Characterization of the molecular packing, thermotropic phase behaviour and critical micellar concentration of a homologous series of N-acyltaurines (n = 9–18). PXRD, DSC and fluorescence spectroscopic studies

Article abstract of DOI:10.1016/j.chemphyslip.2020.104929

N-acyltaurines (NATs) are amides of fatty acids that can be structurally related to endocannabinoids. They show interesting physiological and pharmacological properties. We have synthesized a homologous series of NATs with saturated acyl chains (n = 9–18) and investigated their supramolecular structure and thermotropic phase transitions by powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC). The d-spacings obtained from PXRD increase linearly with chain length with an increment of ～0.847 ? per additional CH₂ moiety suggesting that NATs adopt a tilted bilayer structure with similar packing in crystal lattice. Results obtained from DSC studies indicate that the endothermic transition temperature (T_t) of NATs showed a gradually increasing trend with increasing acyl chain length. The enthalpy (ΔH_t) and entropy (ΔS_t) of transition show odd-even alternations with odd-chain compounds having higher values than the even-chain compounds. The critical micellar concentration (CMC) of NATs was determined in water at room temperature by fluorescence spectroscopy by monitoring the spectral changes of 8-anilinonaphthalene-1-sulfonic acid (ANS). The CMCs of NATs were found to decrease with increase in acyl chain length. The present results provide a thermodynamic and structural basis for investigating the interaction of NATs with other membrane lipids and proteins, which in turn can shed light in understanding how they function in vivo (in biological membranes).

Full text of DOI:10.1016/j.chemphyslip.2020.104929

Journal Pre-proof
Characterization of the molecular packing, thermotropic phase behaviour
and critical micellar concentration of a homologous series of
N-acyltaurines (n = 9-18). PXRD, DSC and fluorescence spectroscopic
studies
Sukanya Arul Prakash, Ravi Kanth Kamlekar
PII:
S0009-3084(20)30060-8
DOI:
https://doi.org/10.1016/j.chemphyslip.2020.104929
CPL 104929
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Chemistry and Physics of Lipids
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Revised Date:
Accepted Date:
26 January 2020
30 April 2020
12 May 2020
Please cite this article as: Prakash SA, Kamlekar RK, Characterization of the molecular
packing, thermotropic phase behaviour and critical micellar concentration of a homologous
series of N-acyltaurines (n = 9-18). PXRD, DSC and fluorescence spectroscopic studies,
Chemistry and Physics of Lipids (2020),
doi: https://doi.org/10.1016/j.chemphyslip.2020.104929
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Characterization of the molecular packing, thermotropic phase behaviour and
critical micellar concentration of a homologous series of N-acyltaurines (n = 9-18).
PXRD, DSC and fluorescence spectroscopic studies
Sukanya Arul Prakash, Ravi Kanth Kamlekar*
*Corresponding Author: Ravi Kanth Kamlekar
Department of Chemistry,
School of Advanced Sciences,
VIT, Vellore - 632014, Tamil Nadu, India
Ph: +91-9787084667
Email: ravikanth.k@vit.ac.in; rkkamlekar@gmail.com
Highlights:
 N-acyltaurines (NATs) are structurally related to endocannabinoids.
 Hydrated homologous NATS show a single broad transition and their transition
temperature increases non-linearly with increase in chain length. Transition enthalpies
and entropies of NATs exhibit linear dependence on chain length.
 PXRD measurement indicated that NATs have linear dependence in d-spacings and they
pack in a tilted bilayer fashion.
 CMCs of homologous NATs indicate the linearity and follow Traube’s rule.
 The above observations provides thermodynamic and structural basis for understanding
NATs role in biological process.
Abstract
1

N-acyltaurines (NATs) are amides of fatty acids that can be structurally related to
endocannabinoids. They show interesting physiological and pharmacological properties. We
have synthesized a homologous series of NATs with saturated acyl chains (n = 918) and
investigated their supramolecular structure and thermotropic phase transitions by powder X-ray
diffraction (PXRD) and differential scanning calorimetry (DSC). The d-spacings obtained from
PXRD increase linearly with chain length with an increment of ~0.847 Å per additional CH2
moiety suggesting that NATs adopt a tilted bilayer structure with similar packing in crystal
lattice. Results obtained from DSC studies indicate that the endothermic transition temperature
(Tt) of NATs showed a gradually increasing trend with increasing acyl chain length. The
enthalpy (ΔHt) and entropy (ΔSt) of transition show odd-even alternations with odd-chain
compounds having higher values than the even-chain compounds. The critical micellar
concentration (CMC) of NATs was determined in water at room temperature by fluorescence
spectroscopy by monitoring the spectral changes of 8-anilinonaphthalene-1-sulfonic acid (ANS).
The CMCs of NATs were found to decrease with increase in acyl chain length. The present
results provide a thermodynamic and structural basis for investigating the interaction of NATs
with other membrane lipids and proteins, which in turn can shed light in understanding how they
function in vivo (in biological membranes).
2

Keywords: NATs: N-acyltaurines; DSC: Differential Scanning Calorimetry; odd-even
alteration; PXRD: Powder X-Ray Diffraction; d-spacing; tilted chain packing; CMC: Critical
Micellar Concentration.
1. Introduction
The endocannabinoid system is an endogenous signaling system that has pleiotropic effects
on metabolism, cell proliferation, appetite, pain sensation and neuromodulatory effects [Luciano,
et al., 2009]. Endocannabinoids have been implicated in cancer development and cause
heterogenous effect in tumor cells, by inducing apoptosis, reducing migration, causing anti-
angiogenic activity and alterations in the cell cycle resulting in growth arrest. Recently, several
novel amides of fatty acids that are structurally related to endocannabinoids have been isolated
from mammalian sources, although the functions of these fatty amides are not well studied [Bo,
et al., 2010]. One group of these novel lipids identified is the N-acyltaurines (NATs) (fatty acids
conjugated to the amino acid, taurine). Many long chain saturated and monounsaturated acyl
chains NATs were detected in brain (C24:0 ~115 pmol/g), spinal cord (C24:0 ~320 pmol/g) and
polyunsaturated NATs were predominant in liver (C20:4 ~20pmol/g, in kidney (C20:4
~160pmol/g) and other tissues in mouse [Saghatelian, et al., 2004; 2005; 2006]. The rate and
magnitude of accumulation of NATs in the liver and kidney of FAAH inhibitor-treated mice can
be associated with the elevation of NAEs in the brain and spinal cord [Saghatelian, et al., 2006].
Transient receptor potential (TRP) proteins are non-selective cation channels carry out a number
of tasks which mainly involving sensory signalling [Taberner, et al., 2015]. Along with
perception of external excitations they also assist individual cells in animals to respond to
internal stimuli [Liu, and Montell, 2015]. NATs are able to activate members of the transient
receptor, potential family of calcium ion channels in a cellular system [Saghatelian, et al., 2006].
Recently, it was also shown that N-arachidonoyltaurine (Fig.1) can be oxidized by lipoxygenase
enzymes, resulting in oxidative metabolites of N-arachidonoyltaurine which may have their own
biological function [Turman, et al., 2008]. N-arachidonoyltaurine activates members of the
transient receptor potential (TRP) channels, specifically the TRP receptors, TRPV1 and TRPV4
ion channels [Waluk, et al., 2013] and it also binds to G protein-coupled receptor, GPR72 at
submicromolar concentrations [Roy and Hannedouche, 2007]. The possible effect of N-
3

acyltaurines, in regulating insulin secretion from pancreatic β-cells is due to the activation of
TRPV1 [Waluk, et al., 2013]. N-arachidonoyltaurine and N-oleoyltaurine could function in a
similar way to endocannabinoids and result in cell cycle alterations or growth arrest in the human
prostate adenocarcinoma cell line PC-3 [Chatzakos et al., 2012]. A new report states the presence
of NATs in Langerhans islets in mouse and human tissue which also reveals their role as insulin
secretagogues alongside acetylcarnitines [Aichler et al., 2017]. Accumulated N-palmitoyltaurine
and N-oleoyltaurine in β cells enhanced the insulin secretion in mature granules without affecting
the number of premature insulin granules.
Figure 1. (A) Structure of N-arachidonoyltaurine, (B) Anandamide, (C) N-
arachidonoyldopamine (NADA).
During the last 15 years or so, studies on the interaction of cholesterol with other lipids have
received a new thrust in view of its presence in membrane rafts, which are rich in cholesterol and
sphingolipids, resulting in the identification of novel ‘condensed complexes’ of simple integral
stoichiometry between cholesterol and phospholipids [Radhakrishnan et al., 1999, 2000, 2000,
2001]. The rapid delivery of N-acylethanolamines (NAEs) to FAAH takes place through a
calveolae/lipid raft mediated endocytotic process [Lambert and Fowler, 2005]. This is certainly
consistent with the known ability of NAEs to bind with cholesterol [Ramakrishnan et al. 2002]
and the ability of cholesterol depletion to prevent NAE induced apoptosis in hepatocytes [Biswas
et al., 2003]. Since cholesterol is a major lipid of the plasma membrane of eukaryotes and NATs
4

with long, saturated acyl chains, e.g., palmitoyl and stearoyl chains have been found to
accumulate in a variety of membranes under stress, specific interactions could be important in
the cytoprotective and stress combating actions of NATs.
Since, NATs are structurally related to endocannabinoids and show interesting biological
properties (activating members of the transient receptor potential (TRP) channels, potential
stimulative role in insulin secretion, putative role in the stress-combating response of organisms
and potential in developing liposomal drug-delivery vehicles), it is important to carry out
systematic investigations on NATs directed at understanding their physicochemical properties,
phase behavior, and interaction of these compounds with other membrane components, i.e.,
phospholipids & cholesterol and proteins. As an initial step in this direction, in the present study
the thermotropic phase transitions of the hydrated NATs have been characterized by differential
scanning calorimetry (DSC), which revealed an unusual odd-even alternation in the transition
temperatures, enthalpies and entropies. The powder X-ray diffraction studies suggest that NATs
are most likely packed in a tilted bilayer format. In addition, the CMC measurements of NATs
by following fluorescence spectra of 8-anilinonapthalene-1-sulfonic acid (ANS) have shown
decrease with increase in acyl chain length.
2. Materials and methods
2.1. Materials
Long chain fatty acids and 8-anilinonapthalene-1-sulfonic acid (ANS) were obtained from
TCI, Tokyo, Japan and oxalyl chloride was purchased from Sigma Aldrich, USA. Taurine is
purchased from Spectrochem Pvt. Ltd., Mumbai, India. All other chemicals and solvents were
purchased from local suppliers. All solvents were distilled and dried before use. For fluorescence
studies all the solutions were prepared using MilliQ water.
2.2. Synthesis of NATs:
NATs were synthesized by the reaction of acid chlorides with taurine. For this, long-chain
fatty acids were converted to the corresponding acid chlorides by treating with 4 mol equivalents
of oxalyl chloride at room temperature for 2 h under nitrogen atmosphere [Kamlekar et al.,
2010]. After completion of the reaction, the excess oxalyl chloride was removed under a stream
5

of dry nitrogen gas. NATs were synthesized by the drop-wise addition of about 1 mol equivalent
of the acid chloride in dichloromethane to a solution of 1.2 mol equivalent of taurine in 1M
NaOH (to maintain pH 9-10.5) under constant stirring at room temperature. After addition of all
the reagents, the reaction was allowed to continue for two hours (Scheme 1).
Scheme 1. Synthesis of N-acyltaurines (NATs).
The crude NATs obtained as slurry was dissolved in ethanol by refluxing. The solution was
filtered and the filtrate was stored in freezer (ca. 4oC) overnight. This led to precipitation of the
NATs and then later recovered by filtration. The obtained NATs were further purified by column
chromatography on silica gel with ethyl acetate/methanol/ammonium hydroxide (80/18/2)
mixture as elution solvent. The overall yield for different NATs ranged around ~70%. The final
products were characterized by FT-IR, 1H and 13C NMR spectroscopy as well as by LC-MS.
2.3. Differential Scanning Calorimetry DSC:
DSC measurements of hydrated NATs sample were done using Nano DSC differential
scanning calorimeter from TA instruments. Accurately weighed samples (~1.5 mg) were
dissolved in dichloromethane with traces of methanol and then the solvent was removed under a
stream of dry nitrogen gas. Final traces of the solvent were removed by vacuum desiccation for
at least two hours. Then the obtained sample was hydrated with 10 mM phosphate buffer pH-7.4
containing 150 mM NaCl and subjected to at least five freeze-thaw cycles with intermittent
6

vortexing in order to get a homogeneous mixture. Samples were sonicated for about 30s before
using in DSC studies. Three heating scans and two cooling scans between 0 and 90°C at a scan
rate of 1°C per minute were recorded for all the hydrated samples. In all cases, the first heating
scan was considered for further analysis. Transition enthalpies (ΔHt) were determined by
integrating the area under the transition curve. Transition entropies (ΔSt) were determined from
the transition enthalpies assuming a first order transition according to the expression [Marsh,
2013]:
ΔHt = Tt. ΔSt …(1 )
where, Tt is transition temperature. The ΔHt values are taken at this temperature in order to
calculate the corresponding ΔSt values. Transition enthalpies were calculated by integrating peak
area under the transition curve after buffer subtraction, normalization, and baseline correction
using the software provided with the instrument. Transition entropies were calculated from the
transition enthalpy values according to Eq. (1).
2.4. Powder X-ray diffraction:
Powder X-ray diffraction patterns of NATs were recorded on Bruker D8 Advance X-ray
diffractometer using Cu-Kα radiation (λ=1.5406 Å). All the compounds were ground with mortar
and pestle to give a homogeneous powder, which were then used for PXRD measurements. The
obtained powder was placed on a circular rotating disc of the sample holder and then pressed
with a glass slide to prevent spillage during the measurements. Diffraction patterns were
collected at room temperature over a 2θ range of 1-50° with a step size of 0.0197°. Bragg’s
equation was used to calculate d-spacing values.
2.5. Determination of critical micellar concentration (CMC):
The CMC of NATs was determined by fluorescence spectroscopy using the fluorescent
probe, 8-Anilinonaphthalene-1-sulfonic acid (ANS) [Kamlekar and Swamy, 2006]. Fluorescence
measurements were performed on a JASCO spectrofluorometer FP8200 with a 1.0 cm quartz
cell. The fluorescence of ANS (10 μM) has been recorded between 450 and 600 nm, keeping the
excitation wavelength at 370 nm and the slit width of excitation and emission at 5nm and 10 nm
respectively. Briefly, 2.5 mL of fluorescent probe (ANS) was titrated by adding small aliquots of
NATs from a concentrated stock solution (in water) and the fluorescence spectrum was measured
7

after an equilibration period of two min. The fluorescence intensity at fixed wavelength was then
plotted as a function of NATs concentration and the CMC was determined as the concentration
where a distinct break in the slope was observed. Prior to analysis, fluorescence intensities were
corrected for volume changes.
3. Results and discussion
3.1. Synthesis and characterization of NATs:
The homologous series of NATs (n = 9-18) synthesized by reaction of acid chloride with
taurine were characterized by FT-IR, 1H and 13C NMR spectroscopy. The FT-IR spectrum was
recorded using Affinity-1 FT-IR (Shimadzu instrument, Japan). IR spectra of all NATs (see in
supporting documents, Fig.1S and Table 1S) showed absorption bands due to the amide linkage
at ~1640 cm-1 (C=O, amide-I) and ~1540-70 cm-1 (NH deformation, amide-II), whereas bands
corresponding to the carbonyl resonances of free fatty acid, acid chloride and ester were absent.
The amide N−H stretching is seen around ~3305-70 cm-1. The C-H symmetric and asymmetric
stretching is seen at ~2916 and ~2848 cm-1. CH2 bending and rocking bands were observed near
~1469 cm-1 and ~719 cm-1[Kamlekar et al., 2010]. NMR spectra of NATs were recorded on a
Bruker Advance III 400 MHz NMR spectrometer using DMSO-D6 as the solvent. The 1H-NMR
spectra of NATs (see in supporting documents, Fig. 2S and Table 2S) have showed resonances at
~2.00 δ (2H, t) and ~1.45 δ (2H, m) for the methylene groups next to the carbonyl moiety on
long acyl chain side, at ~7.70 δ (1H, t) for the amide N-H. Resonance at ~3.29 δ (2H, m), 2.54 δ
(2H, t) is for the methylene groups present next to carbonyl / amide N-H and SO3H moiety
respectively [Zhou et al., 2010]. Resonance of the terminal methyl group was seen at~ 0.837-
0.854 δ (3H, t), whereas the resonance of the polymethylene group was seen at ~1.235 δ (10-
26H, bs). The 13C NMR spectra (see in supporting documents, Fig. 3S and Table 3S) shows a
resonance at ~13.90 δ corresponding to the terminal methyl group of the acyl chain. Resonances
corresponding to the methylene groups are seen at ~22.12, 25.23, 31.32 and 50.64 δ as well as
between ~28.69 and 29.05 δ and between ~35.44 and 35.61 δ. A single peak corresponds to
carbonyl carbon is seen at ~171.86 δ. The mass spectra of NATs were recorded using LCMS-
2010A, Shimadzu instrument. The base peak of the LCMS mass spectra matches well with the
molecular ion peak for all the synthesized NATs (see in supporting documents, Fig. 4S). The
8

results obtained from FTIR, 1H, 13C NMR spectroscopy and LCMS are in full agreement with
the structures of NATs and indicate that they are of high purity.
3.2. Differential scanning calorimetry of hydrated N-acyltaurines:
DSC heating thermograms of NATs with odd and even chain lengths hydrated in 10mM
phosphate buffer pH-7.4 containing 150 mM NaCl are shown in Fig.2. All thermograms of
NATs exhibit a single, slightly broader phase transition. In case of shorter chain length NATs,
they did not show the heating transition within the region of analysis. Data from the first heating
thermograms were used for further DSC analysis. The phase transition temperatures (Tt,),
transition enthalpies (Ht) and transition entropies (St) values obtained from the first heating
DSC scan are listed in Table 1. Also, it is clearly seen from Fig. 2 and Table 1 that the half width
of the phase transition (T1/2) also varies with acyl chain length. The half width of the phase transition
(T1/2) is in the range of 1.90–2.98°C for the NATs investigated in this study. The T1/2 values provide
information about the cooperativity of the phase transition. For the N-lauroyltaurine (NLT, n =
12) the transition is slightly broad with higher T1/2 value (2.08°C) when compared to N-
tridecanoyltaurine (NTDT, n = 13) and N-myristoyltaurine (NMT, n = 14) i.e., 1.90°C and
1.95°C respectively. For longer chain length NATs (i.e., n =15-18), the phase transition is much
broader with increased T1/2 values (2.13 -2.98°C). These results suggest that the phase properties
of NATs depend on the acyl chain length.
Table 1: Transition temperature (Tt), Half width of the phase transition (T1/2), Enthalpy (ΔHt) and
Entropy (ΔSt) values obtained from hydrated DSC thermograms of NATs from C-12 to C-18 in
10mM phosphate buffer pH-7.4 containing 150 mM NaCl.
Acyl chain length (n) Tt (°C)
T1/2 (°C)
ΔHt (k.cal/mol) ΔSt (cal/mol/K)
12
13
14
15
16
17
18
26.57(0.41) 2.08 (0.04) 4.17 (0.41)
42.69(0.62) 1.90 (0.16) 6.65(0.68)
13.9(1.41)
21.05(2.05)
19.55(0.07)
25.45(0.92)
23.1(0.07)
28.55(0.49)
27.8(0.07)
1.95 (0.06)
2.13 (0.03)
2.56 (0.54)
2.33 (0.37
2.98 (0.29)
46.55(0.39)
57.90(0.23)
59.04(0.57)
69.63(0.03)
68.59(0.11)
6.25(0.01)
8.42(0.31)
7.65(0.01)
9.78(0.18)
9.49(0.03)
9

Figure 2: DSC thermogram of hydrated N-acyltaurines with acyl chains having odd and even
number of C-atoms. The number of C-atoms in acyl chain is indicated against each thermogram.
3.3. Odd-even alteration in the thermodynamic properties of NATs:
It has been observed from the DSC heating thermogram (Fig.2), that the transition
temperatures of hydrated NATs with both odd and even acyl chains increase with increase in
acyl chain length. Although the phase transition temperature is increasing, it is not linearly
proportional to acyl chain length (both odd and even chains). For better understanding, the
transition temperatures were plotted as function of acyl chain length (Fig 3A). It is observed that
the Tt values in each series increases in smooth progression but with a slightly lower increment
as chain length is increasing, resulting in a nonlinear fit. The observed trend is comparable to that
found in a number of single-chain amphiphiles such as NAEs, N-acyldopamines(NADAs), O-
acylcholines(OACs), N-acylglycines(NAGs), N-acyl-L-alanines(NAAs), N-acyl-L-alanine alkyl
10

esters(NAAEs), N-acylseratonins(NASTs) and N-acylglycine alkyl esters (NAGEs)
[Ramakrishnan et al., 1997; Reddy et al., 2013; 2014; 2015; Reddy and Swamy, 2017; Tarafdar
et al, 2013; Sivaramakrishna et al., 2015; Sivaramakrishna and Swamy, 2015]. From Fig 3A, it
clear that Tt values exhibit an even-odd alteration, with the odd chain length series showing
slightly higher transition temperatures than the even chain length NATs just like in case of
NAAs and NAAEs.
3.4. Chain length dependence of transition temperature, enthalpy and transition entropy:
The transition enthalpy and transition entropy for the phase transition of hydrated NATs
against the chain length are shown in Fig 3B & 3C, respectively. In even and odd series,
transition enthalpy and transition entropy were linearly proportional to acyl chain length.
Nevertheless, when the data obtained with odd and even acyl chain lengths are viewed together,
a zig-zag pattern is seen with the values of enthalpy and entropy for the odd acyl chain lengths
being slightly higher than those of even acyl chain lengths.
The transition enthalpies and entropies of NATs with odd and even acyl chain lengths could be
fit well to expressions (2) and (3) given below as observed earlier for N-acylethanolamines, N-
acyldopamines, N-acyl-L-alanines, N-acylglycines and N-acylserotonins with even and odd acyl
chain lengths, O-acylcholines with even chain lengths as well as N-,O-diacylethanolamines with
matched as well as mixed acyl chains[Lambert et al 2005; Zhou et al., 2010; Ramakrishnan et al.,
1997; Reddy et al., 2013; 2014; 2015; Reddy and Swamy, 2017; Tarafdar et al, 2013;
Sivaramakrishna et al., 2015; Sivaramakrishna and Swamy, 2015].
ΔHt = ΔH0 + (n − 2) ΔHinc …(2)
ΔSt = ΔS0 + (n − 2) ΔSinc …(3)
where, ΔH0 and ΔS0 are the end contributions to ΔHt and ΔSt, respectively, arising from the
methyl group of the acyl chain and the head group region of N-acyltaurines and n is the number
of C-atoms in the acyl chains. ΔHinc and ΔSinc are the average incremental values of transition
enthalpy and transition entropy contributed per CH2 group to ΔHt and ΔSt and the values of these
parameters obtained from linear least-squares analysis are listed in Table 2.
11

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 to [Gunstone et al., 1986]
ΔHt = (n − 2) ΔHinc …(4)
ΔSt = (n − 2) ΔSinc …(5)
Then the transition temperature at infinite chain length, Tt∞ can be determined from
Tt∞ = ΔHinc/ΔSinc …(6)
From the data presented in Table 2, the Tt∞ values for hydrated NATs of even and odd acyl chain
lengths have been estimated using eq 6 as 384 K and 417 K, respectively.
Table 2: Chain Length Dependence of Incremental Values (ΔHinc, ΔSinc) and End Contributions
(ΔH0, ΔS0) to Phase Transition Enthalpy and Entropy of N-acyltaurines.
Thermodynamic parameters
NAT (Even)
NAT (Odd)
ΔHinc (kcal/mol)
ΔH0 (kcal/mol)
ΔSinc (cal/mol/K)
ΔS0 (cal/mol/K)
0.867 (0.044)
4.39 (0.58)
2.26 (0.14)
0.784 (0.0585)
1.90 (0.765)
1.88 (0.19)
8.30 (1.84)
0.642 (2.45)
#Average values of transition enthalpy and transition entropy given in Table 2 have been used for the
linear fitting of the data. Errors shown in parentheses are fitting errors obtained from the linear least-
square analysis.
For a number of single-chain as well as two-chain lipids, which exhibit linear dependence of ΔHt
and ΔSt on chain length, it has been shown that the transition temperature data can be fit to the
following equation [Reddy et al., 2013].
Tt = (ΔHt/ΔSt) = Tt∞ [1 − (n0 – n′0) / (n – n′0)] …(7)
Where, no (= −ΔH0/ΔHinc) and n′0 (= −ΔS0/ΔSinc) are the values of n at which the transition
enthalpy and transition entropy extrapolate to zero.
From Figure 3, it can be observed that the transition temperatures of hydrated NATs 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.
12

Figure 3: (A) Chain length dependence of transition temperatures (Tt) of N-acyltaurines, showing
even-odd alternation. Solid lines correspond to nonlinear least squares fits of the transition
temperatures of even chain length and odd chain length series to equation 7. (B) Transition
enthalpies (Ht) and (C) transition entropies (St) obtained for the thermotropic phase transitions
of N-acyltaurines are plotted against the number of methylene units (n-2), where ‘n’ is the number
of C-atoms in each acyl chain.
3.5. Powder X-ray diffraction:
Additional insights into the molecular packing of NATs with varying acyl chain length
were obtained from powder X-ray diffraction studies. The PXRD data acquired for NATs of
even and odd chain lengths at room temperature are shown in Fig. 4A and B. All NATs (n = 9-
18) gave several sharp peaks in the 2θ range of 1- 30°. From the position of these diffraction
peaks the d-spacing were calculated. In each case ~4-6 peaks were used for estimating the d-
spacing. The values determined are given in Table 4S (see in supporting documents, Table 4S).
The corresponding plot of chain length dependence of the d-spacing of N-acyltaurines is shown
in Fig 4C.
13

Figure 4: Powder X-ray diffraction patterns of N-acyltaurines with even and odd saturated acyl
chains (A, B) and dependence of d-spacing on the acyl chain length (C). The number of C-atoms
in the saturated, unbranched acyl chain is indicated against each PXRD profile. The solid straight
line 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 1.6936 Å.
The d-spacing values show a linear dependence on the acyl chain length with a slope of 1.6936
Å/CH2, which correspond to increments of 0.847 Å per additional CH2 moiety, respectively. If
the molecules were to pack in a normal bilayer format, this value would correspond to the
projected distance for two C–C bonds. In such a case the d-spacing value would be 2.54 Å, when
the acyl chain length is increased by one CH2 unit and each CH2 group increases the d-spacing
by 1.27 Å. In comparison, the incremental d-spacings estimated from the PXRD data are
significantly smaller. Therefore, it indicates that the acyl chains in both odd-chain and even-
chain NATs must be tilted with respect to the bilayer normal. The tilt angle with respect to
bilayer normal is deduced from the slope (in Fig 4C) as 48.18° [Katsaras et al., 1992]. This is
close to the tilt angle of 49.3° predicted for N-acylserinols (NASOHs) based on similar PXRD
14

measurements [Sivaramakrishna, et al., 2019]. However, in order to understand the structural
origin, the 3-dimensional structures of NATs need to be determined.
3.6. Critical micellar concentration of NATs:
The dual character of surfactants is responsible for their peculiar behaviour in the presence
of water. The micelle forming ability of NATs was studied by using fluorescence spectroscopy.
The CMCs of NATs were determined by using 8-anilinonapthalene-1-sulfonic acid (ANS) as the
fluorescent probe. The fluorescence intensity of ANS was studied as a function of concentration
of NATs. A distinct break in the slope can be observed from the plot which is the CMC of NAT.
The fluorescence intensity of ANS at 490nm plotted against concentration of N-decanoyltaurine
(NDT) is given in Fig. 5A. The successive addition of small aliquots of NATs from concentrated
stock solution to ANS solution showed an increase in fluorescence intensity. This suggests that
the ANS surrounding environment has changed from hydrophilic one to lipophilic one. From the
Fig.5A, it can be seen that the fluorescence intensity follows a particular trend up to a particular
concentration and then a different trend is taken up after this concentration. The intercept of the
linear least squares fits of the two parts yield the CMC for NDT in water. The determined CMC
values for all NATs are reported in Table-3 (also see in supporting documents, Fig. 5S), which
indicates that the CMC values of NATs decrease with increase in acyl chain length.
Figure 5: (A) CMC of N-decanoyltaurine in water. (B) A plot of chain length (n) versus the
ln[CMC], the ln[CMC] values were calculated from CMC values taken in Molar units.
15

Table-3: Critical micellar concentration of various NATs at 25°C.
Acyl chain length (n)
CMC (mM)
9
8.311 (0.065)
5.063 (0.44)
3.68 (0.67)
1.98 (0.22)
1.21 (0.25)
0.2824 (0.02)
0.0562 (0009)
0.0174 (0.0001)
0.0104 (0.00036)
0.0025 (0.00026)
10
11
12
13
14
15
16
17
18
Plot of log (CMC) values against the carbon number of the alkyl chain in N-acyltaurine is shown
in Fig. 5B. A linear dependence was observed in the plot and it followed Traube's rule [David
and Alexander, 2012]. The log (CMC) values of NATs decrease with increase in acyl chain
length and a distinct break in the slope can be observed from the plot which may be due to
formation of two different type of micelles by shorter and longer chain length NATs (i.e., n =9-
13 and n = 14-18). The slope of the individual line yields a value for ΔG/RT and the energy of
micellar formation deduced from the slope is of −0.48 and −1.154 kcal·mol−1 per methylene
group of the alkyl chain, which is comparable to Lysophosphatidylcholine (−0.61 and −1.16
kcal·mol−1/ methylene group) [Marsh, 2013; Masayuki et al., 1986].
4. Conclusions
In the presented work, a homologous series of N-acyltaurines (n = 9-18) have been
synthesized and characterized. DSC studies of hydrated homologous N-acyltaurines show a
single broad transition and their transition temperature increases non-linearly with increase in
chain length. Transition enthalpies and entropies of NATs exhibit linear dependence on chain
length. It is complemented by PXRD measurement, which indicated that NATs have linear
dependence in d-spacings and they pack in a tilted bilayer fashion. CMC exhibited by
homologous NATs has been investigated by fluorescence spectra. The results obtained indicate
that linearity in CMCs of NATs and follow Traube’s rule. The calculated energy of micellar
formation of two different type of micelles are −0.48 and −1.154 kcal.mol-1 per methylene group
16

of the alkyl chains n =9–13 and n = 14–18 respectively. These observations provides
thermodynamic and structural basis for understanding their role in biological process. To
understand their putative biological role, further investigation of NATs interaction with other
membrane components i.e., PCs, PEs, cholesterol and proteins is essential.
Declaration of interests:
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper but
we do respectfully request that Dr. Amitabha Chattopadhyay not review our manuscript.
Acknowledgement
We acknowledge University of Hyderabad for the access of DSC facility and support to
AS under UGC-NRC program. RKK acknowledges DST-SERB (YSS/2014/000021),
Government of India for funding. We thank VIT, Vellore for research and infrastructural facility.
Appendix A. Supplementary data
Supplementary material related to this article can be found in supplementary documents.
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