Polymorphism of N-stearoylethanolamine: Differential scanning calorimetric, vibrational spectroscopic (FTIR), and crystallographic studies

Article abstract of DOI:10.1016/S0009-3084(02)00031-2

Based on a series of physicochemical properties (differential scanning calorimetry, powder X-ray crystallographic studies and Fourier-transform infra red spectroscopic analysis) determined for N-stearoylethanolamine (NSEA) (C18:0) at different temperatures, evidence has been given that this compound can exist in (at least) three polymorphic forms. Powder X-ray crystallography clearly demonstrates the presence of three distinct molecular packings at distinct temperatures while spectral changes in the vibrational spectra reveal that the geometry of the CH₂-CO functional group of the molecule is affected during the polymorphic transitions. Rationalization of the thermal physicochemical behavior of NSEA in terms of molecular packing is also proposed. It supposes rearrangement of the hydrocarbon chains upon heating of the molecule.

Full text of DOI:10.1016/S0009-3084(02)00031-2

Chemistry and Physics of Lipids 119 (2002) 13ꢁ21
/
www.elsevier.com/locate/chemphyslip
Polymorphism of N-stearoylethanolamine: differential
scanning calorimetric, vibrational spectroscopic (FTIR), and
crystallographic studies
J. Wouters a,1, S. Vandevoorde b, C. Culot a, F. Docquir a, D.M. Lambert b,*
a Faculte´ des Sciences, Faculte´s universitaires Notre Dame de la Paix, Rue de Bruxelles, 61, 5000 Namur, Belgium
b Laboratoire de Chimie pharmaceutique et de Radiopharmacie, Ecole de Pharmacie, Faculte´ de Me´decine, Universite´ catholique de
Louvain, Avenue E. Mounier 73, UCL-CMFA 7340, B-1200 Brussels, Belgium
Received 17 September 2001; received in revised form 14 March 2002; accepted 12 April 2002
Abstract
Based on a series of physicochemical properties (differential scanning calorimetry, powder X-ray crystallographic
studies and Fourier-transform infra red spectroscopic analysis) determined for N-stearoylethanolamine (NSEA)
(C18:0) at different temperatures, evidence has been given that this compound can exist in (at least) three polymorphic
forms. Powder X-ray crystallography clearly demonstrates the presence of three distinct molecular packings at distinct
temperatures while spectral changes in the vibrational spectra reveal that the geometry of the CH2Ã
/CO functional
group of the molecule is affected during the polymorphic transitions. Rationalization of the thermal physicochemical
behavior of NSEA in terms of molecular packing is also proposed. It supposes rearrangement of the hydrocarbon
chains upon heating of the molecule. # 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Polymorphism; N-acylethanolamines; Differential scanning calorimetry; Vibrational spectroscopy; Powder diffraction
1. Introduction
variety of structures depending on environmental
conditions as well as on the thermal histories they
have passed through. Such a complexity in struc-
ture is closely related to their functional activities
and physicochemical properties (Hafez and Cullis,
2001). To know the molecular-level structures and
the molecular mechanisms that control the stabi-
lity of particular structures and phase changes is of
fundamental importance in understanding the
physicochemical behavior of such complicated
systems.
Lipids and related organic long-chain com-
pounds such as n-alkanes, n-alcohols, and fatty
acids and their derivatives, appear in a wide
* Corresponding author. Tel.: ꢀ
764-73-63
/
32-2-764-73-47; fax: ꢀ32-2-
/
E-mail address: lambert@cmfa.ucl.ac.be (D.M. Lambert).
1
Present address: Institut de Recherches Microbiologiques
Wiame - Av E Gryzon 1, 1070 Brussels, Belgium.
0009-3084/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 0 9 - 3 0 8 4 ( 0 2 ) 0 0 0 3 1 - 2

14
J. Wouters et al. / Chemistry and Physics of Lipids 119 (2002) 13ꢁ21
/
Among the lipids of interest in molecular
spectroscopic analysis. A putative rationalization
of the thermal physicochemical behavior of that
compound in terms of molecular packing is
proposed.
pharmacology and physiology, N-acylethanola-
mines (NAES) are a class of lipid derivatives
obtained from the condensation between fatty
acids and ethanolamine. Terms of this family
with a fatty acid longer than sixteen carbon atoms
are known in mammals since some decades
but their interest in pharmacology increases with
the discovery of the C20:4 unsaturated homolo-
gous, N-arachidonoylethanolamine (C20:4), better
known as anandamide (Devane et al., 1992).
Anandamide is one of the endogenous ligands of
the cannabinoid receptors (for a review Di Marzo
et al., 1999) and it was recently proposed that it
may act as endogenous ligand of the ligand-gated
calcium channel, the vanilloid receptor VR1 (Zyg-
munt et al., 1999, 2000). Both unsaturated and
saturated fatty acid ethanolamides probably share
common biosynthetic pathways (for a review,
Hansen et al., 2000).
2. Experimental procedures
2.1. Synthesis of N-stearoylethanolamine
NSEA was synthesized in one step by the
addition of stearoyl chloride to a solution of
freshly distilled ethanolamine in excess in dichlor-
omethane at 4 8C. The reaction mixture was
stirred at room temperature and finally washed
with water, 10% citric acid solution and brine. The
organic layer was collected and dried over magne-
sium sulfate. After filtration, the solvent was
removed under reduced pressure and the resulting
powder was recrystallized in ethanolꢁwater.
/
Melting points were determined on a Buchi
¨
N-stearoylethanolamine (NSEA, C18:0, Fig. 1)
has been selected in this study. This molecule is
produced in mammals, significant amounts have
been detected in several tissues (Maccarrone et al.,
2001). Little is known about the pharmacology of
NSEA in mammals (Watanabe et al., 1999; Bueb
et al., 2001) as most of the studies focused the
three major species of NAEs, i.e. N-palmitoyletha-
nolamine, N-oleoylethanolamine and anandamide
(Hansen et al., 2000).
NSEA does neither bind to CB1 nor to CB2
cannabinoid receptors (Lambert and Di Marzo,
1999; Lambert et al., 1999) but is recognized by the
fatty acid amide hydrolase (Jonsson et al., 2001).
In our biological experiments, the solubility of this
saturated fatty acid amide can vary from prepara-
tion to preparation. Therefore, the aim of our
study was to investigate the possible existence of
polymorphism of NSEA, by differential scanning
calorimetry (DSC), powder X-ray crystallographic
studies and Fourier-transform infra red (FTIR)
SMP 20 capillary apparatus and were uncorrected.
The purity of the compound was first checked by
running TLC on aluminum-backed sheets of silica
gel Merck 60F254 using KMnO4 visualization.
Elemental analyses were performed on a Carlo
Erba EA 1108 Analyzer (Carlo-Erba, Milano,
Italy) and are within 90.4% of the theoretical
/
values. 13C NMR were recorded at ambient
temperature on a AC-300 Bruker spectrometer.
The chemical shifts are reported as d (ppm) values
relative to tetramethylsilane. Mass spectra were
recorded on a Varian MAT 44 S (E.I. 70 eV,
source 200 8C), with the grateful help of Professor
G.K.E. Scriba (Iena University, Germany).
NSEA m.p. 100ꢁ
104 8C, MS [Mꢀ] 327, 13C-
NMR (CDCl3) 14.33 (CH3), 23.42, 26.67, 30.06,
30.14, 30.30, 30.44, 32.73, 37.00, 42.68, 61.56
/
(CH2), 176.35 (CÄ
/O), El. Analysis C, H, N for
C20H41NO2.
2.2. Differential scanning calorimetry
Differential scanning calorimetric (DSC) studies
were performed on a Perkin-Elmer DSC-4 differ-
ential scanning calorimeter equipped with a data
station. The calorimeter was calibrated with a
sample of indium (99.99% pure, melting point
Fig. 1. Chemical diagram of NSEA.

J. Wouters et al. / Chemistry and Physics of Lipids 119 (2002) 13ꢁ
/21
15
156.6 8C). Aluminum pans were used for mea-
surements. 1ꢁ3 mg of NSEA were weighted into
interactions analysis, vibrational spectroscopic
spectra and powder diffraction patterns were
performed with the Cerius suite of programs
(MSI San Diego) on an Octane2 Silicon Graphics
Workstation running Irix 6.5.
/
the sample pan, covered with an aluminum lid and
sealed by crimping. Reference pans were prepared
without any sample in them. Heating and cooling
scans were made from room temperature to
120 8C at different scan rates.
3. Results
2.3. Powder X-ray crystallographic studies
3.1. Differential scanning calorimetry
For the X-ray powder diffraction measurements
(PXRD), a Philips PW 1050 diffractometer (Ka
Differential scanning calorigrams for the heat-
˚
Cuꢂ
/
1.54178 A; 2u precisionꢂ
/
0.058) equipped
ing scans of solidꢁfluid phase transitions of NSEA
/
with a temperature control system (Philips PM
2522 A digital VA V meter and Pt probe; Philips,
Eindhoven, The Netherlands) was used. The heat-
are illustrated in Fig. 2.
In a single first heating scan, three peaks are
observed in the differential scanning calorigrams
(Fig. 2, top). The third peak coincides to the
melting point of the compound and therefore this
transition corresponds to the solidꢁliquid phase
transition. This transition occurs at 102.5 8C for
NSEA and confirms the melting point determined
ing (ꢀ20 8C) was performed with a TTK Anton
/
Paar system (Anton Paar, Graz, Austria). The
aluminum sample holder contained about 100 mg
of material. The thermal conditioning was deter-
mined on the basis of the DSC measurements.
Detector scan speed was fixed to 18 2u/min.
/
on a Buchi SMP 20 capillary apparatus. The
¨
observed melting point (III*Liq transition) is in
/
2.4. Fourier-transform infra red spectroscopic
analysis
agreement with earlier reports from the literature
(Lambert et al., 1999; Ramakrishnan et al., 1997).
The two other transitions which occur at tempera-
Infra red measurements were realized on a self-
supported sample wafer prepared by mixing 3 mg
of NSEA with 150 mg of potassium bromide and
pressing with a loading of 5 tons/cm2. The sample
wafer locked in a holder was placed in the infra red
cell, which is made of Pyrex with two CaF2
windows and equipped with a system of oil
circulation. A temperature control circuit had
also been installed. The spectra were recorded
using a Perkin-Elmer Fourier Transform Spectrum
2000 spectrometer. The sample was maintained at
the desired temperature for 15 min for allowing it
to reach an equilibrium before recording the
spectra.
tures below the main melting transitions (T Iꢁ
/
II at
83 8C and T IIꢁIII at 93 8C) correspond to
/
transitions in the solid phase and allow the
identification of (at least) three distinct poly-
morphic forms (termed I, II, and III) for NSAE.
Ramakrishnan (Ramakrishnan et al., 1997) al-
2.5. Packing analysis and molecular modeling
Coordinates of the crystal structure of N(2-
hydroxyethyl)octadecanamide, i.e. NSEA, were
retrieved from the Cambridge Structure Databank
(Allen and Kennard, 1993): reference code
HXEODA. Molecular packing and intermolecular
Fig. 2. Differential scanning calorigrams for the heating scans
of solidꢁfluid phase transitions of NSEA. First heating (top)
/
and second heating (bottom) scans are represented as a function
of the temperature (8C). Positive peaks correspond to en-
dothermic transitions.

16
J. Wouters et al. / Chemistry and Physics of Lipids 119 (2002) 13ꢁ21
/
ready noticed that all NAEs with acyl chains of
14ꢁ20 carbon atoms display two smaller transi-
excluded that some sample impurities were present
in the samples studied by Ramakrishnan et al.
leading to the additional transition. Indeed, in the
case of saturated monoacid triglycerides for
/
tions before the main melting transition in the first
heating scan while N-decanoylethanolamine
(C10:0) and N-lauroylethanolamine (C12:0) dis-
play only one additional transition that is rather
broad. Our data are in complete agreement with
this earlier report.
example, it is known that as little as 2ꢁ3%
/
impurities can markedly affect polymorphism, by
stabilizing some forms in complex mixtures
(Hoerr, 1964).
When the samples are subjected to further cycles
of heating (and cooling) after the first heating and
cooling scan, NSEA gives only a single sharp
transition corresponding to the melting of the
3.2. Powder X-ray crystallographic studies
In order to further approach structural aspects
of the polymorphism of NSEA, powder X-ray
diffractograms were recorded at different tempera-
tures, corresponding to the I (80 8C), II (85 and
90 8C), or III (95 8C) polymorphic forms. Those
diffractograms are compared in Fig. 3.
sample (III*Liq transition). This is contrary to
/
what has been proposed by Ramakrishnan et al.
Indeed this group reported that NEAs having acyl
chain lengths of 16ꢁ20 C-atoms still present a
/
second transition before the main melting transi-
tion. However, in their study, NEAs containing
The diffractograms collected at 80, 85 and
95 8C present different patterns of diffraction in
shorter acyl chains (8ꢁ14 C-atoms) gave only a
/
single sharp transition, as we also observe for
NSEA (C18:0).
the short spacing region (16B
/
2u B268) of the
/
spectra. This region corresponds to short reticular
distances between hydrocarbon chains and/or
polar groups. As will be discussed later in the
text, those distances depend on the subcell and are
directly related to the arrangement of the mole-
cules in the solid state. The differences in the
diffractograms clearly demonstrate the presence of
Different scanning rates (1ꢁ2.5 8C/min) were
/
tested in our study in the hope to detect any other
transition before the melting point. But in all
calorigrams, only one single sharp transition
occurs after the first heating/cooling scan. No
clear explanation is available. It is however not
Fig. 3. X-ray diffraction profile of NSEA in the range of 16B
/
2u B
/
268 (short spacing region of the spectra) at different temperatures.

J. Wouters et al. / Chemistry and Physics of Lipids 119 (2002) 13ꢁ
/21
17
˚
Table 1
X-ray powder diffraction values characteristic of NSEA for
different temperatures
to a longer value of the long spacing (about 44 A)
for this form. No defined diffraction pattern is
observed at 95 8C.
˚
d (A)
Temperature
2u (8)
3.3. Fourier-transform infra red spectroscopic
analysis
80 8C
(Form I)
5.3 (w)a
5.1 (w)
4.3 (s)
16.7
17.5
20.5
22.4
24.3
3.9 (m)
3.6 (m)
Along with X-ray and neutron diffraction
methods, vibrational spectroscopy is a powerful
tool for the investigation of polymorphism (Ka-
bayashi, 1988). In the case of fatty acids and
related compounds having a polar head group and
a chain end, the infra red (IR) series due to
85 8Cꢂ
(Form II)
/90 8C
5.1 (w)
4.5 (m)
4.2 (m)
3.8 (w)
17.5
19.7
20.9
23.3
95 8C
(Form III)
(5.1 (vw))
4.1 (m)
17.5
21.5
methylene wagging modes (1150ꢁ )
1400 cmꢃ1
/
and the methylene rocking-twisting modes (700ꢁ
/
a
1050 cmꢃ1) are the most commonly used. In
particular, the appearance of the well-defined
band series indicates the all-trans conformation
of the alkyl chains.
s, strong; m, medium; w, weak; vw, very weak.
three distinct molecular packings, existing at given
temperatures.
The following high-intensity diffraction peaks
are characteristic to each polymorphic form at:
The FTIR spectra of NSEA between 4000 and
600 cmꢃ1 have been recorded at different tem-
perature: 50, 75, 80, 85, 90, 95, and 102 8C. A
2uꢂ
/
16.78, 17.58, 20.58, 22.48, 24.38 for Form I;
2uꢂ
/
17.58, 19.78, 20.98, 23.38 for Form II; 2uꢂ
/
partial view of those spectra in the 1500ꢁ
cmꢃ1 region is given in Fig. 4.
The 1500ꢁ
1200 cmꢃ1 region, corresponding to
CH2ÃCO vibrations, presents the most marked
/1200
17.58 for Form III. The corresponding reticular
distances are given in Table 1.
Spectra were also recorded in the long spacing
region (2u values under 108) at 80, 85, and 95 8C.
For Form I (80 8C) diffraction peaks are observed
/
/
differences. In particular, a band around 1460
cmꢃ1 appears as a doublet below 80 8C and
becomes a singlet above that temperature. This is
again related to the conformational changes that
occur at the molecular level and gives rise to
distinct polymorphic forms. The spectra of Form
for 2u values of 40.48 (nꢂ
/
1) and 9.38 (nꢂ4)
/
corresponding to a value of long spacing of about
˚
39 A. For Form II (85 8C) peaks are present at
2uꢂ
/
41.68 (nꢂ
/
1) and 15.68 (nꢂ3) corresponding
/
Fig. 4. IR absorption spectra of NSEA recorded at different temperatures in the 1500ꢁ
1200 cmꢃ1 region.
/

18
J. Wouters et al. / Chemistry and Physics of Lipids 119 (2002) 13ꢁ21
/
II (spectra at 85 and 90 8C) more closely resem-
bles the one of Form III than of Form I. This
means that the conformational change is more
important for the Iꢁ
/
II transition than for the IIꢁ
/
III transition, as already suggested by the crystal-
lographic study.
3.4. Molecular packing analysis of NSEA
The coordinates of the crystal structure of
NSEA obtained by single crystal X-ray diffraction
(Dahlen et al., 1977) are available from the Cam-
bridge Structure DataBase (Refcode HXEODA).
N-(2-hydroxyethyl)octadecanamide (NSEA) crys-
tallized in the Pc space group with unit cell
˚
4.886, cꢂ 9.005 A,
dimensions: aꢂ
/
47.588, bꢂ
/
/
bꢂ93.868. There are two molecules in the asym-
/
metric unit leading to four molecules in the
monoclinic unit cell.
The hydrocarbon chain-packing of NSEA be-
Fig. 5. Packing diagrams of NSEA viewed in the ac plan (a)
and schematic representation of the molecular packing (b).
longs to an orthorhombic perpendicular (OÞ
chain packing, meaning that the zig-zag planes of
the different chains are perpendicular (Þ) and
/)
served in the crystallographic structure reported
/
for NSEA. Indeed the closest methylꢁ
/
methyl
contact distances between the opposing layers is
arranged in an orthorhombic subcell. The mole-
cules are organized in a tail-to-tail fashion, similar
to the arrangement expected in a bilayer mem-
brane. A similar arrangement is also found in the
structure of N-myristoylethanolamine (Ramak-
rishnan and Swamy, 1999). The hydrophobic
saturated hydrocarbon chain of NSEA chains
adopts an all-trans zig-zag conformation in the
˚
4.2 A. The distance between the methyl groups of
adjacent molecules in the same layers is about 5.8
˚
A. This is consistent with classical short spacing
values characterizing orthorhombic chain packing
subcells (Hernqvist, 1988).
In the polar heads, strongly connected by a
network of H bonds, the carbonyl O binds the
˚
amide N (2.81 A) while the distance between the
˚
hydroxyl groups is about 2.7 A. This suggests that
the molecular packing deduced from the single
crystal structure corresponds to the Form I poly-
morphic structure. This is reasonable, as it is the
form that is stable at room temperature and that
will first crystallize.
crystalline phase, except for the CÃ
close to the polar group. In particular, torsion
angle C3ÃC4ÃC5ÃC6 is 71.14 and ꢃ67.718 for the
two molecules in the asymmetric unit respectively.
As a consequence, the hydrophobic chains are
tilted with respect to the bilayer as illustrated in
Fig. 5. The methyl ends of the stacked bilayers are
in van der Waals contacts.
/
C bonds very
/
/
/
/
In addition to the van der Waals interactions,
each hydroxyl group forms two hydrogen bonds
with the OH groups of related molecules. These
OH. . .O hydrogen bonds form an extended zig-zag
type network in the crystal packing. There are also
4. Discussion
4.1. Experimental evidence of the existence of
polymorphic structures
strong hydrogen bonds between the amide NÃ
/
H
and carbonyl oxygen of adjacent NSEA molecules.
The short spacing values measured at 80 8C
(Fig. 3, Table 1) correspond to geometries ob-
On the basis of the calorimetric (i.e. chain
melting) properties of NSEA it can be observed

J. Wouters et al. / Chemistry and Physics of Lipids 119 (2002) 13ꢁ
/21
19
that additional endothermic transitions (transi-
tions IꢁII and IIꢁIII, Fig. 2) take place at
temperatures below the main transition (III*Liq
mental data obtained for the different poly-
morphic forms.
/
/
/
The polymorphic form (I), stable at room
temperature, is the one that is observed in the
single crystal structure of NSEA. Short spacing
values measured at 80 8C (Fig. 3 and Table 1)
correspond to interatomic distances in the crystal-
lographic structure reported for NSEA.
transition at 102 8C, Fig. 2). These transitions
must be related to polymorphic crystalline transi-
tion. Similar transitions are observed also for fatty
acids and alkanes (Small, 1986; Marsh and
Swamy, 2000). The transition temperatures there-
fore serve to delineate temperature ranges of
stability for the distinct polymorphic forms:
Form I below 83 8C, Form II between 83 and
93 8C, Form III between 93 and 102 8C. Above
102 8C NSEA melts.
Powder X-ray diffraction patterns (Fig. 3) and
IR vibrational spectra (Fig. 4) have been recorded
for the three polymorphic forms. The spectra of
Form II more closely resembles those of Form III
than Form I. This means that the conformational
The existence of alternative crystal structures is
a characteristic property of long chains com-
pounds (fatty acids, triglycerides,. . .). This is due
to the possibilities for different chain packing
arrangements, and furthermore, for variations in
the tilt of the chains within the layers. In the
particular case of triglycerides, polymorphism is
associated to crystal packing changes from a
perpendicular subcell to a parallel one upon
heating (Culot, 1999). Heating thus allows rear-
rangement of the hydrocarbon chains. A similar
behavior is proposed in the case of NSEA and is
illustrated in Fig. 6. The geometry of the polar
heads of the molecules would not be very much
affected in the beginning of the heating process as
they are held by a network of H bondings. This is
reflected by the presence of fixed short spacing
values common to all three I, II and II poly-
morphic forms in the PXRD patterns. From our
structural interpretation, changes in tilt angles of
the hydrocarbon chains relative to the layer plane
must lead to changes in the small-angle region of
the PXRD patterns (long spacing). Indeed, differ-
change is more important for the Iꢁ
/
II transition
than for the IIꢁIII transition. Those conforma-
/
tional changes directly affect the arrangement of
the molecules in the solid state (subcell) as
reflected by the changes in the short spacing region
(16B
polymorphic transition also deeply affects the
vibrational spectra of NSEA, the 1500ꢁ1200
cmꢃ1 region, corresponding to CH2Ã
CO vibra-
/
2u B
/
268) of the diffractograms (Fig. 3). The
/
/
tions, presenting the most marked differences (Fig.
4). The IR spectral changes induced by the heating
can be interpreted as follows; doublet to single
bands correspond respectively to vibrational
modes in either two distinct or only one plane.
In Form I (B
/
83 8C), the vibration of the CH2Ã
/
˚
ences in values of long spacings (39 and 44 A at 80
CO occurs in two distinct planes, suggesting two
possible arrangements for this form in the crystal
packing. In Form III (spectra at 95 8C), the
vibration at 1460 cmꢃ1 gives rise to a single
band, suggesting that the vibrational mode of the
and 85 8C respectively) are observed between
CH2Ã
/
CO function group lies in only one plane.
4.2. Possible mechanism of polymorphic transitions
Rationalization of the conformational changes
that affect the molecules and their crystal packing
upon heating is proposed in terms of molecular
packing analysis of the crystal structure of NSEA.
Those changes can explain most of the experi-
Fig. 6. Hypothetical molecular arrangements associated to the
different polymorphic forms observed for NSEA.

20
J. Wouters et al. / Chemistry and Physics of Lipids 119 (2002) 13ꢁ21
/
Forms I and II and they confirm a different
longitudinal packing for those two forms.
Most of the rearrangement would affect the
polymorphic forms: Form I below 83 8C, Form II
between 83 and 93 8C, Form III between 93 and
102 8C.
CH2Ã
chain upon heating. This suggestion is in good
agreement with the FTIR data. Indeed, the 1500ꢁ
CO
vibrations presents the most marked differences.
The deeper rearrangement of the hydrocarbon
/
CO function following rotation of the acyl
The diffractograms collected at 80, 85 and
95 8C on powder samples of NSEA are different
and clearly demonstrate the presence of three
distinct molecular packings at distinct tempera-
tures.
The spectra of Form II more closely resembles
the one of Form III than Form I suggesting that
the conformational change is more important for
/
1200 cmꢃ1 region corresponding to CH2Ã
/
chains should be associated to the Form IꢁII
/
transition as the vibrational band around 1460
cmꢃ1 appears as a doublet below 80 8C and
becomes a singlet above that temperature. In
Forms II and III, all lateral hydrocarbon chains
are parallel in the crystal packing and vibrate in a
single plane.
the Iꢁ
/
II transition than for the IIꢁ/III transition.
Spectral changes in the vibrational spectra in the
1500ꢁ
1200 cmꢃ1 region, appear upon heating of
/
NSEA. In particular, a band around 1460 cmꢃ1
appears as a doublet below 80 8C and becomes a
singlet above that temperature. This suggests that
We further propose that Form IIꢁIII transition
/
destabilized the molecular packing leading to a
‘liquid crystalline’ structure for Form III, where
the lateral hydrocarbon chains are free to rotate.
This could explain why the powder diffraction
pattern (Fig. 3 and Table 1) exhibits less lines at
95 8C. At that temperature short spacing values
correspond to mean distances between free rotat-
ing hydrocarbon chains.
Form III thus approaches the liquid state that
would be obtained after further heating above the
melting point. During the whole hypothetical
process, from Form I to the liquid state, entropy
is increased.
the geometry of the CH2Ã
/
CO functional group of
the molecule is affected during the polymorphic
transitions.
A putative rationalization of the thermal physi-
cochemical behavior of NSEA in terms of mole-
cular packing has been proposed. It supposes
rearrangement of the hydrocarbon chains upon
heating of the molecule. This hypothesis is based
on an in depth analysis of the crystal packing of
the compound obtained by single crystal crystal-
lography. It is in agreement with the experimental
data obtained for NSEA and incorporates obser-
vations deduced for related long chains com-
pounds (fatty acids, triglycerides,. . .).
5. Conclusions
Acknowledgements
Based on a series of physicochemical properties
(DSC, powder X-ray crystallographic studies and
FTIR spectroscopic analysis) determined for
NSEA (C18:0) at different temperatures, evidence
has been given that this compound can exist under
(at least) three polymorphic forms.
On the basis of the calorimetric (i.e. chain
melting) properties it can be concluded that
additional endothermic transitions take place at
temperatures below the main transition (102 8C)
and that these transitions must be related to
polymorphic crystalline transition. The observed
transition temperatures therefore serve to delineate
temperature ranges of stability for the distinct
Authors acknowledge Professor Bao Lian Su for
access to FTIR spectroscopy facilities and exper-
tise. Part of this work received financial support
from the DGTRE (Region Wallonne) by First
Spin off Project 001/4602.
References
Allen, F.H., Kennard, O., 1993. Systematic analysis of struc-
tural data as a research technique in organic chemistry. Acc.
Chem. Res. 6, 146ꢁ153.
/

J. Wouters et al. / Chemistry and Physics of Lipids 119 (2002) 13ꢁ
/21
21
Bueb, J.L., Lambert, D.M., Tshirhart, E.J., 2001. Receptor-
independent effects of natural cannabinoids in rat peritoneal
Lambert, D.M., DiPaolo, F.G., Sonveaux, P., Kanyonyo,
M.R., Govaerts, S.J., Hermans, E., Bueb, J.L., Delzenne,
N.M., Tschirhart, E.J., 1999. Analogues and homologues of
mast cells in vitro. Biochem. Biophys. Acta 1538, 252ꢁ
Culot, C., 1999. Molecular modeling of triglycerides poly-
morphism. Recent Res. Devel. Oil Chem. 3, 107ꢁ139.
/
259.
N-palmitoylethanolamide,
agonist: synthesis and binding potency to the rat spleen
cannabinoid receptors. Biochem. Biophys. Acta 1440, 266ꢁ
a putative CB2 endogenous
/
/
Dahlen, B., Pascher, I., Sundell, S., 1977. The crystal structure
274.
of N(2-hydroxyethyl)octadecanamide. Acta Chem. Scand.
Maccarrone, M., Attina, M., Cartoni, A., Bari, M., Finazzi-
Agro, A., 2001. Gas chromatography-mass spectrometry
analysis of endogenous cannabinoids in healthy and
tumoral human brain and human cells in culture. J.
Ser. A 31, 313ꢁ320.
/
Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Steven-
son, L.A., Griffin, G., Gibson, D., Mandelbaum, A.,
Etinger, A., Mechoulam, R., 1992. Isolation and structure
Neurochem. 76, 594ꢁ601.
/
of
a
brain constituent that binds to the cannabinoid
1949.
Marsh, D., Swamy, M.J., 2000. Derivatised lipids in mem-
branes. Physicochemical aspects of N-biotinyl phosphati-
dylethanolamines, N-acyl phosphatidylethanolamines and
receptor. Science 258, 1946ꢁ
/
Di Marzo, V., Bisogno, T., De Petrocellis, L., Melck, D.,
Martin, B.R., 1999. Cannabimimetic fatty acid derivatives:
the anandamide family and other endocannabinoids. Curr.
N-acyl ethanolamines. Chem. Phys. Lipids 105, 43ꢁ69.
/
Ramakrishnan, M., Sheeba, V., Komath, S.S., Swamy, M.J.,
1997. Differential scanning calorimetric studies on the
thermotropic phase transitions of dry and hydrated forms
of N-acylethanolamines of even chain lengths. Biochim.
Med. Chem. 6, 721ꢁ
Hafez, I.M., Cullis, P.R., 2001. Roles of lipid polymorphism in
intracellular delivery. Adv. Drug Delivery Rev. 47, 139ꢁ
148.
Hansen, H.S., Moesgaard, B., Hansen, H.H., Petersen, G.,
/
744.
/
Biophys. Acta (Biomembranes) 1329, 302ꢁ310 (Corrigen-
/
dum appears in 1998 Biochim. Biophys. Acta (Biomem-
branes) 1371, 382).
2000. N-acylethanolamines and precursor phospholipids*
/
relation to cell injury. Chem. Phys. Lipids 108, 135ꢁ150.
/
Ramakrishnan, M., Swamy, M.J., 1999. Molecular packing and
intermolecular interactions in N-acylethanolamines: crystal
structure of N-myristoylethanolamine. Biochim. Biophys.
Hernqvist, L., 1988. Crystal structures of fats and fatty acids.
In: Garti, N., Sato, K. (Eds.), Crystallization and Poly-
morphism of Fats and Fatty Acids. Marcel Dekker, Inc,
Acta 1418, 261ꢁ267.
/
New York, pp. 97ꢁ137.
/
Small, D.M., 1986. The physical chemistry of lipids: from
alkanes to phospholipids. In: Small, D.M. (Ed.), Handbook
Hoerr, C.W., 1964. X-ray diffraction of fats. J. Am. Oil Chem.
Soc. 41 (Suppl. 7), 4.
of Lipid Research. Plenium Press, New York, pp. 245ꢁ392.
/
Jonsson, K.O., Vandevoorde, S., Lambert, D.M., Tiger, G.,
Fowler, C.F., 2001. Effects of homologues and analogues of
palmitoylethanolamide upon the inactivation of the endo-
Watanabe, K., Matsunaga, T., Nakamura, S., Kimura, T., Ho,
I.K., Yoshimura, H., Yamamoto, I., 1999. Pharmacological
effects in mice of anandamide and its related fatty acid
ethanolamides, and enhancement of cataleptogenic effect of
anandamide by phenylmethylsulfonyl fluoride. Biol. Pharm.
cannabinoid anandamide. Br. J. Pharmacol. 133, 1263ꢁ
/
1275.
Kabayashi, M., 1988. Vibrational spectroscopic aspects of
polymorphism and phase transitions of fats and fatty acids.
In: Garti, N., Sato, K. (Eds.), Crystallization and Poly-
morphism of Fats and Fatty Acids. Marcel Dekker, Inc,
Bull. 22, 366ꢁ
Zygmunt, P.M., Petersson, J., Andersson, D.A., Chuang, H.,
Sørga˚rd, M., Di Marzo, V., Julius, D., Hogestatt, E.D.,
/
370.
¨
¨
1999. Vanilloid receptors on sensory nerves mediate the
vasodilator action of anandamide. Nature 400, 452ꢁ457.
Zygmunt, P.M., Julius, D., Di Marzo, V., Hogestatt, E.D.,
New York, pp. 139ꢁ187.
/
/
Lambert, D.M., Di Marzo, V., 1999. The palmitoylethanola-
¨
¨
mide and oleamide enigmas: are these two fatty acid amides
2000. Anandamide*/the other side of the coin. Trends
cannabimimetic? Curr. Med. Chem. 6, 739ꢁ
/
755.
Pharmacol. Sci. 212, 43ꢁ
/44.