Structure and thermotropic phase behavior of a homologous series of n -Acylglycines: Neuroactive and antinociceptive constituents of biomembranes

Article abstract of DOI:10.1021/cg500481u

N-Acylglycines (NAGs) with different acyl chains have been found in the mammalian brain and other tissues. They exhibit significant biological and pharmacological properties and appear to play important roles in communication and signaling pathways within and between cells. In view of this, a homologous series of NAGs have been synthesized and characterized in the present study. Differential scanning calorimetric (DSC) studies show that the transition enthalpies and entropies of dry as well as hydrated NAGs exhibit a linear dependence on the acyl chain length. Most of the NAGs show a minor transition below the chain-melting phase transition, suggesting the presence of polymorphism in the solid state. Structures of N-myristoylglycine (NMG) and N-palmitoylglycine (NPG) were solved in monoclinic system with C2/c and P2₁ space groups, respectively. Analysis of the crystal structures show that NAGs are organized in a bilayer fashion, with head-to-head (and tail-to-tail) arrangement of molecules. The acyl chains in both structures are essentially perpendicular to the bilayer plane, which is consistent with a lack of odd-even alternation in the thermodynamic properties. The bilayer is stabilized by strong hydrogen bonding interactions between COOH groups of the molecules from opposite leaflets as well as N-H···O hydrogen bonds between the amide groups of adjacent molecules in the same leaflet and dispersion interactions among the acyl chains. Powder X-ray diffraction data show that the d-spacings for the NAGs with different acyl chains (n = 8-20) exhibit a linear dependence on the chain length, suggesting that all the NAGs investigated here adopt a similar packing arrangement in the crystal lattice. These observations are relevant for understanding the role of N-acylglycines in biological membranes.

Full text of DOI:10.1021/cg500481u

Article
pubs.acs.org/crystal
Structure and Thermotropic Phase Behavior of a Homologous Series
of N‑Acylglycines: Neuroactive and Antinociceptive Constituents of
Biomembranes
S. Thirupathi Reddy, Krishna Prasad Krovi, and Musti J. Swamy*
School of Chemistry, University of Hyderabad, Hyderabad 500 046, India
S
* Supporting Information
ABSTRACT: N-Acylglycines (NAGs) with different acyl chains have been found in the
mammalian brain and other tissues. They exhibit significant biological and
pharmacological properties and appear to play important roles in communication and
signaling pathways within and between cells. In view of this, a homologous series of
NAGs have been synthesized and characterized in the present study. Differential
scanning calorimetric (DSC) studies show that the transition enthalpies and entropies
of dry as well as hydrated NAGs exhibit a linear dependence on the acyl chain length.
Most of the NAGs show a minor transition below the chain-melting phase transition,
suggesting the presence of polymorphism in the solid state. Structures of N-
myristoylglycine (NMG) and N-palmitoylglycine (NPG) were solved in monoclinic
system with C2/c and P21 space groups, respectively. Analysis of the crystal structures
show that NAGs are organized in a bilayer fashion, with head-to-head (and tail-to-tail)
arrangement of molecules. The acyl chains in both structures are essentially
perpendicular to the bilayer plane, which is consistent with a lack of odd−even alternation in the thermodynamic properties.
The bilayer is stabilized by strong hydrogen bonding interactions between −COOH groups of the molecules from opposite
leaflets as well as N−H···O hydrogen bonds between the amide groups of adjacent molecules in the same leaflet and dispersion
interactions among the acyl chains. Powder X-ray diffraction data show that the d-spacings for the NAGs with different acyl
chains (n = 8−20) exhibit a linear dependence on the chain length, suggesting that all the NAGs investigated here adopt a similar
packing arrangement in the crystal lattice. These observations are relevant for understanding the role of N-acylglycines in
biological membranes.
endogenous cannabinoid anandamide, suggesting that it is a
precursor of the former.18
INTRODUCTION
■
Conjugates of lipids and amino acids (lipoamino acids) were
identified in biological systems over 50 years ago.1 A family of
lipids similar to acyl amino acids containing only one amide
bond connecting a fatty acid with ethanolamine, dopamine, or
amine has been identified in mammalian tissues.2−5 Much
interest has been generated in such conjugates with the
identification of the endocannabinoid N-arachidonylethanol-
amine, also referred to as anandamide, which acts as an
endogenous ligand to the cannabinoid receptor CB1, inhibits
gap-junction conductance, and reduces the fertilizing capacity
of sperm.6−8 N-Arachidonylglycine (NArG), the first lipoamino
acid, was synthesized as a structural analog of anandamide with
differences of CB1 agonist activity.9 Huang et al.10 predicted
that NArG would be present in mammalian tissues because it is
made up of the naturally occurring compounds glycine and
arachidonic acid and demonstrated its presence in bovine and
rat brain as well as in other tissues. NArG produces
antinociceptive and anti-inflammatory effects, exhibits high-
affinity for the orphan GPCR, GPR18, and inhibits the
hydrolytic activity of fatty acid amide hydrolase (FAAH) on
anandamide as well as glycine transport by GLYT2a.11−17 It has
been reported that NArG is an oxidative metabolite of
Since mammalian tissues also contain saturated fatty acids, it
would be expected that corresponding saturated lipoamino
acids would be synthesized by the enzymes that conjugate fatty
acids and glycine. Indeed, N-palmitoylglycine was identified in
rat brain and was shown to act as a modulator of calcium influx
and nitric oxide production in sensory neurons.19 Several other
NAGs, namely, N-stearoylglycine, N-oleoylglycine, N-linoleoyl-
glycine, and N-docosahexaenoylglycine were also found in
different parts of the body at different levels, suggesting region-
specific functionality.20 Recent research has shown that besides
NAGs, a variety of N-acyl amino acids (NAAs) are present in
mammalian brain as well as other tissues.21,22 These and other
studies led to considerable interest in investigating the
physiological roles and pharmacological potential of N-acyl
amino acids and neurotransmitters.5
While a significant amount of work has been carried out on
the biological and pharmacological properties of NAGs, there
have been no studies characterizing their physical properties
Received: April 9, 2014
Revised: August 16, 2014
Published: August 22, 2014
© 2014 American Chemical Society
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about 75−80% yield, which were characterized by melting point,
and interaction with other membrane components. In view of
their amphiphilic nature, it would be expected that NAGs with
long acyl chains will be incorporated into biomembranes, which
in turn may affect their biophysical properties. In order to
understand how NAGs interact with lipid membranes and
modulate their structure and dynamics, it is imperative to
investigate their physical properties and phase behavior, and
characterize their interaction with other membrane compo-
nents such as phospholipids and cholesterol. In view of this, in
the present study, we synthesized a homologous series of NAGs
(n = 8−20) and investigated their thermotropic phase behavior
in dry and hydrated states by DSC, which showed that the
transition enthalpy and entropy exhibit a linear dependence on
the acyl chain length, without the well-known odd−even
alternation observed in other amphiphiles such as long chain
carboxylic acids, N-acylethanolamines, N,O-diacylethanol-
amines and N-acyldopamines.23−26 The lack of odd−even
alternation could be explained on the basis of the three-
dimensional structure of N-myristoylglycine and N-palmitoyl-
glycine, which indicated that NAGs form a bilayer structure in
the crystal lattice with the acyl chains oriented perpendicular to
the bilayer plane.
1
FTIR, and H and 13C NMR spectroscopy.
Capillary melting points of NAGs were recorded on a Superfit
(Mumbai, India) melting point apparatus.26 IR spectra (KBr pellet)
1
were recorded on a Jasco FTIR 5300 spectrometer, and H and 13C
NMR spectra were obtained on a Bruker Avance NMR spectrometer
operating at 400 and 100 MHz, respectively. Samples were dissolved in
CDCl3 containing a trace of CD3OD for 1H NMR studies, whereas for
13C NMR spectroscopy, samples were dissolved in CD3OD.
Differential Scanning Calorimetry. DSC studies on dry NAGs
were carried out on a PerkinElmer PYRIS Diamond differential
scanning calorimeter.26 Heating and cooling scans were performed
with 1−2 mg samples taken in aluminum pans from room temperature
(∼25 °C) to about 140 °C at a scan rate of 1.5°/min. Each sample was
subjected to three heating scans and two cooling scans. Transition
enthalpies were determined by integrating the area under the
transition curve. Transition entropies were calculated from the
transition enthalpies according to eq 1, applicable to first order
transitions:29
ΔHt = TΔSt
(1)
where T is the transition temperature and ΔHt values were taken at
this temperature in order to calculate the corresponding ΔSt values.
Hydrated samples for DSC studies were prepared with ca. 4−5 mg
of each NAG using the procedure described earlier for N-acyldop-
amines,26 excepting that in the present study double distilled water was
used to hydrate the samples rather than phosphate buffer. DSC studies
were carried out on a VP-DSC equipment (MicroCal LLC,
Northampton, MA, USA), essentially as described earlier.26 All
samples were subjected to three heating and two cooling scans
between 10 and 110 °C at a scan rate of 1°/min. Transition enthalpies
were calculated by integrating the area under the transition curve after
blank (water) subtraction, normalization, and baseline correction.
Transition entropies were determined from the transition enthalpies
using eq 1.
Crystallization, X-ray Diffraction, and Structure Solution.
Colorless, thin plate-type crystals of N-myristoylglycine and N-
palmitoylglycine were grown at room temperature from their solutions
in ethyl acetate containing traces of methanol and toluene containing
traces of methanol, respectively. X-ray diffraction data were collected at
room temperature (ca. 25 °C) on a Bruker SMART APEX CCD area
detector system using a graphite monochromator and Mo Kα (λ =
0.717073 Å) radiation, obtained from a fine-focus sealed tube. The
minimum resolution of X-ray diffraction measurements is 0.84 Å. Data
reduction was done using the Bruker SAINTPLUS program.
Absorption correction was applied using the SADABS program, and
refinement was done using the SHELXTL program.30 Mercury 3.0 and
Diamond 2.1 softwares were used for preparing molecular packing and
hydrogen bonding diagrams, respectively.
EXPERIMENTAL SECTION
■
Materials. Long chain fatty acids (n = 8−20), glycine methyl ester
hydrochloride, and lithium hydroxide were purchased from Sigma-
Aldrich (Milwaukee, WI, USA). Oxalyl chloride was obtained from
Spectrochem (Mumbai, India). Solvents and other chemicals used
were of analytical grade and purchased locally. Milli-Q water was used
in all experiments.
Synthesis of N-Acylglycines. NAGs were synthesized by a minor
modification of a reported procedure adopted earlier for the synthesis
of N-hexadecanoylserine (Scheme 1).27 Briefly, glycine methyl ester
Scheme 1. Synthesis of N-Acylglycines
Powder X-ray Diffraction. Powder X-ray diffraction patterns of
NAGs were recorded on a Bruker SMART D8 Advance powder X-ray
diffractometer (Bruker-AXS, Karlsruhe, Germany) using Cu Kα
radiation (λ = 1.5406 Å) at 40 kV and 30 mA. Samples were placed
and pressed on a circular rotating disk of the sample holder. The
diffracted beam from the sample was detected by a LynxEye PSD data
collector. Diffraction patterns were collected for all the NAGs at room
temperature over a 2θ range of 2−50° with a step size of 0.0198°, with
a measuring time of 10 s for each step. To investigate polymorphism in
NPG, PXRD data were recorded at different temperatures with
samples placed in a Anton Paar TTK 450 sample chamber stage with
reflection but without movement. An equilibration time of ca. 10 min
was given at each temperature before data collection. In these
measurements, a 2θ range of 5−42° with a step size of 0.0198° s−1 was
used. The appearance of a new polymorph was identified by the
appearance of new diffraction peaks. Origin 7 was used for overlaying
the experimental PXRD pattern and the simulated PXRD pattern
obtained from the single-crystal X-ray diffraction.
hydrochloride (1 mmol) was dissolved in water (4 mL), and sodium
bicarbonate (2 mmol) and chloroform (12 mL) were added with
vigorous stirring. To this mixture, a chloroform solution of the acid
chloride (1 mmol), prepared by the reaction of fatty acid with oxalyl
chloride,28 was added, and the mixture was kept under stirring for 2 h
at room temperature. Then the organic phase was washed successively
with saturated sodium chloride solution, 0.1 N HCl, and distilled water
(twice). The solution was dried over anhydrous sodium sulfate,
filtered, and evaporated to dryness under reduced pressure.
Recrystallization from hexane at −20 °C yielded N-acylglycine methyl
ester in 80−85% yield. The methyl ester was converted to the free acid
by base-catalyzed hydrolysis for 2 h with lithium hydroxide (10 mmol)
in a methanol/water (3:1, v/v) mixture. The pH of the reaction
mixture was adjusted to 3.0 by dropwise addition of 2 N HCl, and the
NAGs were extracted into ethyl acetate. The extract was then washed
with 1 N HCl and twice with distilled water, dried over anhydrous
sodium sulfate, filtered, and evaporated to dryness under reduced
pressure. Recrystallization from ethyl acetate yielded pure NAGs in
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RESULTS AND DISCUSSION
■
Characterization of N-Acylglycines. The homologous
series of NAGs (n = 8−20) synthesized in this study have been
1
characterized by FT-IR and H and 13C NMR spectroscopy.
1
Representative FTIR and H NMR spectra of N-decanoylgly-
cine are given in Figure S1 and Figure S2, Supporting
Information, respectively. IR spectra of all NAGs showed
absorption bands corresponding to the amide linkage at 1645−
1644 cm−1 (amide-I), 1561−1557 cm−1 (amide-II), and 3333−
3310 cm−1 (N−H stretching). Two bands were seen in the
carbonyl stretching region, at 1748−1740 and 1707−1700
cm−1, which could be due to differences in hydrogen bonding
in different polymorphic states. The methylene groups of the
acyl chain (and the glycine moiety) show stretching, scissoring
and rocking bands at 2922−2849, 1470−1462, and 696−693
cm−1, respectively. The values of resonances corresponding to
various absorption bands for all the NAGs are given in Table
S1, Supporting Information. 1H NMR spectra of NAGs showed
resonances at 2.15−2.24 δ (2H, t) and 1.55−1.62 δ (2H, m) for
the methylene groups at α and β positions to the amide
carbonyl, respectively. Resonances were seen at 3.91−4.22 δ
(2H, s) for the methylene group in the glycine moiety and at
6.16−6.92 δ (1H, bs) for the amide N−H. Resonance
corresponding to the terminal methyl group was seen at
0.80−0.99 δ (3H, t) whereas resonance for the polymethylene
group was observed at 1.17−1.27 δ (8−32, m). These data for
different NAGs are given in Table S2, Supporting Information.
A representative 13C NMR spectrum of N-decanoylglycine is
given in Figure S3, Supporting Information. The spectrum
shows a resonance at 13.11δ, which corresponds to the methyl
group of the acyl chain. Resonances arising from methylene
carbons are seen at 22.38, 25.50, 31.70, 35.46, and 40.36 δ as
well as between 28.92 and 29.25 δ (three peaks with the peak at
29.13 being more intense than the others due to two methylene
carbons giving resonances at the same δ value). Resonances
corresponding to the carbonyl moieties of carboxylic acid and
amide groups are observed at 171.74 and 175.39 δ, respectively.
These values are consistent with the structure of N-
decanoylglycine. All other NAGs gave similar spectra, except
that the intensity of the peak at ∼29.13 δ increased with
increase in the acyl chain length, which can be attributed to
several carbon atoms in the middle of the acyl chain having the
same chemical shift. 13C NMR data for all the NAGs are given
in Table S3, Supporting Information.
Figure 1. DSC heating thermograms of dry NAGs with (A) even and
(B) odd number of carbon atoms in the acyl chains. The number of C
atoms in the acyl chain is indicated against each thermogram.
Thermograms of hydrated NAGs with even and odd acyl
chains are shown in Figure 2A,B, respectively. For each NAG a
single sharp peak was seen at ca. 20−40 °C below the phase
transition temperature of the dry sample. When the samples
were subjected to a second heating scan, small decreases were
observed in the transition enthalpies compared with the first
scan. Therefore, in all cases, only the first heating scan was
considered for further analysis. Values of Tt, ΔHt, and ΔSt
obtained for hydrated NAGs are also given in Table 1.
Chain Length Dependence of Thermodynamic Pa-
rameters of Dry and Hydrated NAGs. When the total
enthalpy and entropy of the phase transitions (major + minor)
of dry NAGs are plotted against the acyl chain length a linear
dependence was observed (Figure 3A,B). These data could be
fit well to expressions 2 and 3 given below as observed earlier
for N-acylethanolamines (NAEs) and N-acyldopamines with
even and odd acyl chain lengths, O-acylcholines with even chain
lengths, and N,O-diacylethanolamines (DAEs) with matched as
well as mixed acyl chains.23−26,28,31,32
ΔHt = ΔH0 + (n − 2)ΔHinc
(2)
DSC Studies on the Thermotropic Phase Transitions
of N-Acylglycines. Heating thermograms of dry NAGs
containing even and odd acyl chains are shown in Figure
1A,B, respectively. Each NAG exhibits a major transition that
matches well with its melting point. All the NAGs except NMG
showed one minor transition before the melting transition,
which is most probably due to polymorphism and not because
of any impurity since impurities would normally broaden the
transition rather than giving rise to separate transitions. The
minor transitions disappeared in all cases in the second heating
scans and small decreases were noticed in the transition
enthalpies. This suggests that during the cooling scans NAGs
do not come back to their original structure. Therefore, the first
heating scan was considered for further analysis in all cases, and
the total area under minor and major transitions was integrated
to get the transition enthalpies. Transition temperatures (Tt),
enthalpies (ΔHt), and entropies (ΔSt) of dry NAGs, obtained
from the DSC studies, are presented in Table 1.
ΔSt = ΔS0 + (n − 2)ΔSinc
(3)
where n is the number of C atoms in the acyl chains and ΔH0
and ΔS0 are the end contributions to ΔHt and ΔSt, respectively,
arising from the terminal methyl group and the polar region of
the NAG molecule. ΔHinc and ΔSinc are the average
incremental values of ΔHt and ΔSt contributed by each CH2
group. Linear least-squares analysis of the chain length-
dependent values of ΔHt and ΔSt for homologous series of
NAGs yielded the incremental values (ΔHinc and ΔSinc) and
end contributions (ΔH0 and ΔS0), which are listed in Table 2.
The ΔHinc and ΔSinc values observed for the dry NAGs are 0.92
0.03 kcal·mol−1 and 2.23 0.08 cal·mol−1·K−1, respectively.
A linear chain length dependence of the transition enthalpy
and transition entropy observed here for the NAGs suggests
that structures of the homologous series of NAGs would be
quite similar in the solid state. Thus, the molecular packing and
intermolecular interactions (e.g., hydrogen bonding) are likely
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Table 1. Average Values of Transition Temperatures (Tt), Transition Enthalpies (ΔHt), and Transition Entropies (ΔSt) of
a
NAGs in Dry and Hydrated States
dry NAGs
hydrated NAGs
acyl chain length
T (°C)
ΔHt (kcal·mol−1
)
ΔSt (cal·mol−1·K−1
)
T (°C)
ΔHt (kcal·mol−1
)
ΔSt (cal·mol−1·K−1
)
8
9
106.4 0.1
110.1 0.1
115.6 0.3
117.3 0.2
120.4 0.1
121.6 0.1
123.8 0.2
124.6 0.4
124.9 0.2
125.7 0.2
127.3 0.1
127.6 0.1
128.8 0.1
5.65 0.03
6.28 0.07
7.82 0.13
8.58 0.33
9.25 0.01
10.19 0.59
10.07 0.7
12.02 0.09
13.40 0.14
14.41 0.25
14.68 0.02
15.29 0.45
16.98 0.16
14.9 0.1
16.4 0.1
20.1 0.3
21.1 0.8
23.5 0.1
25.8 1.5
25.8 1.8
30.2 0.2
32.7 0.4
36.1 0.6
36.7 0.1
38.2 1.1
42.3 0.4
66.15 0.03
74.07 0.09
81.47 0.09
84.86 0.04
89.21 0.08
92.31 0.08
95.19 0.11
97.71 0.06
99.83 0.27
102.16 0.16
103.90 0.30
105.73 0.06
107.67 0.05
1.40 0.58
3.61 0.47
5.56 0.61
5.80 0.89
7.28 0.41
8.89 0.85
9.15 0.52
10.47 0.38
11.47 0.44
12.91 0.75
14.03 0.58
15.18 1.21
16.02 1.73
4.2 1.7
10.4 1.4
15.7 1.7
16.2 2.5
20.1 1.1
24.3 2.3
24.9 1.4
28.3 1.0
30.8 1.2
34.4 2.0
37.2 1.5
40.1 3.2
42.1 2.5
10
11
12
13
14
15
16
17
18
19
20
a
The standard deviations correspond to at least three independent measurements.
Figure 2. DSC heating thermograms of hydrated NAGs with (A) even
and (B) odd number of carbon atoms in the acyl chain. The number of
C atoms in the acyl chain is indicated against each thermogram.
Figure 3. Chain length dependence of (A) transition enthalpies and
(B) transition entropies of dry NAGs. Values of ΔHt and ΔSt were
plotted against the number of methylene (CH2) units (n − 2) in
panels A and B, respectively. Solid lines correspond to linear least-
squares fits of the data.
to be very similar in all NAGs in the homologous series.
Therefore, determination of the crystal structure of any one
NAG should give a reasonably good idea of the molecular
packing and intermolecular interactions present in the crystal
lattice for the entire homologous series. In the present study,
we determined the three-dimensional structure of two different
NAGs by single-crystal X-ray diffraction and found that they are
isostructural (see below).
Similar to the dry state, upon hydration also NAGs exhibit a
linear dependence of transition enthalpy and entropy on their
acyl chain length (Figure 4A,B), although the magnitude of
these parameters are lower in the hydrated state compared with
the dry state. The enthalpy and entropy data of hydrated NAGs
also fit well to expressions 2 and 3, as observed earlier for
hydrated N-acylethanolamines with even and odd acyl chain
lengths and N-acyldopamines.24,26,28
results obtained with a number of long-chain compounds
including hydrocarbons, fatty acids, N-acylethanolamines, N-
acyldopamines, and N,O-diacylethanolamines, all of which
exhibit odd−even alternation in the transition enthalpies and
entropies.23−26,32 Such alternation in long chain fatty acids has
been explained on the basis of packing of hydrocarbon chains,
where differences in the packing of terminal methyl groups
between the even and odd acyl chain lengths.23 Such
differences do not arise if the hydrocarbon chains are
perpendicular to the methyl group plane. However, if the
chains are tilted with respect to the bilayer normal, their
packing modes can differ, leading to alternation in the physical
properties.23 Consistent with this, both NAEs and DAEs, which
exhibit odd−even alternation, show significant N-acyl chain tilt
(∼34−37° with respect to the bilayer normal).25,32−35 On the
other hand, the acyl chains in NAGs are nearly perpendicular to
the bilayer plane with very small tilt angles of 0.03° and 0.84°
Thermodynamic Properties and Acyl Chain Tilt. From
Figure 3, it is clear that the enthalpy and entropy corresponding
to the chain melting phase transitions of dry NAGs exhibit a
linear dependence on the chain length. This is in contrast to the
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Table 2. Incremental Values (ΔHinc, ΔSinc) of Chain Length Dependence and End Contributions (ΔH0, ΔS0) to Phase
a
Transition Enthalpy and Entropy of NAGs
ΔHinc (kcal·mol−1
)
ΔH0 (kcal·mol−1
)
ΔSinc (cal·mol−1·K−1
)
ΔS0 (cal·mol−1·K−1
)
dry NAGs
0.92 0.03
1.15 0.03
0.42 0.41
2.23 0.08
2.95 0.08
1.29 1.05
hydrated NAGs
−4.34 0.35
−10.06 1.05
a
Average values of transition enthalpy and transition entropy given in Table 1 have been used for the linear fitting of the data. Errors are fitting errors
obtained from the linear least squares analysis.
Chain Length Dependence of Phase Transition
Temperatures of NAGs. The transition temperatures of dry
as well as hydrated NAGs increase with increasing chain length
as shown in Figure 5, but the magnitude of the change
Figure 4. Chain length dependence of (A) transition enthalpies and
(B) transition entropies of hydrated NAGs. Values of ΔHt and ΔSt
were plotted against the number of methylene (CH2) units (n − 2) in
panels A and B, respectively. Solid lines correspond to linear least-
squares fits.
Figure 5. Chain length dependence of chain-melting phase transition
for NMG and NPG, respectively (see below). These
observations are consistent with the observed linear depend-
ence of transition enthalpies and entropies of the homologous
series of dry NAGs.
●
○
temperatures of NAGs in the dry ( ) and hydrated ( ) states. Solid
lines correspond to nonlinear least-squares fit of the transition
temperatures to eq 7.
Incremental Values of Transition Enthalpy and
Entropy. Linear least-squares analysis of transition enthalpies
and transition entropies of NAGs in the dry and hydrated states
yielded incremental values (ΔHinc and ΔSinc) and end
contribution (ΔH0 and ΔS0). It is observed that the value of
ΔHinc of dry NAGs is slightly lower than the ΔHinc
corresponding to the hydrated state. But, in case of NAEs it
was found that ΔHinc of dry state is higher than that of the
hydrated state. The end contribution of entropy, ΔS0, is also
more negative for hydrated samples compared with the dry
samples.24,28,36 The higher ΔHinc of hydrated NAGs compared
with the dry samples and larger negative ΔS0 of higher NAGs
are indicative of the hydrophobic effect playing a role in the
packing of NAGs, which could arise due to the ordering of
water molecules around the carboxylic acid moiety of the
headgroup.37 Therefore, it appears that the hydrophobic effect
in hydrated form brings the NAG molecules closer, resulting in
a tight packing of the acyl chains.
decreases with increase in the chain length. When the acyl chain
length is increased, the total contribution from the poly-
methylene portion toward the total enthalpy and entropy of the
phase transition also increases, whereas the end contributions
remain constant. Therefore, at infinite acyl chain length, the
end contributions become insignificant compared with the
contribution from the polymethylene portion. Consequently,
the end contributions can be neglected, which reduces eqs 2
and 3 to23
ΔHt = (n − 2)ΔHinc
(4)
ΔSt = (n − 2)ΔSinc
(5)
Then the transition temperature for infinite chain length, T∞t ,
will be given by
Tt∞ = ΔHinc/ΔSinc
(6)
The acyl chain tilt with respect to the bilayer normal in
NAGs is in the neighborhood of zero, whereas in NAEs it is
around 35−37°,36 which is in agreement with the higher ΔHinc
for NAGs, in the dry and hydrated states. This suggests that the
acyl chains are packed more closely in NAGs than in NAEs.
From the data presented in Table 2 and using eq 6, the Tt∞
values for NAGs in the dry and hydrated state have been
estimated as 412.6 and 389.8 K, respectively.
For a number of single-chain as well as two-chain lipids,
which exhibit linear dependence of ΔHt and ΔSt on chain
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Figure 6. ORTEPs showing the molecular structures of (A) N-myristoylglycine and (B) N-palmitoylglycine.
length, it has been shown that the transition temperature data
bond angles involving all non-hydrogen atoms are given in
Table S5, Supporting Information, and selected torsion angles
are given in Table S6, Supporting Information. Corresponding
data for NPG are given in Tables S7−S9, Supporting
Information. The ORTEPs in Figure 6 indicate that both
NMG and NPG adopt an essentially linear geometry. This is
consistent with the torsion angles observed in the hydrophobic
region (Tables S6 and S9, Supporting Information), which are
very close to 180°, clearly indicating that the nonpolar portion
(C3−C18) of the acyl chain is in all-trans conformation in both
these compounds. In addition, the carbonyl oxygen and amide
N−H are also in trans geometry in both compounds.
can be fit to the following equation:38
∞
′
′
T = ΔHt/ΔSt = Tt [1 − (n0 − n0)/(n − n0)]
(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. Figure 5 shows that the Tt values of
NAGs investigated here fit very well with eq 7, in the dry and
hydrated states. The fitting parameters also yielded the Tt∞
values for NAGs in the dry and hydrated states as 411.4 1.4 K
and 406.6
2.1 K, respectively. These values are in good
agreement with the T∞t values estimated using eq 6.
Molecular Packing, Hydrogen Bonding, and Inter-
molecular Interactions. A packing diagram of NPG viewed
down the c-axis is shown in Figure 7A and close-up views of the
hydrogen bonding interactions as seen down the c-axis and b-
axis are shown in Figure 7B,C, respectively. From this figure, it
can be seen that NPG molecules are packed in a head-to-head
(and tail-to-tail) fashion in stacked bilayers. The carboxylic acid
groups from opposite leaflets associate via O−H···O hydrogen
bonds, leading to the formation of eight-membered cyclic
structures connecting two leaflets from adjacent bilayers.
Additionally, the amide groups of adjacent molecules in the
same leaflet are also involved in hydrogen bonding. The methyl
ends of stacked bilayers are in van der Waals’ contacts, and the
methyl ends from opposite leaflets face each other with the
closest methyl−methyl distance between opposite layers being
4.008 Å and that within the same leaflet being 4.868 Å. The
bilayer thickness (i.e., O1−O1 distance) in the NPG crystal
lattice is 49.954 Å, whereas the monolayer (single leaflet)
thickness (O1−C18 distance) is 23.387 Å. The repeat distance
(d-spacing) is 51.28 Å. The all-trans acyl chains are tilted by
0.84° with respect to the bilayer normal.
Crystal Structures of NMG and NPG. ORTEPs of N-
myristoylglycine and N-palmitoylglycine are shown in Figure
6A,B, respectively, along with the atom numbering for all non-
hydrogen atoms. The crystal parameters of NMG are given in
Table 3, and the atomic coordinates and equivalent isotropic
displacement parameters for all non-hydrogen atoms are given
in Table S4, Supporting Information. The bond distances and
Table 3. Crystallographic Data for N-Myristoylglycine and
N-Palmitoylglycine at 298 K
crystal parameters
NMG
NPG
formula
formula wt
cryst syst
space group
a (Å)
C16H31NO3
285.42
monoclinic
C2/c
C18H35NO3
313.47
monoclinic
P21/c
92.388(16)
4.86(8)
7.6485(13)
90.00
51.343(9)
4.8679(8)
7.6195(13)
90.00
b (Å)
c (Å)
α (deg)
β (deg)
γ (deg)
Z
V (Å3)
Dcalc (g cm−3
91.702(4)
90.00
92.830(3)
90.00
Packing diagrams of NMG viewed down the c-axis and b-axis
are shown in Figure S4A,B, Supporting Information,
respectively. Similar to NPG, NMG molecules are also packed
in a head-to-head fashion, akin to that found in phospholipid
bilayers. The molecular packing and intermolecular interactions
are essentially similar to those found in the crystal structure of
NPG, described above. Only minor differences are seen as
indicated below. The closest methyl−methyl distances between
8
4
3432.7(10)
1.105
1902.0(6)
1.095
)
R1
8.05
8.74
wR2
GOF
20.72
24.4
1.115
1.167
data completeness
99.0%
96.1%
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Figure 7. Packing diagrams of N-palmitoylglycine indicating the bilayer arrangement and hydrogen bonding networks. (A) A view of the bilayer
arrangement in NPG. (B) A view down the c-axis depicting the O−H···O and N−H···O type hydrogen bonds. (C) A view down the b-axis, depicting
the O−H···O and C−H···O type hydrogen bonds. Atom code: carbon, large open circle; nitrogen, blue circle; oxygen, red circle; hydrogen, small
open circle.
opposite layers and within the same leaflet in NMG are 3.73
and 4.86 Å, respectively. The bilayer thickness (O1−O1
distance) and monolayer thickness (O1−C16 distance) are
44.61 and 20.86 Å, respectively. The repeat distance (d-
spacing) is 46.17 Å. The all-trans acyl chains are tilted with
respect to bilayer normal by 0.03°, which is very close to that
observed in the crystal structure of NPG. The acyl chain tilt
observed in the NAGs is negligible compared with that
observed in N-acylethanolamines (ca. 35−37°).33−35 These
observations are consistent with a lack of odd−even alternation
in the thermodynamic parameters determined from DSC
studies, discussed above.
acid group being involved in two hydrogen bonds with an NPG
molecule from the opposite leaflet. Here, each carboxylic acid
group acts both as a proton donor and as a proton acceptor
with the carboxylic acid group of another molecule in the
opposite layer. The H···O distance in the O−H···O hydrogen
bonds is 1.742 Å, whereas the distance between the donor and
acceptor oxygen atoms is 2.686 Å, and the bond angle
subtended at the hydrogen atom is 156.34°. Besides the
hydrogen bonds between the carboxyl groups, strong hydrogen
bonds are also formed between the amide N−H and carbonyl
oxygen of adjacent NPG molecules (Figure 7B). While the N−
H group of each NPG molecule forms a hydrogen bond with
another NPG molecule in the adjacent unit cell on one side, the
carbonyl oxygen of the same molecule forms a hydrogen bond
An examination of Figure 7B shows that the O−H···O
hydrogen bonds form a cyclic structure, with each carboxylic
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with the amide N−H of another NPG molecule in the adjacent
unit cell on the other side. All these hydrogen bonds are also
identical, with a N−O distance of 2.974 Å, a H···O distance of
1.808 Å, and a N−H···O angle of 177.99°. Similar N−H···O
hydrogen bonds have been observed in the crystal structures of
a number of amphiphiles including N-acylethanolamines, N,O-
diacylethanolamines, and N-acyldopamines and in N,N,N″-
tris(n-octyl)benzene-1,3,5-tricarboxamide, a C3-symmetric tri-
amide with three alkyl chains.25,26,32−35,39
The hydrogen bonding pattern in the crystal lattice of NMG
viewed down the c-axis and b-axis are shown in Figure S5A,B,
Supporting Information, respectively. From this figure, it can be
clearly seen that the hydrogen bonding patterns in the NMG
crystal lattice are very similar to those observed in the crystal
lattice of NPG. All of the O−H···O hydrogen bonds in NMG
are identical, with an O−O distance of 2.684 Å, an O···H
distance of 1.721 Å, and a hydrogen bond angle of 168.34°.
Similar to that observed in the crystal structure of NPG, all of
the N−H···O hydrogen bonds in NMG are also identical, with
an O···H bond length of 2.091 Å, a N−O distances of 2.963 Å,
and a hydrogen bond angle of 164.77°. In both cases, donut
shaped structures are formed by the hydrogen bonded
carboxylic acid dimers. This is shown for NPG in Figure S6,
Supporting Information.
whose three-dimensional structures have been determined
earlier, namely, N-myristoylethanolamine (21.95 Å2), N-
palmitoylethanolamine (polymorphs α and β, 21.99 Å2 and
22.03 Å2, respectively), and N-stearoylethanolamine (21.99 Å2),
as well as some other single-chain lipids such as 3(11-
bromoundecanoyl)-D-glycerol, 3-lauroyl-D-glycerol, and 3-
stearoyl-D-glycerol, whose molecular areas are in the range of
21.5−22.7 Å2.33−35,45−48 The smaller molecular area of NAGs
is due to their small headgroup size and negligible acyl chain tilt
with respect to the bilayer normal. Other single chain lipids
such as lysophosphatidic acid and lysophosphatidylethanol-
amine have somewhat larger areas (33.6 and 34.8 Å2,
respectively). Such large molecular areas observed in these
two molecules are in part due to the very high tilt in the acyl
chains with respect to bilayer normal and large hydrophilic
head groups.49,50
Powder X-ray Diffraction Studies. Additional insights
into the molecular packing of NAGs with different acyl chains
were obtained from powder X-ray diffraction studies. PXRD
profiles of NAGs with different acyl chain lengths investigated
here are shown in Figure 8A,B. All the NAGs gave sharp
In addition to the O−H···O and N−H···O hydrogen bonds,
weak C−H···O hydrogen bonds have been observed in the
crystal structures of NMG and NPG. Hydrogen bonds with
interaction energies up to −5 kcal·mol−1 are generally
considered weak hydrogen bonds, and many examples of this
type have been described earlier.40−43 Figure 7C gives a view of
four sets of C−H···O type hydrogen bonds observed in the
crystal lattice of NPG. The C−H···O hydrogen bonds between
C2−H and O2 are of two types. These bonds are formed
between O2 (oxygen atom of carboxylic carbonyl group) of one
molecule with two different H atoms (H2A and H2B) on C2
(carbon atom α to the carboxylic carbonyl) of the adjacent
molecule in the same layer. All the C2(H2A)···O2 bonds are
identical, with a C2(H2A)−O2 distance of 3.743(3) Å, an
H2A···O2 distance of 3.074 Å, and a C2−H2A···O2 angle of
129.8°. Similarly, all the C2(H2B)···O2 bonds are also
identical, with a C2(H2B)−O2 distance of 3.248 Å, an
H2B···O2 distance of 2.406 Å, and C2−H2B···O2 angle of
144.8°. These C2(H2B)···O2 hydrogen bonds are considerably
stronger than the C2(H2A)···O2 hydrogen bonds. Stabilization
of lipid membrane structure by C−H···O hydrogen bonds has
been reported earlier for the paraffinic ylide trimethylammonio
n-hexadecylsulfonamidate.44
Figure 8. Powder X-ray diffraction patterns of N-acylglycines with
different saturated acyl chains (A, B) and dependence of d-spacings on
the acyl chain length (C). The number of carbon atoms in the
saturated unbranched acyl chains is indicated against each PXRD
profile. The solid line in panel C is a linear least-squares fit of the data.
The slope of this line yielded increase in the d-spacing per each
additional CH2 unit as 1.323 Å.
Other C−H···O hydrogen bonds between C4−H and O3 are
of two types. These bonds are formed between O3 (oxygen
atom of amide carbonyl group) of one molecule with H atoms
on C4 (carbon atom α to the amide carbonyl) of the adjacent
molecule in the same layer (Figure 7C). All the C4(H4A)···O3
bonds are identical, with a C4(H4A)−O3 distance of 3.487 Å
(3), an H4A···O3 distance of 2.62 Å, and a C4−H4A···O3
angle of 149°. Similarly, all the C4(H4B)···O3 bonds are also
identical, with a C4(H4B)−O3 distance of 5.228 Å, an H4B···
O3 distance of 4.59 Å, and a C4−H4B···O3 angle of 126.6°.
Very similar C−H···O hydrogen bonds were observed in the
crystal lattice of NMG (see Figure S5B and associated
description in the Supporting Information).
diffraction peaks that were consistent with lamellar packing of
the acyl chains. From the position of the peaks, the d-spacings
were calculated, and in each case 4−6 peaks were used for
estimating the d-spacing. The values obtained are presented in
Table S10, Supporting Information. The d-spacings obtained
for NMG and NPG from the PXRD data (47.69 and 52.31 Å,
respectively) are in good agreement with the values obtained
from the single crystal X-ray diffraction (46.17 and 51.28 Å,
respectively).
A plot depicting the chain length dependence of the d-
spacing (Figure 8C) is linear with a slope of 2.645 Å, which
corresponds to an increment of 1.323 Å per additional CH2
moiety in each chain. This is consistent with the observation of
untilted bilayer packing observed in the crystal structures of
NMG and NPG. The linear dependence of the d-spacings on
the acyl chain length suggests that all the NAGs pack in a
Molecular Area. The area per molecule in the bilayer plane
in the crystal lattice of NMG and NPG is 18.59 and 18.55 Å2,
respectively. These values are less than those found for NAEs
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similar manner in the solid state. That is, in the solid state all
the NAGs would adopt a bilayer structure with chains packed in
an essentially untilted fashion (with respect to the bilayer
normal).
Polymorphism in N-Acylglycines in the Solid State. As
indicated in the section DSC Studies on the Thermotropic
Phase Transitions of N-Acylglycines, DSC studies show that all
the NAGs (except NMG) studied here show a minor
endothermic phase transition in addition to the major
transition, which corresponds to the capillary melting point
of the compound. This additional transition is most likely due
to conversion of one solid form to another and suggests the
presence of structural polymorphism in NAGs. In order to
investigate this in greater detail, we carried out variable
temperature powder X-ray diffraction (VT-PXRD) and FTIR
studies. PXRD patterns of NPG recorded at different
temperatures are shown in Figure 9A. It may be noted that
the DSC thermogram of NPG shows a minor transition at 107
°C and a melting transition at 124.95 °C (see Figure 1 and
Table 1). The PXRD patterns recorded at 27 and 105 °C are
essentially identical indicating that no changes occur in the
solid state structure of this compound below 105 °C. However,
upon heating to 110 °C, additional peaks are seen in the PXRD
data, suggesting that significant changes occur in the solid state
structure of NPG during the minor transition. No additional
changes are seen upon further heating to 120 °C, whereas all
the peaks in the PXRD pattern disappear when the sample was
further heated to 125 °C, consistent with the liquid state of the
sample above the phase transition. We refer to the phase
present below 107 °C as form-I and that seen above 107 °C as
form-II. A comparison of the simulated PXRD pattern derived
from the structure of NPG determined by single-crystal X-ray
diffraction with the PXRD patterns obtained at 27 and 115 °C
(Figure 9B) shows a good correlation between the simulated
PXRD pattern and that of form-II. This shows that the single
crystal of NPG used in the X-ray diffraction study corresponds
to form-II. Since single crystals of form-I could not be obtained
for any of the NAGs, we carried out FT-IR spectroscopic
studies in order to obtain additional insights into their solid-
state polymorphism, with NPG as a representative example.
FTIR spectra of NPG recorded at different temperatures are
shown in Figure S7, Supporting Information The spectrum
recorded at room temperature (ca. 25 °C), shows two IR bands
around ∼1705 and ∼1740 cm−1 for the carboxylic acid moiety,
whereas the spectrum recorded after the sample was heated to
115 °C and cooled to room temperature (which is above the
minor solid-to-solid phase transition; see Figure 1) shows only
one band in the carbonyl stretching region at ∼1705 cm−1,
which is consistent with a change in the crystal form. A similar
spectrum was obtained after heating the sample to 135 °C
followed by cooling to room temperature, suggesting that only
form-II is obtained when the molten NPG undergoes liquid−
solid phase transition. This observation is in good agreement
with DSC and VT-PXRD studies.
Figure 9. (A) Variable temperature powder X-ray diffraction patterns
(VT-PXRD) of dry N-palmitoylglycine. The temperature at which
each scan was recorded is indicated in the figure. (B) Comparison of
simulated PXRD pattern of NPG obtained from single-crystal XRD
with experimental VT-PXRD pattern of NPG.
corresponds to the melting transition (Figure 1). The VT-
PXRD pattern of NMG does not show any detectable changes
when heated from room temperature to the phase transition
temperature (data not shown). In addition to these, the FTIR
spectrum of NMG gave only one carbonyl band at ∼1700 cm−1
corresponding to the carboxylic acid moiety (Figure S9,
Supporting Information). These observations suggest that
polymorphism could not be detected in the solid state of NMG.
Biological Implications and Future Directions. Results
obtained in the present studies show that NAGs exhibit
significantly higher phase transition temperatures compared
with diacylphospholipids such as phosphatidylcholines and
phosphatidylethanolamines of similar acyl chain lengths and
The DSC thermogram of N-pentadecanoylglycine (NPDG)
shows a minor transition at 109.5 °C, in addition to the melting
transition at 124.6 °C. The minor transition therefore
corresponds to a solid−solid phase transition and clearly
indicates that NPDG also exhibits polymorphism. This
interpretation is further supported by VT-PXRD, which showed
additional diffraction peaks at 120 °C (Figure S8, Supporting
Information). In contrast to other even acyl chain length
NAGs, NMG gives only one phase transition, which
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that they pack in a bilayer format. This suggests that NAGs
would most likely stabilize the membrane structure when
incorporated into phospholipid bilayers. The crystal structures
of NMG and NPG show that the acyl chains in these molecules
are oriented essentially perpendicular to the bilayer plane,
which would be the least disturbing way to accommodate them
into the host matrix. The hydrogen bonding capability of the
amide moiety at the hydrophobic−hydrophilic interface of the
NAG molecule could provide potential interaction with other
membrane consitituents such as phospholipids, glycolipids,
cholesterol, and integral proteins. The carboxyl group of the
glycine moiety can potentially exhibit electrostatic interactions
with peripheral proteins. Such interactions could be relevant to
the biological roles of NAGs, for example, antinociceptive
activity.
ACKNOWLEDGMENTS
■
This work was supported by a research grant from the
Department of Science and Technology (India) to M.J.S. S.T.R.
is a Senior Research Fellow of the Council of Scientific and
Industrial Research (India). K.P.K. was supported by a D. S.
Kothari postdoctoral fellowship from the University Grants
Commission (India). Use of the National Single Crystal
Diffractometer Facility at the School of Chemistry, University
of Hyderabad, funded by the DST (India), is gratefully
acknowledged. The UGC (India) is acknowledged for their
support through the UPE and CAS programs to the University
of Hyderabad and School of Chemistry, respectively.
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SUMMARY AND CONCLUSIONS
■
In the present study, we have synthesized a homologous series
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ASSOCIATED CONTENT
■
S
* Supporting Information
1
Assignment of IR resonances, H and 13C NMR data, and d-
spacings for NAGs, atomic coordinate and isotropic displace-
ment parameters, selected bond distances and angles, and
torsion angles for NMG and NPG, representative IR, NMR,
PXRD, and hydrogen bonding data for select NAGs, and
crystallographic information in CIF format. This material is
available free of charge via Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
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Burstein, S.; Walker, J. M. J. Lipid Res. 2010, 51, 112−119.
(23) Larsson, K. In Physical Properties−Structural and Physical
Characteristics. The Lipid Handbook; Gunstone, F. D., Harwood, J.
*Prof. Musti J. Swamy. Tel: +91-40-2313-4807. Fax: +91-40-
2301-2460. E-mail: mjssc@uohyd.ernet.in; mjswamy1@gmail.
com.
Notes
The authors declare no competing financial interest.
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