Differential scanning calorimetric and powder X-ray diffraction studies on a homologous series of N-acyl-L-alanine esters with matched chains (n = 9-18)

Article abstract of DOI:10.1007/s12039-015-0928-5

A homologous series of two chain derivatives of L-alanine, namely N-acyl L-alanine alkyl esters (NAAEs), bearing matched, saturated, acyl and alkyl chains (n= 9-18) have been synthesized. The thermotropic phase transitions and supramolecular structure of NAAEs were investigated by differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD). Results obtained from DSC studies indicate that the transition temperatures (T _t), enthalpies (ΔH _t) and entropies (ΔS _t) exhibit odd-even alternation with compounds bearing odd acyl and alkyl chains showing higher values of T _t, ΔH _t and ΔS _t as compared to NAAEs with even acyl and alkyl chains. However, the transition enthalpies and entropies of the odd- and even chain length series independently exhibit a linear dependence on the chain length. The d-spacings obtained from PXRD increase linearly with chain length with an increment of 1.76 ?/CH ₂, suggesting that NAAEs adopt either a tilted bilayer structure or a bent structure. The present results provide a thermodynamic and structural basis for investigating the interaction of NAAEs with other membrane lipids, which in turn can shed light in understanding how they can enhance the transdermal permeability of stratum corneum.

Full text of DOI:10.1007/s12039-015-0928-5

J. Chem. Sci. Vol. 127, No. 9, September 2015, pp. 1627–1635. ꢀc Indian Academy of Sciences.
DOI 10.1007/s12039-015-0928-5
Differential scanning calorimetric and powder X-ray diffraction studies
on a homologous series of N-acyl-L-alanine esters with matched chains
(
n = 9-18)
∗
D SIVARAMAKRISHNA and MUSTI J SWAMY
School of Chemistry, University of Hyderabad, Hyderabad 500 046, India
e-mail: mjswamy@uohyd.ac.in; mjswamy1@gmail.com
MS received 9 May 2015; revised 22 June 2015; accepted 23 June 2015
Abstract. A homologous series of two chain derivatives of L-alanine, namely N-acyl L-alanine alkyl esters
(
NAAEs), bearing matched, saturated, acyl and alkyl chains (n = 9-18) have been synthesized. The ther-
motropic phase transitions and supramolecular structure of NAAEs were investigated by differential scanning
calorimetry (DSC) and powder X-ray diffraction (PXRD). Results obtained from DSC studies indicate that
the transition temperatures (T ), enthalpies (ꢀH ) and entropies (ꢀS ) exhibit odd-even alternation with com-
t
t
t
pounds bearing odd acyl and alkyl chains showing higher values of Tt, ꢀHt and ꢀSt as compared to NAAEs
with even acyl and alkyl chains. However, the transition enthalpies and entropies of the odd- and even chain
length series independently exhibit a linear dependence on the chain length. The d-spacings obtained from
PXRD increase linearly with chain length with an increment of 1.76 Å/CH , suggesting that NAAEs adopt
2
either a tilted bilayer structure or a bent structure. The present results provide a thermodynamic and structural
basis for investigating the interaction of NAAEs with other membrane lipids, which in turn can shed light in
understanding how they can enhance the transdermal permeability of stratum corneum.
Keywords. N-acyl-L-alanine esters; DSC; odd-even alteration; tilted chain packing; PXRD; d-spacing.
1
. Introduction
and alkyl chains bearing 12 C-atoms enhanced the per-
meability by ∼12.5 fold, the corresponding serine ana-
log showed <3 fold increase in the permeability. The
lower ability of the serine analog to increase perme-
ability of stratum corneum was explained due to its
hydrogen bonding (through the hydroxyl group) with
the hydroxyl group of ceramides present in the stratum
The stratum corneum, which is the outermost layer of
the skin, is composed of 15-20 layers of flattened, dead
cells with no nuclei and cell organelles. The main
composition of the stratum corneum is free fatty acids,
sterols and ceramides. Ceramides are considered to be
the key molecules for lipid lamellar organization, resis-
tance to chemical and physical stress and environmen-
tal changes. Transdermal drug action depends on the
percentage of the permeable drug through the stratum
corneum. N-acyl amino acid esters are a new class of
lipids, which are structurally similar to the ceramides
with a small polar head group and two saturated,
unbranched and long N-acyl chain and alkyl ester. N-
acyl glycine alkyl esters (NAGEs) and N-acyl serine
alkyl esters (NASEs) have been investigated to evalu-
ate their ability to enhance the permeability of the stra-
1
2
4
corneum. Replacing the hydroxyl group with a hydro-
gen (which would convert the serine analogs to alanine
analogs) would remove the hydrogen bonding ability
of these molecules which in turn increase their ability
to enhance the permeability of the stratum corneum. In
view of this, in the present study, we have synthesized
N-acyl L-alanine esters (NAAEs) with matched acyl
and alkyl chains (n = 9-18), which are homologous to
NAGEs and exhibit similar hydrogen bonding ability
with a slightly increased head group size as compared
to the glycine derivatives.
The above observations suggest that NAAEs may be
useful in the formulation of transdermal drug delivery
systems. In order to understand how NAAEs can be
used in such an application it is necessary to explore the
properties of these molecules in a systematic manner.
Most useful in this context would be studies aimed at
understanding the phase behavior and supramolecular
3
4
tum corneum. When present in the drug formulation,
such compounds can facilitate transdermal drug deliv-
ery by increasing the permeability of stratum corneum.
It has been found that while NAGE with matched acyl
∗
For correspondence
1627

1628
D Sivaramakrishna and Musti J Swamy
organization and intermolecular interactions exhibited 10% sodium carbonate and then dried with anhydrous
by them. In the present study the thermotropic phase Na SO . The product obtained was recrystallized from
2
4
transitions of NAAEs have been characterized by dif- acetone containing HCl followed by only acetone at
◦
ferential scanning calorimetry (DSC), which revealed −20 C.
an unusual odd-even alternation in the transition tem-
peratures, enthalpies and entropies. In addition, powder
N-acylation of L-alanine ester: L-alanine esters were
X-ray diffraction studies suggest that NAAEs are most
prepared according to the procedure reported by Sivara-
likely packed either in a tilted bilayer structure, sim-
ilar to the organization found in the crystal structure
of phosphatidylglycerols, or a bent structure with the
acyl and alkyl chains facing each other as seen in the
7
makrishna et al. First, the fatty acyl chlorides were
prepared from the corresponding fatty acids by react-
8
ing with oxalyl chloride. The acid chloride was then
reacted with L-alanine ester hydrochloride (1.0 mmol)
in the presence of sodium bicarbonate (2 mmol) dis-
solved in a mixture of chloroform and water (2:1). After
stirring for 2-3 h, the chloroform layer was washed suc-
cessively with the following: double distilled water, sat-
urated brine solution, 0.1 M HCl and double distilled
water. The product obtained was recrystallized from
3
-dimensional structure of N, O-diacylethanolamines
(
DAEs).
2. Experimental
2.1 Materials
◦
dichloromethane at −20 C. Overall yields of different
Long chain alcohols (C9-C18), p-toluenesulphonic NAAEs ranged around 80-90%. The final product thus
acid monohydrate and long chain fatty acids (C9- obtained was filtered and dried by vacuum desiccation,
1
13
C18) were purchased from Aldrich (Milwaukee, WI, and characterized by FTIR, H-NMR, C-NMR and
USA). L-alanine and oxalyl chloride were purchased HRMS.
from Merck (Germany). Other chemicals and solvents
IR spectra were recorded on a Jasco FTIR 5300 Spec-
1
13
(analytical grade) were purchased from local chemical trometer using KBr pellets of the samples. H- and C-
suppliers.
NMR spectra were recorded on a Bruker Avance NMR
spectrometer operating at 400 and 100 MHz, respec-
tively, using CDCl as the solvent. Capillary melt-
ing points of the NAAEs were recorded on a Superfit
3
2.2 Synthesis of N-acyl-L-alanine esters
(
Mumbai, India) melting point apparatus as described
N-acyl-L-alanine esters were synthesized in two steps.
First L-alanine was converted to the ester, and the prod-
uct was N-acylated to yield the N-acyl-L-alanine ester
7
earlier.
2
.3 DSC of dry NAAEs
(scheme 1).
DSC studies with dry NAAEs were carried out on
Ester preparation: L-alanine esters were prepared a Perkin–Elmer Pyris Diamond differential scanning
5
,6
according to a procedure reported earlier.
L- calorimeter. Samples (1-2 mg) were weighed accu-
alanine (1.0 mmol), 1-alcohol (1.0 mmol) and p- rately into aluminum sample pans and covered with
toluenesulphonic acid monohydrate (1.2 mmol) were aluminum lids and sealed by crimping. Reference pans
◦
taken in about 30 mL of toluene and refluxed at 110 C. were prepared similarly, but without any sample in
After 5-6 h of refluxing, the solvent was removed them. For long chain NAAEs (n = 11-18), heating
◦
by rotary evaporation under reduced pressure. The scans were performed between 25 and 100 C, whereas
residue was dissolved in chloroform and washed with for N-nonanoyl-L-alanine nonyl ester (NNANE) and
H N Cl
O
3
O
H O:CHCl
H
N
2
3
R
R
O
O
RT, 2-3 hrs
R
Cl
O
O
R
L-alnine alkyl
ester.HCl
Acid chloride
N-acyl-L-alanine alkyl ester
Scheme 1. Synthesis of NAAEs.

DSC and PXRD studies on N-acyl-L-alanine esters
1629
6
N-decanoyl-L-alanine decyl ester (NDADE) heating sodium dodecyl sulfate. Interestingly, N-acyl amino
◦
scans were performed between 10 and 65 C. A scan acid esters such as N-acyl glycine esters and N-acyl
◦
rate of 2.0 /min was used in all DSC experiments. For serine esters were found to increase the permeability
each sample three heating and two cooling scans were of stratum corneum to topically applied drugs, which
recorded. Except for NNANE and NDADE, all heating increases the drug efficacy. In particular, N-acyl glycine
scans gave similar results; therefore, in all cases the first ester with matched, saturated acyl and alkyl chains was
heating scan was considered for further analysis. Tran- reported to exhibit the highest effect in enhancing the
4
sition enthalpies (ꢀH ) were determined by integrat- permeability. In view of this, in the present study we
t
ing the peak area under the transition curve. Transition have synthesized a homologous series of N-acyl L-
entropies (ꢀS ) were determined from the transition alanine alkyl esters with matched alkyl and acyl chains
t
enthalpies assuming a first order transition according to and characterized their phase transitions and structural
9
the expression:
properties in detail. The results obtained are presented
below.
ꢀ
H = T .ꢀS
(1)
t
t
t
where T is the transition temperature and the ꢀH val-
ues were taken at this temperature in order to calculate
t
t
3.1 Synthesis and characterization of NAAEs
ꢀSt values.
FTIR spectrum of N-decanoyl-L-alanine decyl ester
(NDADE) is shown in figure S1, in Supplementary
Information. The spectra obtained with other NAAEs
were qualitatively very similar. IR spectra of NAAEs
contained absorption bands due to ester carbonyl group
2
.4 Powder X-ray diffraction studies
Powder X-ray diffraction patterns of NAAEs were
recorded on a Bruker SMART D8 Advance powder X-
ray diffractometer (Bruker-AXS, Karlsruhe, Germany)
with Cu-Kα radiation at 40 kV and 30 mA. Fine pow-
ders of NAAEs, obtained by grinding with mortar and
pestle, were placed on a circular rotating disk of the
sample holder. Diffraction patterns were collected at
−1
−1
at 1736-1747 cm , amide linkage at 1638-1654 cm
−1
and C-H stretching at 2843-2958 cm . C-H scissoring
−
1
and rocking bands were observed at 1468-1473 cm
−1
and 717-723 cm , respectively. The N-H stretching
−1
bands were observed at 3314-3320 cm . The IR reso-
nances obtained for the homologous series of NAAEs
investigated here are listed in table S1.
◦
room temperature (∼25 C) using a LynxEye PSD data
◦
collector over a 2θ range of 1-50 with a step size of
1
H-NMR spectrum of N-decanoyl-L-alanine decyl
◦
0.0198 and a measuring time of 1.5s for each step.
ester (NDADE) is shown in figure S2. Since all the
NAAEs investigated here are chemically very similar
and differ only in the number of methylene units in the
acyl/alkyl chain, their NMR spectra were almost iden-
3
. Results and Discussion
A large number of amphiphiles derived from amino tical except for the integration value of the peak cor-
1
acids such as N-acyl amino acids bearing both responding to a part of the polymethylene moiety. H
saturated and unsaturated chains in acyl moiety NMR spectra of the NAAEs gave the following res-
(e.g., N-arachdonyl alanine, N-palmitoylglycine, N- onances: 0.869-0.903δ (6H, t), 1.256-1.281δ (nH, m),
oleoylserine) have been reported to be present in mam- 1.391-1.413δ (3H, d), 1.59-1.65δ (4H, m), 2.172-2.22δ
10–12
malian tissues.
In view of this, it is of inter- (2H, t), 4.13-4.161 δ (2H, t), 4.59-4.618δ (1H, q), 6.01-
est to investigate their physicochemical properties and 6.151δ (1H, d). These resonances are consistent with
1
interaction with other membrane constituents. In pre- the structure of NAAEs. The H NMR chemical shifts
vious work, we have synthesized and characterized obtained for the NAAEs with matched chains are listed
the self assembly, supramolecular organization and in table S2.
1
3
thermotropic phase behavior of N-acylalanines and
N-acylglycines.
A C NMR spectrum of NDADE is given in figure
7
,13
While N-acylation of glycine and S3. Resonances corresponding to the N-acyl chain and
alanine yields anionic amphiphiles, esterification of O-alkyl chains are seen in the spectrum at: ∼14.09δ
these amino acids with long chain alcohols would give (both terminal methyls), ∼22.66δ (methylenes α to
cationic amphiphiles which may find use in develop- terminal methyls), 36.58δ (C-atom α to the amide
ing catanionic lipid based drug delivery systems. There- carbonyl), 65.63δ (C-atom adjacent to the ester oxy-
fore, we have also synthesized a homologous series gen), and 4-5 closely spaced resonances of varying
of L-alanine alkyl esters and characterized them with intensity between 25 and 32δ for the remaining
respect to aggregation properties in aqueous disper- methylene groups. Resonances of the alanine moi-
sion, 3-dimensional structure as well as interaction with ety are seen at ∼47.97δ (chiral C-atom) and ∼18.70δ

1630
D Sivaramakrishna and Musti J Swamy
(
a)
(b)
C 17
C 18
C 16
C 14
C 15
C 13
C 11
C 9
C 12
C 10
40
50 60 70 80 90
c)
40 50 60 70 80 90
(
(d)
C 17
C 15
C 13
C 18
C 16
C 14
C 11
C 9
C 12
C 10
20
40
60
80
20
40
60
80
100
o
Temperature ( C)
Figure 1. DSC heating (a, b) and cooling (c, d) thermograms of dry NAAEs
with odd (a, c) and even (b, d) number of C-atoms in the acyl chain. The number
of C-atoms is indicated against each thermogram.
(
methyl group). The ester and amide carbonyl reso- the corresponding cooling thermograms are shown in
13
nances are seen around 172.69δ and 173.35δ. The C figures 1c and 1d, respectively. This figure shows that
NMR spectra of other NAAEs were qualitatively very both odd and even chain NAAEs exhibits single sharp
similar and were consistent with the expected struc- transitions, which matched well with the capillary melt-
tures. The C NMR chemical shifts of different NAAEs ing points of the compounds. When the same samples
are listed in table S3. were subjected to second and third heating scans, except
13
A high resolution mass spectrum of NDADE is pre- the compounds with 9 and 10 C-atoms in the matched
sented in figure S4. The two most intense peaks seen chains, all heating scans gave similar results with minor
at m/z = 384.3462 and 406.3296 match well with the decreases being observed in the transition enthalpies.
+
molecular ion of the compound ([M+H] , calculated Therefore, in all cases the first heating scan was consid-
+
mass = 384.61) its sodium adduct ([M+Na] , calcu- ered for further analysis and the transition temperatures
lated mass = 406.60). Other NAAEs with different (T ), enthalpies (ꢀH ) and entropies (ꢀS ) obtained
t
t
t
matched acyl and alkyl chains also yielded essentially from the first heating thermograms are presented in
similar results and the mass spectrometric data for them table 1.
are listed in table S4.
3.3 Chain length dependence of transition enthalpy
3.2 Differential scanning calorimetry
and transition entropy
Heating thermograms of dry NAAEs with odd and The effect of varying the chain length on the transition
even acyl chains are shown in figures 1a and 1b and enthalpies (ꢀH ) and transition entropies (ꢀS ) of dry
t
t

DSC and PXRD studies on N-acyl-L-alanine esters
1631
Table 1. Average values of transition temperatures (Tt),
transition enthalpies (ꢀHt) and transition entropies (ꢀSt) of
NAAEs in the dry state. Values in parentheses correspond to
standard deviations from three independent measurements.
Table 2. Incremental values (ꢀHinc, ꢀSinc) of chain
length dependence and end contributions (ꢀHo, ꢀSo) to
phase transition enthalpy and entropy of NAAEs in the
dry state. Values in parentheses correspond to fitting errors
obtained from the least squares analysis.
Acyl chain
Dry NAAEs
o
Thermodynamic
parameter
Dry NAAEs
length (n)
Tt ( C)
ꢀHt (kcal/mol) ꢀSt(cal/mol/k)
Odd chain length Even chain length
9
43.4 (0.4)
47.9 (0.1)
59.9 (0.1)
62.8 (0.2)
71.2 (0.1)
72.9 (0.4)
79.5 (0.2)
80.6 (0.2)
85.7 (0.2)
86.2 (0.1)
12.43 (0.01)
13.33 (0.15)
16.28 (0.29)
17.68 (0.24)
20.88 (0.18)
21.33 (0.39)
24.14 (0.23)
25.01 (0.09)
28.59 (0.14)
28.46 (0.19)
39.3 (0.1)
41.5 (0.4)
48.9 (0.9)
52.8 (0.8)
60.6 (0.5)
61.7 (1.1)
68.5 (0.7)
70.7 (0.2)
79.7 (0.4)
78.8 (0.6)
10
11
12
13
14
15
16
17
18
ꢀHinc (kcal/mol)
2.01 (0.05)
−1.64 (0.59)
5.02 (0.15)
4.18 (1.67)
1.88 (0.05)
−1.39 (0.57)
4.62 (0.16)
5.60 (2.02)
ꢀH (kcal/mol)
o
ꢀSinc (cal/mol/K)
ꢀS (cal/mol/K)
o
series such as long chain fatty acids, hydrocarbons,
alcohols, N-acylethanolamines, N-acyldopamines, N-
acylserotonins and N, O-diacylethanolamines values
of thermodynamic parameters for the even chain-
length series are higher as compared to those of the
14–18
(b)
odd chain length series.
However, we recently
7
6
5
0
reported this kind of odd-even alteration in a homol-
ogous series of N-acyl-L-alanines. Similar results
7
were observed earlier with phosphatidylcholines con-
taining ω-tertiary-butyl fatty acyl chains and dl-methyl
anteisobranched fatty acyl chains in the hydrated
4
3
5
0
19,20
state.
The ꢀH and ꢀS data for odd and even
t t
acyl chain length NAAEs could be fit well to expres-
(a)
14
sions (2) and (3) given below as reported previously
with N-acylethanolamines, N-acyldopamines, and N-
acylserotonins with even and odd acyl chain lengths,
O-acylcholines with even chainlengths as well as N-,
O-diacylethanolamines with matched as well as mixed
2
2
1
5
0
5
15–18,21–23
acyl chains.
ꢀ
H = ꢀH + (n − 2)ꢀH
(2)
(3)
t
o
inc
6
8
10
12
14
16
Chain length (
n
-2)
ꢀS = ꢀS + (n − 2)ꢀS
t
o
inc
where ꢀH and ꢀS are the end contributions to ꢀH
t
o
o
Figure 2. Chain length dependence of transition enthalpy
and ꢀS , respectively, arising from the terminal methyl
t
(a) and transition entropy (b) of dry NAAEs. Values of ꢀHt
and ꢀSt were plotted against the number of methylene (CH2) group of the acyl/alkyl chain and the head group region.
units (n-2). Solid lines correspond to linear least squares fits ꢀHinc and ꢀSinc are the incremental values of ꢀH and
t
of the data.
ꢀ
S per CH group. Values of ꢀH , ꢀS , ꢀH and
t 2 inc inc o
ꢀ
So corresponding to NAAEs with odd and even acyl
NAAEs is shown in figures 2a and 2b, respectively. It chains, obtained from linear least squares analysis, are
is seen that, both ꢀH and ꢀS exhibit an essentially lin- given in table 2.
t
t
ear dependence on the chain length independently for
When the transition enthalpies and transition entro-
even and odd chain length series. However, when all the pies of NAAEs are compared with those corresponding
data of odd and even chain NAAEs are viewed together, to the single chain amphiphiles derived from L-alanine
a zig-zag pattern is seen in the enthalpy and entropy (N-acyl-L-alnines and L-alanine alkyl esters),6 it is
values. The ꢀH and ꢀS for the even chain length seen that NAAEs exhibit higher values of ꢀH and
,7
t
t
t
series are found to be slightly lower than those of odd ꢀS for the solid to liquid transition, which could be
t
chain length series, i.e., the calorimetric data exhibit attributed to the high molecular weight of the NAAEs.
odd-even alteration. This sort of odd-even alteration On the other hand, when compared to the N, O-diacy-
is a rare observation since in many other homologous lethanolamines (DAEs, which differ from the NAAEs

1632
D Sivaramakrishna and Musti J Swamy
only in the head group region, due to the presence of
the α-methyl group and the reversal of the connectiv-
ity between the ester carbonyl and oxygen) the corre-
sponding values are lower, which could be due to the
bulkier head group (due to methyl substitution), which
could prevent tight packing of the NAAE molecules in
the crystal lattice.
8
0
60
40
A linear chain length dependence of the ꢀH and
t
ꢀSt observed here for the dry NAAEs of odd and
even chain lengths indicate the structures of NAAEs
within each series are likely to be very similar in the
solid state. Therefore, three dimensional structure deter-
mination of any one molecule in each series of NAAEs
can give a good idea of the molecular packing and inter-
molecular interactions present in the crystal structure
10
12
14
16
18
Chain length (n)
Figure 3. Chain length dependence of chain-melting phase
transition temperatures of NAAEs in the dry state. Solid lines
correspond to nonlinear least-squares fit of the transition
temperatures to eq. 7.
in each series. Odd-even alternation in the ꢀH and
t
ꢀSt can be explained based on differences in molecu-
lar packing between even and odd chain length com-
pounds. When the long alkyl/acyl chains are tilted with
respect to methyl end group planes then the van der
Waals interactions between methyl groups from oppo-
site layers are usually different for the even- and odd
chain length series.2 Therefore, odd-even alternation
is clearly observed when the alkyl chain packing is
inclined with respect to the methyl end planes, but not
when they are packed perpendicular to the methyl end
series exhibits higher values of calorimetric properties
as compared to the even-chain-length series as seen
with N-acyl-L-alanines and L-alanine alkyl esters.6
As the acyl/alkyl chainlength increases, the end con-
tributions towards the total enthalpy and entropy of the
phase transition will be very small as compared to the
contributions due to the polymethylene portion, such
that the former are negligible in comparison. Therefore,
at infinite acyl chain length, equations 2 and 3 can be
,7
4,25
14,25
planes.
The odd-even alternation in the calorimet-
ric properties of dry and hydrated NAAEs as observed
here suggests that the acyl and/or alkyl chains are tilted
with respect to the respective methyl end planes in
them. In addition, our results suggest that the termi-
nal methyl groups of opposing layers of the acyl and/or
alkyl chains are more closely packed in the NAAEs
with odd chain lengths as compared to those with even
chain lengths.
14
reduced to equations (4) and (5):
ꢀH
= (n − 2)ꢀHinc
(4)
(5)
t
ꢀ
S = (n − 2)ꢀS
t
inc
Then the transition temperature at infinite chain-
∞
length, T , can be obtained from:
t
∞
Tt = ꢀH /ꢀS
(6)
inc
inc
3
.4 Chain length dependence of transition
Incorporating the values of ꢀH and ꢀSinc from
table 1 into eq. (6), the Tt values for the NAAEs of
temperature
inc
∞
A plot depicting the chain length dependence of tran- odd and even acyl chain lengths have been estimated as
sition temperatures of NAAEs in the dry state is given 400.4 and 406.9 K, respectively.
in figure 3. This plot shows a zig-zag pattern with the
For a variety of one-chain and two-chain amphi-
odd-chain length NAAEs displaying higher T values philes/lipids, whose transition enthalpy and transition
t
as compared to the even-chain length NAAEs, i.e., the entropy exhibit linear dependence on the chain length,
T values also exhibit odd-even alternation. The T val- it has been shown that the ꢀH
ues increase in a smooth progression in each series but- fit to the following equation:
and ꢀS
Values can be
t
t
t
t
26
the increments between successive values in each series
decrease with the increase in the chain length. Although
∞
ꢁ
ꢁ
T = ꢀH /ꢀS = T [1 − (n − n )/(n − n )] (7)
t
t
t
t
o
o
o
ꢁ
it has been observed that compounds in the even-chain- where n (=–ꢀH /ꢀH ) and n (=–ꢀS /ꢀS ) are
o
o
inc
o
o
inc
length series exhibit higher values of the calorimet- the values of n at which ꢀH and ꢀS extrapolate to
t
t
ric properties (T , ꢀH and ꢀS ) compared to the odd zero. It can be seen from figure 3 that the T values of
t
t
t
t
chain length molecules in many cases, it is quite inter- dry NAAEs containing even as well as odd number of
esting to note that for NAAEs, the odd-chain length C-atoms in the acyl/alkyl chains independently fit quite

DSC and PXRD studies on N-acyl-L-alanine esters
1633
4
3
3
2
2
0
5
0
5
0
(
a)
(b)
(c)
C 17
C 15
C 18
C 16
C 14
C 13
C 11
C 12
C 9
30
C 10
30
1
0
20
10
20
10 12 14 16 18
2
Chain length (n)
Figure 4. Powder X-ray diffraction patterns of N-acyl-L-alanine esters with different saturated acyl
chains (a, b) and dependence of d-spacings on the chain length (c). The number of C-atoms in the satu-
rated, unbranched acyl chain is indicated against each PXRD profile. The solid line in C represents a linear
least squares fit of the data. The slope of this line yielded increase in the d-spacing per each additional CH2
group as 0.88 Å.
well with eq. (7). In addition, the fitting parameters also Table 3. d-Spacing values of NAAEs determined from
∞
PXRD data.
yielded the Tt values for NAAEs of both odd and even
chain lengths in the dry state as 404.8 K and 402 K.
These values are in good agreement with the Tt values
Acyl chain length (n)
d-spacing (Å)
∞
estimated using eq. (6), presented above.
9
22.09 (0.19)
23.87 (0.16)
25.43 (0.08)
27.27 (0.19)
28.89 (0.19)
30.91 (0.23)
32.67 (0.25)
34.11 (0.46)
36.44 (0.81)
37.75 (0.42)
10
11
12
3.5 Powder X-ray diffraction
Our attempts to obtain good quality single crystals of 13
14
15
16
17
18
NAAEs using different solvents/combinations did not
yield much success as we got only crystalline flakes,
which showed very weak diffraction. Therefore, we
carried out X-ray diffraction data on finely powdered
samples in order to gather some insights on the struc-
tural aspects of NAAEs in the solid state. The PXRD
data obtained for NAAEs of different chain lengths
If the molecules were to pack in a normal bilayer
◦
are shown in figure 4a, b (2θ range = 1 − 30 ). All format, with the acyl as well as alkyl chains oriented
NAAEs (n=9-18) gave several sharp diffraction peaks. parallel to each other, then the incremental increase in
It was observed that the diffraction peaks move toward the d-spacing corresponding to the addition of a CH2
lower 2θ values with the increase in the length of the group in each chain would be 2.54 Å. This would then
◦
acyl/alkyl chains from 9 C-atoms to 18 C-atoms (3.95
correspond to an increase in the d-spacing by 1.27 Å
◦
to 2.39 for n = 1). In each case 4-5 peaks were used to for each CH group since there will be two molecules
2
calculate the d-spacings. The average d-spacing values in the two leaflets of the bilayer. The increment in d-
obtained are given in table 3 and a plot of chain length spacing observed for the NAAEs are 0.88 Å, which is
dependence of the d-spacing is shown in figure 4c. The significantly smaller than the above value, suggesting
plot shows that the d-spacings exhibit a linear depen- that the bilayer must be tilted similar to that seen in the
dence on the acyl/alkyl chain length with a slope of 1.76 structure of dimyristoylphosphatidylglycerol (DMPG)
Å/CH , which corresponds to an increment of 0.88 Å and cerebroside, in which the hydrocarbon chains are
2
◦
per additional CH moiety in each acyl chain.
tilted with respect to the bilayer normal by 29 and
2

1634
D Sivaramakrishna and Musti J Swamy
◦
27,28
4
9 , respectively.
From the above slope the chain tilt DS was supported by Senior Research Fellowships
angle with respect to the bilayer normal was calculated from the Council of Scientific and Industrial Research
◦
as 46.1 . This value is closer to the tilt angle observed (India). The University Grants Commission (India) is
in cerebroside and considerably higher than that found acknowledged for its support through the UPE and CAS
in DMPG.
programs, to University of Hyderabad and School of
An alternative packing arrangement for the Chemistry, respectively.
NAAEs would be similar to thatobserved in N, O-
diacylethanolamines with matched chains, which adopt
References
a bent structure with the O-acyl chains being oriented
16
at an angle with respect to the N-acyl chains. In the
1. Bouwstra J A and Honeywell-Nguyen P L 2002 Adv.
Drug Deliv. Rev. 54 S1–S41
supramolecular organization of DAE molecules in the
16
2. Lampe M A, Burlingame A L, Whitney J, Williams M
L, Brown B E, Roitman E and Elias P M 1983 J. Lipid
Res. 24 120
crystal, the N-acyl and O-acyl chains face each other.
The present PXRD data on NAAEs would be consis-
tent with both these packing arrangements and further
studies are required to distinguish between them.
3. Elias P M 1983 J. Invest. Dermatol. 80 44
4. Vavrova K, Hrabalek A, Dolezal P, Holas T and
Zbytovska J 2003 Bioorg. Med. Chem. Lett 13 2351
5
. Minakuchi N, Hoe K, Yamaki D, Ten-no S, Nakashima
K, Goto M, Mizuhata M and Maruyama T 2012
Langmuir 28 9259
3.6 Conclusions
In the current work, we have synthesized and charac-
terized a homologous series of N-acyl-L-alanine esters
bearing saturated acyl and alkyl chains with matched
chainlengths (n = 9-18). An unusual odd-even alter-
nation was observed in the transition temperatures,
enthalpies and entropies of NAAEs in the dry state
with the ꢀH and ꢀS values corresponding to the odd-
6. Sivaramakrishna D and Swamy M J 2015 Langmuir 31
9546
7
. Sivaramakrishna D, Reddy S T, Nagaraju T and Swamy
M J 2015 Colloids and Surfaces A: Physicochem. Eng.
Aspects 471 108
. Akoka S, Tellier C, Le Roux C and Marion D 1988
Chem. Phys. Lipids 46 43
9. Marsh D 1990 In Handbook of Lipid Bilayers (Florida:
CRC Press) p. 135
0. Huang S M, Bisogno T, Petros T J, Chang S-Y,
Zavitsanos P A, Zipkin R E, Sivakumar R, Coop A,
Maeda D Y, De Petrocellis L, Burstein S, Di Marzo V
and Walker J M 2001 J. Biol. Chem. 276 42639
8
t
t
chain-length series being higher than those obtained for
1
the even-chain-length compounds. Values of T , ꢀH
t
t
and ꢀS for both odd- and even-chain-length series
t
exhibit linear dependence on the acyl chain length, sug-
gesting that the molecular packing and intermolecular 11. Rimmerman N, Bradshaw H B, Hughes H V, Chen
J S-C, Hu S S-J, McHugh D, Vefring E, Jahnsen J A,
Thompson E L, Masuda K, Cravatt B F, Burstein S,
Vasko M R, Prieto A L, O’Dell D K and Walker J M
interactions would be rather similar for all the NAAEs
in each series. Powder X-ray diffraction studies sug-
gest that all NAAEs with matched acyl and alkyl chains
2008 Mol. Pharmacol. 74 213
(n = 9-18) adopt either a tilted bilayer structure or a
1
2. Smoum R, Bar A, Tan B, Milman G, Attar-Namdar M,
Ofek O, Stuart J M, Bajayo A, Tam J, Kram V, O’Dell
D, Walker M J, Bradshaw H B, Bab I and Mechoulam R
bent structure. These observations provide a thermody-
namic and structural perspective for understanding the
phase behavior of NAAEs and for investigating their
interaction with other membrane lipids, which in turn
can shed light on how these amphiphies can enhance
the transdermal permeability of stratum corneum.
2010 Proc. Natl. Acad. Sci. U S A 107 17710
1
3. Reddy S T, Krovi K P and Swamy M J 2014 Cryst.
Growth Des. 14 4944
1
4. Larsson K 1986 In The Lipid Hand-book F D Gunstone,
J L Harwood and F B Padley (Eds.) (London: Chapman
and Hall) p. 321
Supplementary Information
15. Ramakrishnan M, Sheeba V, Komath S S and Swamy
M J 1997 Biochim. Biophys. Acta 1329 302
1
13
Representative FTIR, H-NMR, C-NMR and HRMS
spectra of N-decanoyl-L-alanine decyl ester are given
in Figures S1–S4. Corresponding spectral data for all
NAAEs (n = 9-18) are given in tables S1–S4. Sup-
plementary Information is available at www.ias.ac.in/
chemsci.
1
1
1
1
6. Kamlekar R K, Tarafdar P K and Swamy M J 2010 J.
Lipid Res. 51 42
7. Reddy S T, Tarafdar P K, Kamlekar R K and Swamy
M J 2013 J. Phys. Chem. B 117 8747
8. Reddy S T and Swamy M J 2015 Biochim. Biophys. Acta
1848 95
9. Lewis R N A H, Mantsch H H and McElhaney R N 1989
Biophys. J. 56 183
Acknowledgements
20. Lewis R N A H, Sykes B D and McElhaney R N 1987
Biochemistry 26 4036
1. Marsh D and Swamy M J 2000 Chem. Phys. Lipids 105
This work was supported by a research grant from the
Department of Science and Technology (India) to MJS.
2
43

DSC and PXRD studies on N-acyl-L-alanine esters
1635
2
2
2. Tarafdar P K, Reddy S T and Swamy M J 2012 Cryst. 26. Marsh D 1982 In Supramolecular Structure and Func-
Growth Des. 12 1132
3. Tarafdar P K, Reddy S T and Swamy M J 2013 J. Phys.
Chem. B 117 9900
tion G Pifat and J N Herak (Eds.) (Plenum Press: New
York) p. 127
27. Pascher I, Sundell S, Harlos K and Eibl H 1987 Biochim.
Biophys. Acta 896 77
24. Larsson K 1996 Acta Crystallogr. 21 267
5. Larsson K 1966 J. Am. Oil Chem. Soc 43 559
2
28. Pascher I and Sundell S 1977 Chem. Phys. Lipids 20 175