A2.
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