Structure and thermotropic phase behavior of a homologous series of bioactive N-acyldopamines

Article abstract of DOI:10.1021/jp402750m

N-Acyldopamines (NADAs), which are present in mammalian nervous tissues, exhibit interesting biological and pharmacological properties. In the present study, a homologous series of NADAs with varying acyl chains (n = 12-20) have been synthesized and characterized. Differential scanning calorimetric studies show that in the dry state the transition temperatures, enthalpies, and entropies of NADAs exhibit odd-even alternation with the values corresponding to the even chain length series being slightly higher. Both even and odd chain length NADAs display a linear dependence of the transition enthalpies and entropies on the chain length. However, odd-even alternation was not observed in the calorimetric properties upon hydration, although the transition enthalpies and entropies exhibit linear dependence. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-Lauroyldopamine (NLDA) crystallized in the monoclinic space group C2/c with eight symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that NLDA molecules are organized in the bilayer form, with a head-to-head (and tail-to-tail) arrangement of the molecules. Water-mediated hydrogen bonds between the hydroxyl groups of the dopamine moieties of opposing layers and N-H···O hydrogen bonds between the amide groups of adjacent molecules in the same layer stabilize the crystal packing. These results provide a thermodynamic and structural basis for investigating the interaction of NADAs with other membrane lipids, which are expected to provide clues to understand how they function in vivo, e.g., as signaling molecules in the modulation of pain.

Full text of DOI:10.1021/jp402750m

Article
pubs.acs.org/JPCB
Structure and Thermotropic Phase Behavior of a Homologous Series
of Bioactive N‑Acyldopamines
S. Thirupathi Reddy,† Pradip K. Tarafdar,† Ravi Kanth Kamlekar, and Musti J. Swamy*
School of Chemistry, University of Hyderabad, Hyderabad 500 046, India
S
* Supporting Information
ABSTRACT: N-Acyldopamines (NADAs), which are present in mammalian
nervous tissues, exhibit interesting biological and pharmacological properties.
In the present study, a homologous series of NADAs with varying acyl chains
(n = 12−20) have been synthesized and characterized. Differential scanning
calorimetric studies show that in the dry state the transition temperatures,
enthalpies, and entropies of NADAs exhibit odd−even alternation with the
values corresponding to the even chain length series being slightly higher.
Both even and odd chain length NADAs display a linear dependence of the
transition enthalpies and entropies on the chain length. However, odd−even
alternation was not observed in the calorimetric properties upon hydration,
although the transition enthalpies and entropies exhibit linear dependence.
Linear least-squares analyses yielded incremental values contributed by each
methylene group to the transition enthalpy and entropy and the
corresponding end contributions. N-Lauroyldopamine (NLDA) crystallized in the monoclinic space group C2/c with eight
symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that NLDA molecules are organized in
the bilayer form, with a head-to-head (and tail-to-tail) arrangement of the molecules. Water-mediated hydrogen bonds between
the hydroxyl groups of the dopamine moieties of opposing layers and N−H···O hydrogen bonds between the amide groups of
adjacent molecules in the same layer stabilize the crystal packing. These results provide a thermodynamic and structural basis for
investigating the interaction of NADAs with other membrane lipids, which are expected to provide clues to understand how they
function in vivo, e.g., as signaling molecules in the modulation of pain.
immune system.6 Another important class of lipid molecules are
N-acylethanolamines (NAEs), which have been studied for
INTRODUCTION
■
Lipids are simple amphiphilic molecules which generally do not
possess complex structures like proteins and nucleic acids,
although in the presence of excess water they form a variety of
supramolecular assemblies depending on their structure and
other factors such as ionic strength, pH, and temperature.
Among the various aggregated structures adopted by hydrated
lipid dispersions, the bilayer assembly is found in the
membranes of cells and organelles. However, for long lipids
have been considered to be dull molecules compared to other
biomolecules such as proteins that catalyze biochemical
reactions and nucleic acids that carry genetic information
necessary for producing proteins. This traditional view of lipids
several decades due to their putative role in combating stress
and interesting biological and medicinal properties.7−9
Among the NAANs, amides of long chain fatty acids with
dopamineN-acyldopamines (NADAs)are a growing group
of neuroactive lipids (neurolipins). Recent studies have
revealed that NADAs with different fatty acyl chains serve as
signaling molecules and play a major role in the modulation of
pain. For example, N-arachidonyldopamine (NArDA) activates
both human and rat vanilloid receptor subtype1 (TRPV1) with
an efficiency equivalent to that of capsaicin.10 It also acts as a
cannabinoid receptor1 (CB1) agonist and induces hypo-
thermia, hypolocomotion, catalepsy, and analgesia in
has changed significantly over the past two decades or so, and
mice.11,12 Apart from the agonist activity toward CB1 and
their role as second messengers in cell signaling, cell function,
and health has been emphasized.1,2 Recently, lipid metabolites
TRPV1 receptors, NArDA also activates a redox-sensitive p38
MAPK pathway that stabilizes COX-2 mRNA, resulting in the
and pathways strategy (LIPID MAPS) initiative in lipidomics
accumulation of the COX-2 protein.13 Analysis of the bovine
has been taken up to identify and quantitate the major and
minor lipid species in mammalian cells.3−5 The goal of this is to
striatal extract indicated the existence of other NADAs, such as
N-palmitoyldopamine (NPDA), N-stearoyldopamine (NSDA),
and N-oleoyldopamine (OLDA) besides NArDA, which was
quantitate the changes in various lipid species in response to
perturbation and to characterize their biochemical and
biophysical roles. Among the various classes of lipids, N-acyl
conjugates of amino acids and neurotransmitters (NAANs)
have attracted considerable attention in recent years because of
their potential roles in the nervous system, vasculature, and the
Received: March 20, 2013
Revised: June 20, 2013
Published: June 24, 2013
© 2013 American Chemical Society
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Scheme 1. Synthesis of N-Acyldopamines
isolated earlier from the same tissue.14 OLDA is a capsaicin-like
lipid with agonist activity for TRPV1 but devoid of affinity to
CB1 receptors. OLDA is also involved in cellular Ca2+
trafficking and behavioral alternations.14 The naturally occur-
ring saturated N-acyldopamines such as NPDA and NSDA did
not activate TRPV1, although they significantly enhanced the
TRPV1-mediated effects of NArDA, called the entourage
effect.15 However, recent findings on the agonist activity of
N-octanoyldopamine suggest that TRPV1 activation might be a
functional property of the whole class of N-acyldopamine
derivatives.16 Interestingly, NPDA, OLDA, and NArDA were
found to inhibit IgE-mediated allergic response in RBL-2H3
cells,17 suggesting potentially therapeutic/preventive applica-
tion for these compounds. In addition, OLDA was found to
cross the blood−brain barrier (BBB) and therefore may serve
as a prodrug of dopamine.18
The above observations suggest that NADAs may represent a
new source of biochemically and pharmacologically important
compounds. Although the structure−activity relationships of
NADAs have not been evaluated in detail, there are possibilities
that NADAs may disrupt lipid packing at the target protein−
lipid interface.6 While the dopamine moiety in the headgroup
may provide specificity, the lipid tail can insert into cell
membranes and alter the membrane dynamics, which could
affect the functional properties of membrane proteins (e.g.,
CB1, TRPV1). In order to understand how NADAs interact
with other membrane lipids and perturb membrane structure
and dynamics, it is important to investigate the physical
properties, phase behavior, and interaction of these compounds
with other membrane components such as phospholipids. As
the first step in this direction, we have performed differential
scanning calorimetric studies on a homologous series of
NADAs with saturated acyl chains in order to characterize
the phase transitions of dry and hydrated forms of NADAs, and
the results obtained are reported here. In addition, we have
determined the 3-dimensional structure of NLDA, which will
be helpful in understanding the physical properties of NADAs
and to establish structure−activity relationship for them.
Synthesis and Characterization of N-Acyldopamines.
N-Acyldopamines (NADAs) were synthesized by a slight
modification of a previous procedure11 involving a simple
condensation between fatty acid and dopamine hydrochloride.
Briefly, to a solution of fatty acid (1.2 equiv) in acetonitrile kept
at 4 °C, triethylamine (1.5 equiv) and isobutyl chloroformate
(1.2 equiv) were added. The mixture was stirred for an hour
under nitrogen atmosphere and diluted with water, and the
mixed anhydride was extracted with ethyl acetate. The mixed
anhydride was allowed to react with a solution of dopamine
hydrochloride (1.3 equiv) in dimethylformamide (DMF)
containing triethylamine (1.5 equiv) overnight at 0 °C under
inert atmosphere. After completion of the reaction, dichloro-
methane was added to the solution followed by water, and the
sample was mixed vigorously to remove DMF. The organic
layer was washed 5−6 times with distilled water to completely
remove DMF, dried over anhydrous sodium sulfate, and
concentrated. The crude product obtained was purified on a
silica gel column by using increasing concentrations of ethyl
acetate in hexane and recrystallized from a mixture of
acetonitrile and acetone at −20 °C. Overall yields ranged
between 60 and 65%. The NADAs obtained were characterized
by TLC, melting point, FTIR, 1H NMR and 13C NMR
spectroscopy, and high-resolution mass spectrometry. IR
spectra (KBr pellet) were recorded on a Jasco FTIR 5300
spectrometer. 1H NMR spectra were obtained at room
temperature in CDCl3 (solvent) on a Bruker Avance NMR
spectrometer operating at 400 MHz, whereas 13C NMR spectra
were recorded at room temperature in CD3OD at 100 MHz on
the same spectrometer. Mass spectra of NADAs were obtained
in the positive ion mode on a Bruker Maxis high-resolution
mass spectrometer, equipped with an electrospray ionization
system.
Capillary melting points of the compounds were recorded on
a Superfit (Mumbai, India) melting point apparatus. Briefly,
solid NADA samples were packed in glass capillaries with one
end sealed, and the melting of the compound was monitored
visually by means of a magnifying lens which was built into the
apparatus. Temperature was measured to 0.1 degree accuracy
with a calibrated thermometer.
Differential Scanning Calorimetry. DSC studies on solid
NADAs were carried out on a PerkinElmer PYRIS Diamond
differential scanning calorimeter. About 1−2 mg of dry
NADAsobtained by recrystallization as described above
was weighed accurately into aluminum sample pans, covered
with an aluminum lid, and sealed by crimping. Reference pans
MATERIALS AND METHODS
■
Materials. Fatty acids and dopamine hydrochloride were
obtained from Sigma (St. Louis, MO), and triethylamine was
obtained from Merck, Germany. Isobutyl chloroformate was
purchased from Lancaster (Eastgate, England). All solvents
were purchased from Sisco Research Laboratory (Mumbai,
India) and were distilled and dried prior to use.
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were prepared similarly, but without any sample in them.
Heating and cooling scans were performed from room
temperature (ca. 25 °C) to about 120 °C at a scan rate of
1.5°/min, and each sample was subjected to three heating scans
and two cooling scans. Transition enthalpies were determined
by integrating the area under the transition curve, and transition
entropies were calculated from the transition enthalpies
assuming a first-order transition according to the equation19,20
(amide-I) and 1556−1548 cm−1 (amide-II). The amide N−H
stretching band was seen at 3308-3296 cm−1, and the O−H
stretching of the catechol moiety was observed around 3470−
3445 cm−1, whereas the C−N stretching band was seen at
1118−1111 cm−1. The stretching, bending, and rocking modes
of the polymythelene portion of the hydrophobic acyl chains
were seen at 2921−2849, 1466−1456, and 719 cm−1,
respectively. The aromatic C−H stretching bands were seen
at 3089−3077 cm−1, and bending vibrations (from ortho and
meta protons) were found around 866−862 and 819−815
cm−1. The FTIR data for all the NADAs are summarized in
Table S1. The 1H NMR spectra of NADAs showed resonances
at 2.17−2.19 δ (2H, t) and 1.58−1.61 δ (2H, m) for the
methylene groups at α and β positions to the carbonyl moiety,
at 5.56−5.82 δ (1H, bs) for the amide N−H, at 3.50−3.57 δ
(2H, q) for the methylene group adjacent to the amide N−H,
and at 2.72−2.74 δ (2H, t) for the methylene group connected
to the aromatic ring. The two hydroxy protons gave broad
singlets at 7.4−8.0 δ and 5.61−6.32 δ, whereas the aromatic
protons gave resonances between 6.58 and 6.84δ. Resonance of
the terminal methyl group was seen at 0.88−0.92 δ (3H, t),
whereas the resonance of the polymethylene group was
observed at 1.24−1.27 δ (16−32H, m). These data are
summarized in Table S2.
ΔHt = TΔSt
(1)
t
where Tt is the transition temperature and ΔHt values taken at
this temperature were used to calculate the corresponding ΔSt
values.
DSC studies of hydrated NADAs were carried out on a VP-
DSC microcalorimeter from MicroCal (Northampton, MA).
Samples of NADAs (3−4 mg) were weighed accurately in
aluminum pans and transferred into clean, dried glass test
tubes. Each sample was dissolved in dichloromethane/
methanol (2:1; v/v) mixture, and a thin film of the lipid was
obtained by blowing a gentle stream of dry nitrogen gas over
the solution. Final traces of the solvent were removed by
vacuum desiccation for at least 3 h. Then 20 mM sodium
phosphate buffer, pH 7.4 was added to the thin film, and the
sample was subjected to several cycles of freeze−thawing with
intermittent vortexing in order to obtain a homogeneous
suspension, which was used for DSC studies. Each sample was
subjected to three heating scans and two cooling scans between
ca. 10 and 110 °C at a scan rate of 1°/min.
Crystallization, X-ray Diffraction, and Structure Sol-
ution. Thin plate type, colorless crystals of N-lauroyldopamine
(NLDA) were grown at room temperature from acetonitrile
containing a trace of acetone. X-ray measurements were carried
out at room temperature (ca. 25 °C) on a Bruker SMART
APEX CCD area detector system using a graphite mono-
chromator and Mo Kα (λ = 0.717 073 Å) radiation obtained
from a fine-focus sealed tube. The minimum resolution of X-ray
diffraction measurements was 0.84 Å. Data reduction was done
using Bruker SAINTPLUS program. Absorption correction was
applied using SADABS program, and refinement was done
using SHEXTL program.21 Structure refinement was carried
out using 1172 [>2σ(Fo)] observed reflections and converged
into a final R1 = 0.1777 and wR2 = 0.4383, with goodness of fit
= 1.597.
A representative 13C NMR spectrum of N-lauroyldopamine
is given in Figure S3. The spectrum shows a resonance at 13.04
δ corresponding to the terminal methyl group of the acyl chain.
Resonances corresponding to the methylene groups are seen at
22.33, 25.68, 29.33, 31.67, 34.57, 35.78, and 40.81 δ as well as
between 28.87 and 29.34 δ (five peaks with the peak at 29.34
being more intense than the others) and six resonances
between 114.92 and 144.86 (corresponding to the aromatic
carbons) and a peak at 174.87 δ (carbonyl carbon). These
values are consistent with the structure of N-lauroyldopamine.
All other NADAs gave similar spectra, except that the intensity
of the peak at ∼29.34 δ increased with increase in the acyl chain
length. The 13C spectral data for all the NADAs are given in
Table S3.
A high-resolution ESI mass spectrum of NLDA is shown in
Figure S4. The most intense peak seen at 336.2536 matches
well with the molecular ion of the compound ([M + H]+, calcd
mass 336.2538). In addition, prominent peaks are also seen at
671.4982 ([M2 + H]+, calcd mass 671.4998) and 1006.7397
([M3 + H]+, calcd mass 1006.7458), which correspond to
proton adducts of dimer and trimer of NLDA. In addition,
sodium ion adducts of M, M2, and M3 are also seen in the
spectrum of NLDA. For both proton adducts and sodium ion
adducts, the intensity of the peaks decreases with increasing size
of the adduct. In each case, the proton adduct is more intense
as compared to the sodium ion adduct. Other NADAs also
yielded essentially similar mass spectra. These data are given in
Table S4.
Crystal Parameters of N-Lauroyldopamine. Molecular
formula, C20H35NO4; molecular weight, 353.49; crystal system,
monoclinic; space group (Sg), C2/c; unit cell dimensions (with
standard deviations in parentheses): a = 77.42(6) Å, b =
4.900(2) Å, c = 10.919(6) Å, α = 90°, β = 90.966(11)°, γ = 90°;
volume of the unit cell, V = 4142(4) Å3; number of molecules
in the unit cell, Z = 8.
RESULTS
■
1
The above results from FTIR, H and 13C NMR spectros-
Synthesis and Characterization of N-Acyldopamines.
A homologous series of N-acyldopamines (NADAs) with
saturated acyl chains containing 12−20 C atoms have been
synthesized in the present study by a simple condensation of
the fatty acids with dopamine hydrochloride. The structure and
copy, and ESI mass spectrometry are fully consistent with the
structures of N-acyldopamines and indicate that they are of
high purity.
Differential Scanning Calorimetry. Heating thermo-
grams corresponding to dry NADAs of even acyl chain lengths
are presented in Figure 1A, and those corresponding to the odd
chain length series are shown in Figure 1B. The thermograms
show that each NADA exhibits a major endothermic transition,
which corresponds to the capillary melting point of the
compound. In some cases, particularly for NADAs with shorter
1
purity of NADAs were characterized by TLC and FTIR, H
NMR and 13C NMR spectroscopy, and ESI mass spectrometry.
Representative FTIR and 1H NMR spectra of N-palmitoyldop-
amine are given in Figure S1 and Figure S2, respectively. FTIR
spectra (KBr pellet) of recrystallized NADAs showed
absorption bands due to amide linkage at 1641−1637 cm−1
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Figure 1. DSC heating thermograms of dry N-acyldopamines. (A)
Thermograms of NADAs with even number of C atoms in the acyl
chains. (B) Thermograms of NADAs with odd number of C atoms in
the acyl chains. The number of carbon atoms in the acyl chain is
indicated against each thermogram.
Figure 2. DSC heating thermograms of hydrated NADAs. (A)
Thermograms of NADAs with even number of C atoms in the acyl
chains. (B) Thermograms of NADAs with odd number of C atoms in
the acyl chains. The number of carbon atoms in the acyl chain is
indicated against each thermogram.
entropies, ΔSt, were calculated from transition enthalpies using
eq 1. The values obtained are listed in Table 1.
acyl chains, an additional minor transition was observed. These
minor transitions suggest the possibility of polymorphism in
NADAs. When the samples were subjected to a second heating
scan, in most cases the thermograms became more complex,
involving multiple transitions (Figure S5), possibly due to
partial degradation of the samples when subjected to heating to
high temperatures. Therefore, in all the cases only the first
heating scan was considered for further analysis, i.e., to estimate
transition temperatures, enthalpies, and entropies, and the
values obtained are presented in Table 1.
DSC studies were also carried out on NADAs upon
hydration with 20 mM phosphate buffer (pH = 7.4). Each
NADA gave a single, sharp phase transition in the hydrated
state and the transitions were reversible, although enthalpy of
the transition decreased slightly when the sample was subjected
to several cycles of heating and cooling. Thermograms from the
first heating scans for NADAs with even and odd acyl chains are
shown in Figures 2A and 2B, respectively. The phase transition
temperatures, Tt, were estimated from the peak positions of the
DSC endotherms, whereas the transition enthalpies, ΔHt, were
obtained by integrating the area under the curve after buffer
subtraction, normalization, and baseline correction. Transition
Chain Length Dependence of Transition Enthalpy and
Transition Entropy. Chain length dependences of transition
enthalpy and transition entropy for the chain-melting phase
transitions of dry NADAs are given in Figures 3A and 3B,
respectively. In both cases, NADAs with even and odd acyl
chains independently exhibit linear dependence of ΔHt and ΔSt
on the chain length. However, when the data obtained with
even and odd acyl chain lengths are viewed together, a zigzag
pattern is seen with the values of enthalpy and entropy for the
odd acyl chain lengths being slightly lower than those of even
acyl chain lengths. That is, the calorimetric data exhibit odd−
even alternation, and this aspect will be discussed in more detail
below. The transition enthalpies and transition entropies of dry
NADAs for even and odd acyl chain lengths could be fit well to
expressions 2 and 3:22
ΔHt = ΔH0 + (n − 2)ΔHinc
(2)
ΔSt = ΔS0 + (n − 2)ΔSinc
(3)
where n is the number of C atoms in the acyl chain and ΔH0
and ΔS0 are the end contributions to the ΔHt and ΔSt,
Table 1. Transition Temperatures (Tt), Transition Enthalpies (ΔHt), and Transition Entropies (ΔSt) of N-Acyldopamines in
Dry and Hydrated States (Average Values of Three Measurements with Standard Deviations Are Given)
dry NADAs
hydrated NADAs
acyl chain length
Tt (°C)
ΔHt (kcal/mol)
ΔSt (cal/mol/K)
Tt (°C)
ΔHt (kcal/mol)
ΔSt (cal/mol/K)
12
13
14
15
16
17
18
19
20
78.0 0.1
78.6 0.1
87.0 0.1
88.1 0.1
94.5 0.1
94.0 0.2
99.2 0.1
97.8 0.1
100.9 0.1
10.27 0.03
9.10 0.10
12.70 0.01
12.50 0.22
14.49 0.31
14.03 0.02
16.01 0.17
15.31 0.01
16.50 0.19
29.3 0.1
28.4 0.3
35.3 0.1
34.6 0.6
39.4 0.8
38.2 0.1
43.0 0.5
41.3 0.1
44.1 0.5
73.9 0.1
78.7 0.1
82.5 0.1
85.8 0.1
88.7 0.1
91.1 0.1
93.3 0.2
94.8 0.1
96.1 0.1
3.60 0.14
5.35 0.21
6.40 0.28
7.35 0.63
9.40 0.91
10.65 0.07
12.60 0.85
13.65 0.92
14.75 0.35
10.4 0.4
15.2 0.6
18.0 0.8
20.5 1.8
26.0 2.5
29.3 0.2
34.4 2.3
37.1 2.5
40.0 1.0
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Figure 4. Chain length dependence of transition enthalpies (A) and
transition entropies (B) of hydrated NADAs. Values of ΔHt and ΔSt
were plotted against the number of methylene (CH2) units (n − 2) in
the acyl chains. Solid line represents linear least-squares fit of the data.
Figure 3. Chain length dependence of transition enthalpies (A) and
transition entropies (B) of dry NADAs. Values of ΔHt and ΔSt are
plotted against the number of methylene (CH2) units (n − 2) in the
acyl chains. Solid and dotted lines represent linear fits of the data for
even- and odd-chain length series, respectively.
values and end contributions of transition enthalpy and entropy
were obtained. These values are also listed in Table 2.
respectively, arising from the terminal methyl group and polar
region of N-acyldopamine molecule. ΔHinc and ΔSinc are the
average incremental values contributed by each CH2 group to
ΔHt and ΔSt, respectively, and the values of these parameters
obtained from linear least-squares analysis are listed in Table 2.
Chain Length Dependence of Transition Temper-
atures. The transition temperatures of both dry and hydrated
NADAs increase with increasing chain length (Figure 5).
However, in both cases magnitude of the change decreases with
increase in the chain length. Additionally, for the dry NADAs
an alternation is seen between the odd-chain length and even-
chain length series, with the even chain length series exhibiting
slightly higher transition temperatures than odd chain length
Table 2. Incremental Values (ΔHinc, ΔSinc) of Chain Length
Dependence and End Contributions (ΔH0, ΔS0) to Phase
a
Transition Enthalpy and Entropy of N-Acyldopamines
dry NADAs
thermodynamic
parameters
even chain
length
odd chain
length
hydrated NADAs
ΔHinc (kcal/mol)
ΔH0 (kcal/mol)
ΔSinc (cal/mol/K)
ΔS0 (cal/mol/K)
0.95 ( 0.07)
1.01 ( 0.97)
2.27 ( 0.2)
7.22 ( 2.62)
0.87 ( 0.1)
0.73 ( 1.42)
2.11 ( 0.26)
6.10 ( 3.64)
1.42 ( 0.04)
−10.55 ( 0.57)
3.76 ( 0.11)
−26.99 ( 1.55)
a
Average values of transition enthalpy and transition entropy given in
Table 1 have been used for the linear fitting of the data. Errors shown
in parentheses are fitting errors obtained from the linear least-squares
analysis.
Linear chain length dependence of transition enthalpy and
transition entropy observed here indicates that structures of the
NADAs of different even chain lengths are very similar in the
solid state. Similarly, the data suggest that structures of all odd-
chain NADAs are also likely to be very similar. This suggests
that the molecular packing and intermolecular interactions in all
the even chain length NADAs are likely to be very similar, and
determination of the 3-dimentional structure of any one of
them is likely to give a good idea of the molecular packing and
intermolecular interactions present in the crystalline state for
the entire homologous series.
Chain length dependences of transition enthalpy and
transition entropy for the thermotropic phase transitions of
hydrated NADAs are shown in Figures 4A and 4B, respectively.
Interestingly, for the hydrated samples no clear-cut even−odd
alternation is seen, and the values of ΔHt and ΔSt for the entire
homologous series could be fitted satisfactorily to a straight
line. From the linear least-squares analysis, the incremental
Figure 5. Chain length dependence of phase transition temperatures
of NADAs in the dry (A) and hydrated (B) conditions. Solid and
dashed lines in (A) correspond to nonlinear least-squares fits of the
transition temperatures of even-chain length and odd-chain length
series to eq 7. Solid line in (B) corresponds to nonlinear least-squares
fit of the Tt values of all hydrated NADAs to eq 7.
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compounds (Figure 5A), whereas such alternation was not
observed with the hydrated samples (Figure 5B). With the dry
samples, both for even series and odd series, the Tt values
increase in a smooth progression but with decreasing
increments as the chain length is increased. As the acyl chain
length increases, the total contribution from the polymethylene
portion toward the total enthalpy and entropy of the phase
transition will be sufficiently large that the end contributions
are negligible in comparison. At infinite acyl chain length, eqs 2
and 3 can be reduced to22
additional water molecule is also seen, which is found in the
same asymmetric unit. The atomic coordinates and equivalent
isotropic displacement parameters for all non-hydrogen atoms
are given in Table S5. The bond distances and bond angles
involving the non-hydrogen atoms are presented in Table S6,
whereas the torsion angles are given in Table S7. These data
show that the hydrocarbon portion (C10−C20) of NLDA
molecule is in an all-trans conformation as all the torsion angles
corresponding to the acyl chain region are in the neighborhood
of 180°. The dihedral angle data presented in Table S5 also
shows that all the carbon atoms of the phenyl ring, the two
oxygen atoms belonging to the catechol moiety, and the C7
carbon atom are in the same plane, which is expected due to the
sp2 hybridization of the ring carbons. The torsion angles of
C1−C7−C8−N1, C7−C8−N1−C9, and C8−N1−C9−-C10
are 171.66°, 129.5°, and 174.9°, respectively. This shows that
geometry of the NLDA molecule is essentially linear until the
C8 atom. The structure of dopamine moiety is similar to that
seen in the crystal structure of dopamine hydrochloride as well
as the MD simulated structure of dopamine bound to its
receptor (dopamine D2 receptor).26,27 Therefore, acylation
does not seem to alter the conformation adopted by the
dopamine moiety in the crystal.
ΔHt = (n − 2)ΔHinc
(4)
ΔSt = (n − 2)ΔSinc
(5)
Then the transition temperature for infinite chain length, Tt∞,
will be given by
Tt∞ = ΔHinc/ΔSinc
(6)
From the data presented in Table 2, the T∞t values for NADAs
of even and odd acyl chain lengths (in the dry state) have been
estimated using eq 6 as 418.5 and 412.3 K, respectively.
Similarly, the T∞t value for the hydrated NADAs was estimated
as 377.7 K.
Molecular Packing and Molecular Area. Packing
diagrams of NLDA viewed down the c-axis and a-axis are
shown in Figures 7A and 7B, respectively. The NLDA
molecules are packed head-to-head (and tail-to-tail) in stacked
bilayers. The O−H···O hydrogen bonds between the head
groups of opposite leaflets mediated by water molecules
stabilize this arrangement (see Figure 8A). In addition, the
amide groups of adjacent molecules are also involved in
hydrogen bonds. The carbonyl oxygen atoms of adjacent
For a number of diacyl lipids as well as single chain
amphiphiles, which exhibit linear dependence of transition
enthalpy and transition entropy on chain length, it has been
shown that the data can be fit to the equation20,23−25
T = ΔHt/ΔSt = Tt∞[1 − (n0 − n′0)/(n − n′0)]
(7)
t
where n0 (= −ΔH0/ΔHinc) and n′0 (= −ΔS0/ΔSinc) are the
values of n at which the transition enthalpy and transition
entropy extrapolate to zero. It can be seen from Figure 5A that
the transition temperatures of NADAs with even number of C
atoms as well as odd-number of C atoms in the acyl chains
independently fit rather well with eq 7. With the hydrated
NADAs the transition temperatures for the entire homologous
series could be satisfactorily fit by nonlinear least-squares
method to eq 7, as seen clearly in Figure 5B. Additionally, the
fitting parameters also yielded the T∞t values for even and odd
NADAs in the dry state as 395.1 7.6 and 390.8 0.9 K and
as 393.7 1.5 K for the hydrated NADAs. These values are in
reasonable agreement with the T∞t values estimated using eq 6.
Description of the Structure. An ORTEP of the crystal
structure of N-lauroyldopamine is shown in Figure 6 along with
the atom numbering for all non-hydrogen atoms. In this plot an
Figure 7. Packing diagrams of NLDA. (A) A view down the c-axis. (B)
A view down the a-axis. Color code: carbon, gray; nitrogen, blue;
oxygen, red.
Figure 6. ORTEP showing the molecular structure of N-lauroyldop-
amine (NLDA).
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Figure 8. (A) Hydrogen-bonding pattern in the crystal lattice of NLDA. A close-up view of the bilayer is shown displaying N−H···O and O−H···O
type hydrogen bonding down the c-axis. The acyl chains are truncated to show an enlarged view of the headgroup region with hydrogen bonds
shown in dashed lines. (B) Packing of the catechol moieties in the crystal lattice. The aromatic rings are laterally displaced, precluding any possibility
of π-stacking interactions. Carbon atoms are shown as open circles, nitrogen atoms are shown in blue, and oxygen atoms are in red.
molecules point in opposite direction, providing appropriate
juxtaposition of the amide carbonyl and N−H moieties of
adjacent molecules to form N−H···OC type hydrogen
bonds. The terminal methyl groups of the acyl chains in
stacked bilayers are in van der Waals contact, with the closest
methyl−methyl distance between opposite layers and same
layer being 3.705 and 4.9 Å, respectively. The bilayer thickness
(C20−C20 distance) for NLDA is 47.96 Å. The layer thickness
(C20−O2 distance) in the crystal structure of NLDA is 22.78
Å, and the all-trans N-acyl chains of the molecule are tilted by
46.16° with respect to the normal to the methyl end plane. This
value is considerably higher than the tilt angles observed in the
crystal structures of several N-acylethanolamines (35°−
37°).9,28−30 The higher tilt angle may arise from the larger
area of the headgroup in NADAs, containing the catechol
moiety. The area per each NLDA molecule in the bilayer plane
is 26.75 Å2, which is higher than the molecular area of different
NAEs (21.95−22.03 Å2)28−30 as well as in diacylethanolamines
with matched chains (21.42−22.13 Å2) whose crystal structures
have been determined earlier.20,31,32 The larger molecular area
and higher tilt angle in NLDA suggest that the acyl chains are
apart from each other.
Hydrogen-Bonding and Intermolecular Interactions.
In order to understand the intermolecular hydrogen-bonding
interactions in the crystal structure of NLDA, the molecular
packing in the crystal was examined from various angles. Figure
8A shows the hydrogen-bonding interactions between NLDA
molecules in the crystal lattice. These include intermolecular
hydrogen bonds between the O−H groups of the catechol
moieties from opposite leaflets mediated via bridging water
molecules as well as H-bonds between amide N−H and
carbonyl oxygen atoms of adjacent NLDA molecules in the
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same side of the bilayer. Each hydroxyl group of the dopamine
moiety forms a water-mediated H-bond with a hydroxyl group
of the dopamine moiety of an NLDA molecule from the
opposing layer. Thus, each NLDA molecule from one leaflet is
connected to an NLDA molecule from the opposite leaflet by
two water-mediated H-bonds. Each water-mediated H-bond
connects the OH group on the C-1 atom of an NLDA molecule
to the OH group on the C-2 atom of an NLDA molecule in the
opposite layer (Figure 8A). While the OH group on the C-1
acts as a hydrogen bond donor, the OH group on the C-2 acts
as a hydrogen bond acceptor, with a water molecule mediating
these interactions. All the O−H···O hydrogen bonds in NLDA
have two types of O···H distances: the O2···H distance is 2.108
Å, whereas the O1···H distance is 2.316 Å. The corresponding
distances between the donor and acceptor oxygen atoms are
2.802 and 2.855 Å, respectively. These hydrogen bonds are
nonlinear with the angle between covalent bond and hydrogen
bond being 142.27° and 121.17°, respectively.
In addition to the hydrogen bonds between the hydroxyl
groups, there are also strong hydrogen bonds between the
amide N−H and carbonyl oxygen atoms of adjacent NLDA
molecules (Figure 8A). The distance between the hydrogen-
bonded carbonyl oxygen and amide hydrogen atom is 2.116 Å,
and the N···O distance is 2.972 Å. The angle between the
covalent and hydrogen bond (N−H···O angle) is 173.23°. The
O−H···O hydrogen bonds form extended networks, which
stabilize the bilayer arrangement in the crystal lattice. The N−
H···O hydrogen bonds connect adjacent NLDA molecules in
the same plane. However, while the N−H group of each NLDA
molecule forms a hydrogen bond with an adjacent molecule in
another unit cell on one side, the carbonyl oxygen atom of the
same NLDA molecule forms a hydrogen bond with the N−H
group of an adjacent NLDA molecule in another unit cell on
the other side.
inside the membrane interior, thus making the amide bond less
accessible to the enzyme. However, there are no reports
regarding the physicochemical properties of NADAs, which can
help to understand the above interesting properties. In the
present study we have investigated the phase behavior of a
homologous series of NADAs bearing saturated fatty acyl
chains and compared their behavior with NAEs, which are also
single-chain amphiphiles that exhibit interesting biological
properties. In addition, we have determined the structure of
NLDA at atomic resolution and analyzed the intermolecular
and packing interactions in the crystal lattice, which will be
helpful in understanding the physical properties of NADAs and
for establishing structure−activity relationships.
Incremental Enthalpy and Entropy. Linear least-squares
analysis of transition enthalpies and entropies of dry as well as
hydrated NADAs yielded ΔH0 and ΔS0 (the end contributions
to transition enthalpy and entropy, respectively, arising from
the terminal methyl group and polar region of N-acyldopamine
molecule) as well as ΔHinc and ΔSinc (the average incremental
contribution of each CH2 group to the transition enthalpy and
entropy). Theoretically, ΔHinc gives a measure of cohesiveness
of the acyl chain packing. It can be seen from Table 2 that
ΔHinc of NADAs in the dry state (for both even and odd acyl
chain lengths) is lower than the ΔHinc in the hydrated state. In
the case of NAEs it was found that ΔHinc of the dry state is
higher than that of the hydrated state.24,25,34 This difference in
the ΔHinc of NADAs and NAEs possibly arises from the
hydrophobic effect and π-stacking of the aromatic rings of the
catechol moiety. Analysis of the packing interactions in the
crystal structure of NLDA shows that the catechol moieties in
adjacent layers are laterally displaced, resulting in a lack of π-
stacking interaction of the aromatic rings in the solid state
(Figure 8B). The end contribution of entropy, ΔS0, is also
more negative for hydrated samples as compared to the dry
state. The negative ΔS0 is also indicative of the hydrophobic
effect playing a role in the packing of NLDA, which could arise
due to the ordering of water molecules around the catechol
moiety of the headgroup.35 Together, these observations
suggest that the hydrophobic effect and possibly π-stacking in
hydrated form bring the NLDA molecules closer, which results
in a more compact packing of the acyl chains. On the other
hand, the acyl chains in NAEswhich do not have any
nonpolar moieties in the headgroup region that can be involved
in hydrophobic/π-stacking interactionsare more tightly
packed in the dry state.28−30 In the solid state of NLDA,
water-mediated hydrogen bonds between two catechol moieties
help them to pack. Each hydrogen bond likely provides an
enthalpic stabilization of 5−6 kcal/mol.36 The enthalpic
stabilization by H-bonding likely puts a geometric constraint
such that π-stacking between catechol moieties would be
difficult to achieve. Also, the acyl chains in the NLDA crystal
are far apart and less tightly packed in the solid state. The
closest and longest methyl−methyl distances in the same layer
of NLDA are 4.9 and 6.2 Å, whereas in NAEs, the closest and
longest methyl−methyl distances in the same layer were 4.88
and 5.4 Å, respectively. Together, these observations further
confirm that the acyl chains are less tightly packed in solid
NLDA. The decrease in the melting transition temperature
upon hydration in NAEs was >15 deg, whereas in NADAs the
decrease is in the neighborhood of ∼5 deg. Altogether, our data
strongly suggest that upon hydration the acyl chains become
more tightly packed in NADAs, and the most likely factors
responsible for this are hydrophobic effect and π-stacking.
DISCUSSION
■
N-Acylated derivatives of amino acids, neurotransmitters, and
ethanolamine constitute an important class of bioactive lipids.
Among these, considerable attention has been focused on N-
acylethanolamines (NAEs) for nearly 5 decades in view of their
putative stress combating role.7−9,25,33 Over the past 15 years
our laboratory has been investigating the structure, phase
behavior, and membrane interactions of NAEs with the
objective of establishing structure−activity relationships for
them.9,25 Recently, N-acyl amino acids and neurotransmitters
(NAANs) have attracted much attention in view of their
biological properties.6 However, structure−activity relation-
ships for these compounds are still largely unexplored. N-
Acyldopamines (NADAs), which are produced by the
condensation of fatty acids with dopamine, possess interesting
biological activity in vivo and in vitro. NADAs with N-
arachidonyl and N-oleoyl chains activate TRPV1 channel
whereas NADAs bearing stearoyl and palmitoyl chains were
found to increase the activity of N-arachidonyl dopamine,15 an
effect termed as entourage effect. It is believed that these
molecules act by direct binding to the receptor or by changing
the physical properties of the membrane.6 The entourage effect
shown by NSDA and NPDA suggests that NADAs alter the
membrane structure. In addition, OLDA has been reported to
cross the BBB with an efficiency that is many times greater than
that of dopamine18 and that it resists hydrolysis by FAAH.11
This retardation in the hydrolysis could be attributed to its
interaction with biomembranes, with the acyl chain embedded
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Odd−Even Chain Alternation in Thermodynamic
Parameters. The data presented in Figures 3 and 5 show
that the phase transition temperatures and thermodynamic
parameters (ΔHt and ΔSt) for dry NADAs exhibit an odd−
even alternation. Similar trends were observed in the transition
temperatures and thermodynamic properties of long-chain
hydrocarbons, fatty acids, N-acylethanolamines and N-, O-
diacyl ethanolamines with matched as well as mixed chains in
the solid state.20,22,25,34 This alternation has been explained on
the basis of packing of hydrocarbon chains. In long-chain fatty
acids, for example, it was shown that differences in the packing
properties between the terminal methyl groups between the
even and odd acyl chain lengths can explain the differences in
the physical properties.22 Such differences do not arise in the
methyl group packing if the chains are perpendicular to the
methyl group plane. However, if the hydrocarbon chains are
tilted with respect to the plane of the methyl groups, their
packing modes can differ, leading to alternation in the physical
properties.22 The hydrocarbon chain tilt seen in the crystal
structure of NLDA is consistent with the alternation in the
thermodynamic parameters between the homologous series of
NADAs containing even and odd acyl chain lengths.
Phase Behavior and Biological Implications. The phase
behavior of amphiphiles is guided by the packing parameter, P
(= v/lca0, where v and lc are the volume and chain length of the
hydrophobic portion, respectively, and a0 is the optimal surface
area occupied by the molecule at the hydrocarbon interface).37
Amphiphiles for which P ≈ 1 favor the formation of a lamellar
phase, with amphiphilic bilayers separated by water layers. The
lamellar phase is the common structure seen in biological
membranes. If P > 1, i.e., amphiphiles with a large effective
chain volume and/or a small headgroup possess a reverse
wedge shape and tend to promote formation of inverse
mesophases. N-Acylethanolamines (NAEs) with small head-
group form lamellar crystalline phase at low temperature,
whereas at high temperature they form lamellar liquid
crystalline phase and at least two isotropic phases.24,38 At
high temperatures, the crystalline chains becomes fluid,
resulting in an effective increased hydrocarbon volume. This
provides a wedge-shaped conformation to small headgroup
NAEs necessary for inverse phases (inverse micelles, inverse
cubic) formation. Addition of an ethanol moiety to NAEs forms
diethanolamide with a relatively large headgroup, which also
has been shown to favor lyotropic lamellar phase formation.39
However, for shorter chain diethanolamides micellar phase is
observed at low temperature also and in excess water.39 The
headgroup area of NADAs is not expected to be too large
because of π-stacking, and they most likely form lamellar
crystalline phase at low temperature and lamellar fluid phase
above transition temperature. However, we do not rule out the
possibility of other nonlamellar phases at high temperatures
and particularly at low hydration. Another popular surfactant,
monoacylglycerides, form a variety of phases in water ranging
from lamellar, inverted hexagonal, bicontinuous cubic phases,
etc.40 Apart from packing parameter, the H-bond plays an
important role in determining the existence of various phases.
For example, N-(2-hydroxydodecyl)-L-alanine, an N-alkyl
amino acid, forms lamellar phase with intermolecular H-bond
interactions,41 whereas in N-alkyl ureas H-bonding results in
high curvature, which favors the formation of inverse micellar
(L2) phase at high temperature.39,42 The π-stacking as well as
H-bonding ability of NADAs makes them an attractive choice
for detailed analysis of binary phase behavior in water.
A knowledge of thermodynamic parameters associated with
the phase transitions of a homologous series of lipids, especially
when they exhibit linear dependence on chain length, allows an
estimation of the end contribution of enthalpy and entropy.
The end contributions give a measure of the enthalpic and
entropic contributions arising from the headgroup moiety to
the overall phase transition enthalpy and entropy.23,32 This was
tested in our earlier report on N,O-diacylethanolamines
(DAEs) of fixed N-acyl chain with different O-acyl chains,
and it was found that the end contributions could be attributed
to N-acyl part of the molecule, with the incremental
contribution coming from polymethylene part of the O-acyl
chain. In the present study, the end contribution of enthalpy,
ΔH0, mainly arises from the dopamine (DA) moiety. It was
observed that upon hydration dopamine moiety gains a
substantial enthalpic stabilization (−10.55 kcal/mol) with the
expense of ordering of water structure (reduction in entropy).
The enthalpic stabilization could arise from hydrophobic, π-
stacking and hydrogen-bonding interactions and provides a
possible explanation as to how dopamine accumulates in a
dense core carotid body.43 This enthalpic stabilization in a
hydrophobic environment also provides further clues on the
interaction of DA with its receptors. It was found that the
receptor binding site of DA comprises of aromatic amino acid
residues, which may help to gain hydrophobic as well as π-
stacking interactions. Apart from the enthalpic stabilization of
dopamine moiety, NADAs were found tightly packed in
aqueous medium, which may explain their ability to cross the
blood−brain barrier and remain sufficiently stable under
hydrolyzing condition. It is possible that such tightly packed
chains in NADAs may be localized in lipid rafts in
biomembranes and may play a role in modulating the activity
of membrane proteins. The entourage effect produced by NPDA
and NSDA20 may arise from the interaction of tightly packed
NADAs with other constituents of biomembranes. Miscibility
studies between NADAs with other lipids, such as phosphati-
dylcholine and phosphatidylethanolamine, would be required
to further investigate their possible role in lipid raft formation.
Such studies are currently underway in our laboratory.
CONCLUSIONS
■
In this study, a homologous series of N-acyldopamineswhich
are biologically relevant and neuroactive lipidshave been
synthesized, and their thermotropic phase transitions were
characterized by differential scanning calorimetry. A linear
dependence was observed in the thermodynamic parameters,
ΔHt and ΔSt, associated with the chain-melting phase
transitions. The 3-dimensional structure of N-lauroyldopamine,
solved by single crystal X-ray diffraction, revealed that the
molecules of this lipid adopt an extended, essentially linear
geometry, with the hydrophobic acyl chains facing each other,
i.e., a head-to-head (and tail-to-tail) arrangement, akin to a
bilayer structure. The acyl chains are tilted considerably (by
∼46°) with respect to the bilayer normal. Studies aimed at
understanding the interaction of these molecules with other
membrane lipids/drugs would be expected to provide clues to
understand their role in biological processes and pharmaceut-
ical applications.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Data from FTIR, 1H NMR, and 13C NMR spectroscopy as well
as ESI MS spectrometry. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel +91-40-2313-4807; Fax +91-40-2301-2460/0145; e-mail
mjssc@uohyd.ernet.in, mjswamy1@gmail.com (M.J.S.).
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■
Author Contributions
†S.T.R. and P.K.T. contributed equally to this study.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by a research grant from the
Department of Science and Technology (India) to M.J.S.
S.T.R., P.K.T., and R.K.K. were supported by Senior Research
Fellowships from the Council of Scientific and Industrial
Research (India). Use of the National Single Crystal
Diffractometer Facility (SMART APEX CCD single crystal X-
ray diffractometer) at the School of Chemistry, University of
Hyderabad, funded by the Department of Science and
Technology (India) is gratefully acknowledged. We thank Dr.
P. Raghavaiah for his help in the solution of the crystal structure
of NLDA. The University Grants Commission (India) is
acknowledged for for their support through the UPE and CAS
programs to the University of Hyderabad and School of
Chemistry, respectively.
ABBREVIATIONS USED
■
NADA, N-acyldopamine; NArDA, N-arachidonyldopamine;
NLDA, N-lauroyldopamine; NPDA, N-palmitoyldopamine;
NSDA, N-stearoyldopamine; DA, dopamine; DSC, differential
scanning calorimetry; MAPK, mitogen-activated protein kinase;
MCF-7, michigan cancer foundation-7; COX-2, cyclooxyge-
nase-2; mRNA, messenger RNA; TRPV1, transient receptor
potential vanilloid-1; Tt, transition temperature; ΔHt, transition
enthalpy; ΔSt, transition entropy; ΔHinc, incremental enthalpy;
ΔSinc, incremental entropy; ΔH0, end contribution of enthalpy;
ΔS0, end contribution of entropy.
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