Synthesis, characterization, and determination of critical micellar concentration and thermotropic phase transition of taurolipids

Article abstract of DOI:10.1016/j.molliq.2017.03.064

Taurolipids (NAT) are a newer class of fatty acid amides (FAA) identified in the central nervous system, liver, and kidney of fatty acid amide hydrolase knockout mice. In this study, we reported a convenient method to synthesize NATs in fair yield and characterized using FTIR, ¹H NMR and ¹³C NMR. Fluorescence spectroscopy study showed that NATs form micelles in an aqueous environment and the critical micellar concentration (CMC) follows a linear trend with respect to fatty acid acyl chain length. Differential scanning calorimetry reveals that NATs undergoes broad endothermic transition with transition temperatures increases with increasing fatty acid acyl chain length though the magnitude of change from one chain length to another chain length decreases as the acyl chain length increase. The transition enthalpy (ΔH_t) and transition entropy (ΔS_t) corresponding to the chain-melting phase transition depends linearly on the acyl chain length and fitted to a linear equation, which yielded the incremental values, ΔH_inc (1.09?±?0.03?kcal·mol^??1) and ΔS_inc (2.96?±?0.16?cal·mol^??1·K^??1), contributed by each methylene unit to ΔH_t and ΔS_t. The end contributions arising from the head group and the terminal methyl group were calculated to be ΔH⁰?=???7.28?±?0.46?kcal·mol^??1 and ΔS⁰?=???17.36?±?2.11?cal·mol^??1·K^??1. Thermodynamic parameters, incremental values of ΔH_inc and ΔS_inc suggest that the acyl chains in NATs are tightly packed and the obtained larger negative ΔS⁰ suggest that the hydration of polar sulfonate group of NATs also play an important role in the self-assembly of NATs.

Full text of DOI:10.1016/j.molliq.2017.03.064

Accepted Manuscript
Synthesis, characterization, and determination of critical micellar
concentration and thermotropic phase transition of taurolipids
Sengan Megarajan, Pothiappan Vairaprakash, Veerappan
Anbazhagan
PII:
S0167-7322(16)33981-2
doi: 10.1016/j.molliq.2017.03.064
MOLLIQ 7099
DOI:
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
10 December 2016
16 March 2017
17 March 2017
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ACCEPTED MANUSCRIPT
Revised version – correction marked
Synthesis, characterization, and determination of critical micellar
concentration and thermotropic phase transition of taurolipids
Sengan Megarajan, Pothiappan Vairaprakash, Veerappan Anbazhagan*
Department of Chemistry, School of Chemical and Biotechnology, SASTRA University,
Thanjavur – 605043, Tamil Nadu, India
*
Corresponding author,
V. A: Phone: +91 4362-264101-3689; Fax: +91ꢀ 4362-264120,,
e-mail: anbazhagan@scbt.sastra.edu
1

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Abstract
Taurolipids (NAT) are a newer class of fatty acid amides (FAA) identified in the central
nervous system, liver, and kidney of fatty acid amide hydrolase knockout mice. In this
study, we reported a convenient method to synthesize NATs in fair yield and
1
13
characterized using FTIR, H-NMR and C-NMR. Fluorescence spectroscopy study
showed that NATs form micelles in an aqueous environment and the critical micellar
concentration (CMC) follows a linear trend with respect to fatty acid acyl chain length.
Differential scanning calorimetry reveals that NATs undergoes broad endothermic
transition with transition temperatures increases with increasing fatty acid acyl chain
length though the magnitude of change from one chain length to another chain length
decreases as the acyl chain length increase. The transition enthalpy (H ) and transition
t
entropy (S ) corresponding to the chain-melting phase transition depends linearly on the
t
acyl chain length and fitted to a linear equation, which yielded the incremental values,
-1
-1 -1

Hinc (1.09 ± 0.03 kcal.mol ) and S (2.96 ± 0.16 cal.mol .K ), contributed by each
inc
methylene unit to H and S . The end contributions arising from the head group and
t
t
0
-1
0
the terminal methyl group were calculated to be H = -7.28 ± 0.46 kcal.mol and S = -
-1
-1
1
7.36 ± 2.11 cal.mol .K . Thermodynamic parameters, incremental values of H and
inc
Sinc suggest that the acyl chains in NATs are tightly packed and the obtained larger
0
negative S suggest that the hydration of polar sulfonate group of NATs also play an
important role in the self-assembly of NATs.
Keywords: N-acyltaurine; taurolipids; critical micellar concentration; phase transition;
fluorescence; differential scanning calorimetry
2

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1
. Introduction
Fatty acid amides (FAA) are well known as an endogenous signaling molecule and
possess numerous physiological functions in mammalian tissues [1]. For example,
anandamide (arachidonoyl ethanolamide) is an endogenous cannabinoid neurotransmitter
[
2], oleoylethanolamide regulates appetite through the activation of G-protein coupled
13
receptor [3], erucamide (22:1 ) is the major angiogenic component stimulates new blood
vessel formation [4], arachidonoylglycine does not bind to the cannabinoid receptors, but
relieves pain [5], virodhamine relaxes arteries through endothelial cannabinoid receptors
[
6]. The advent of mass spectrometry facilitated the field of lipidomics and identified
newer fatty acid amide signaling molecules in mammalian tissues, but their site of action
is largely unknown [7]. Moreover, isolation of FAA from the tissues is largely
complicated because these classes of lipids are often synthesized, released and inactivated
proximal to their sites of action [1]. In order to investigate the biology of newly identified
FAA, researchers required a reasonable quantity of FAA. This will be possible through
developing devoted synthetic strategy and physicochemical characterization of FAA.
Fatty acids that are amidated to taurine are collectively known as N-acyltaurines
or taurolipids (NAT) [8]. NAT was first identified in the central nervous system (CNS),
liver and kidney of fatty acid amide hydrolase (FAAH) knockout mice [8]. NAT isolated
in the liver and kidney were predominantly polyunsaturated compounds (~160 pmol/g in
kidney; ~20 pmol/g in liver), while long chain saturated and monounsaturated N-acyl
chains were the most common in CNS (upto 2 nmol/g) [9]. The enzymes, the acyl-CoA:
amino acid N-acyltransferase (ACNAT1) [10] and the bile acid-CoA: amino acid N-
acyltransferase (BAT) [11] have been proposed for the production of N-acyltaurines.
Long et al., study suggested the existence of a transport system for NAT that transfers
these lipids from liver to other tissues [12]. It has been noted that the endogenous levels
of NAT were elevated following acute or chronic inactivation of FAAH, suggests that
NAT could form a major signaling system similar to other FAA [13]. N-
arachidonyltaurine activates several transient receptor potential (TRP) channels,
including TRPV1 and TRPV4 calcium channels, both of which are expressed in the
kidney [13]. TRP channels are suggested to playing a role in the regulation of osmotic
3

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sensation and blood pressure. Treatment with N-arachidonyltaurine and N-oleoyltaurine
showed a marked reduction in the proliferation of prostate cancer PC-3 cells [14]. NAT
regulates insulin secretion from pancreatic β-cells through TRPV1 channels and other
receptor channels also involved in the insulin secretion response to treatment with NAT
[
15]. The biology of NAT remains elusive, even though studies with mouse tissues
shown their presence with diverse N-acyl chain length in plasma, heart, brown and white
adipose tissue, lung, kidney, testis, liver and spleen [12].
While compared to other FAAs like N-acyl ethanolamine [16, 17], N-acyl
aminoacids [18, 19, 20], N-acyl dopamine [21], there are no reports on the
physicochemical characterization of NAT. As a first step in learning about NAT, we are
reporting a facile method to synthesise N-acyltaurine with saturated acyl chains of even
number of carbon atoms and studied their thermotropic phase behaviour using differential
scanning calorimetry (DSC) and determined the critical micellar concentration (CMC) of
NAT using fluorescence probes. The CMC of NAT was found to decrease with increase
in the acyl chain length. DSC study showed that the transition enthalpies and entropies of
hydrated NATs exhibit a linear dependence on the acyl chain length. Most of the phase
transition occurred over a broad temperature range, suggesting that NAT undergoes a
phase transition from the micellar to the monomeric state.
4

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2
. Material and methods
.1. Materials
2
Decanoylcholoride
(N10),
laurylchloride
(N12),
myristoylchloride
(N14),
palmitoylchloride (N16), stearoylchloride (N18), taurine (T), 8-anilinonapthalene
sulfonic acid (ANS) and N-phenylnapthalamine (NPN) were obtained from Sigma, India.
All other reagents were of reagent grade and were obtained from local suppliers. Solvents
were distilled and dried prior to usage.
2
.2. Synthesis of N-acyltaurines
N-acyltaurines are synthesized in fair yield by a condensation reaction between taurine
and fatty acid chloride. In a typical synthesis, taurine (275 mg, 2.2 mmol) (i) was mixed
with sodium hydroxide (395 mg, 9.8 mmol) and hydrated with 500 L of water and then
added tetrahydrofuran (10 mL). To the resulting mixture, acid chloride (2 mmol) was
added under constant stirring and the reaction was allowed to proceed for 12 h at room
temperature. The progress of the reaction was followed by thin-layer chromatography
(TLC) on silica gel (solvent system: acetic acid: water: n-butanol, 1:1.4:2 v/v) and the
TLC plates were stained with ninhydrin (20 mg ninhydrin dissolved in 9.5 mL of n-
butanol and 0.5 mL of 10% methanol). Noteworthy, taurine showed pink color while
staining with ninhydrin whereas N-acyltaurines appears in yellow color. After the
completion of reaction, the reaction mixture was centrifuged at 3000 rpm for 15 min and
the resultant crude N-acyltaurine (iii) was subjected to column chromatography. Briefly,
the crude NAT was loaded onto silica gel column and washed with 100% ethyl acetate to
remove unreacted fatty acids. Further elution with the mixture of solvent containing
1
methanol and ethyl acetate (20:80 v/v) yielded pure N-acyltaurines as judged by H-NMR
1
3
1
and C-NMR. The isolated yields of different NATs range from 60-65 %. H NMR (300
1
3
MHz), C NMR (75 MHz) were recorded on Bruker-300-Advance II spectrometer, with
D O or CD OD as solvent and tetramethylsilane (TMS) as reference (δ = 0 ppm). The
2
3
chemical shifts are expressed in δ downfield from the signal of internal TMS. FT-IR
spectra were recorded using a PerkinElmer spectrum by adopting KBr pellet method.
5

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H
O
4
.5 eq. NaOH
O
N
R
O
NH2
S
S
+
R
Cl
THF, RT, 12 h
HO
O
HO
O
O
(iii)
(i)
(ii)
R: CH -(CH ) ; CH -(CH ) ; CH -(CH ) ; CH -(CH ) ; CH -(CH )
2 16
3
2 8
3
2 10
3
2 12
3
2 14
3
Scheme 1: Synthesis of N-acyltaurine from taurine and fatty acid chloride
2
.3. Determination of critical micellar concentration
The CMC of NATs was determined by fluorescence spectroscopy using the fluorescent
probe, ANS and NPN [22, 23]. Fluorescence measurements were performed on a JASCO
spectrofluorometer FP8200 with a 1.0 cm quartz cell. The fluorescence of ANS (10 M)
was recorded between 400-650 nm, keeping the excitation wavelength at 370 nm and the
slit widths of both excitation and emission set at 5 nm. The fluorescence of NPN (5 M)
was recorded between 360-600 nm, keeping the excitation wavelength at 340 nm. The
stock solution of 100 mM, 80 mM, 15 mM, 5 mM and 1 mM of N10T, N12T, N14T,
N16T and N18T, respectively was prepared by dissolving exactly weighed NAT in 10
mM sodiumphosphate buffer, pH -7.4. Briefly, 3 mL of fluorescent probe (ANS or NPN)
was titrated by adding small aliquots of NATs from a concentrated stock solution and the
fluorescence spectrum was measured after an equilibration period of 2 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. 10 mM phosphate buffer, pH-7.4 was used throughout the study and
o
experiments were performed at 25 C. All experiments were repeated at least three times
to arrive at the average values.
2
.4. Differential scanning calorimetry
Samples for DSC was prepared by suspending accurately weighed NATs (5-6 mg) with
1
0 mM phosphate buffer, pH-7.4. N12T and N14T were hydrated at room temperature,
o
whereas N16T and N18T were hydrated by heating the suspension to ~65 C. The
hydrated samples and buffer were loaded into the sample and reference cell, respectively,
o
of a Nano DSC-TA instrument, India. A scan rate of 1 C/min was used. Three heating
6

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scans were performed for each sample and the scans were found to be reproducible. All
heating scans were normalized with respect to concentration of NAT, which yields the
heat capacity (C ) in kcal/mol.K. Transition temperature (T ), transition enthalpy (H )
p
t
t
for each transition was estimated by integrating the area under the transition using the
software provided with the instrument. In all cases, only the first heating scans were
considered for further analysis. Transition entropies (S ) were estimated from the H
t
t
assuming a first order transition according to the expression [24]:

H = T . S
(1)
t
t
t
7

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3
. Results and discussion
.1. Synthesis of N-acyltaurine
3
Taurine fatty acid conjugates were synthesized by reacting taurine and fatty acid chloride
in THF in the presence of NaOH at room temperature for 12 h (Scheme 1). Our initial
efforts on pre-activating fatty acid chloride as NHS-ester derivative and reacting with
taurine failed to yield the corresponding product. Then, the condensation between taurine
and fatty acid chloride was carried out utilizing various organic bases and organic solvent
system (Table 1). In chloroform, both inorganic bases and organic bases did not yield the
product (Table 1, entries 1 - 4). In acetonitrile solvent, N-acyltaurine was obtained in
trace quantities using Et N as base (Table 1, entry 5). Adding traces of water to the
3
reaction medium improved the yield to 12 % (Table 1, entry 6). Under same condition,
using NaOH as base further improved the product formation and the corresponding N-
acyltaurine was obtained in 27 % (Table 1, entry 7). Using THF/water mixture as solvent
and NaOH as base increased the product yield to 62 % (Table 1, entry 8).Though the base
NaOH is expected to compete with taurine in the nucleophilic attack in aqueous system,
given solvent system [THF/H O (19:1, v/v)] reduces the availability of NaOH for
2
nucelophilic attack and we could able to get the product in better yields. Taurolipids were
1
13
purified by flash chromatography and characterized by TLC, H-NMR and C-NMR. IR
spectra of all NATs showed absorption bands corresponding to the amide I at 1640-1645
-1
-1
cm and amide II at 1540-1553 cm . The amide N-H stretching was observed at 3337-
-1
-1
3
284 cm and N-H bending was seen at 1552-1540 cm . The stretching, bending and
rocking modes of the polymethylene group of the hydrophobic acyl chain was observed
-1
-1
-1
1
at 2919-2916 cm , 1470-1467 cm and 718-721 cm , respectively (Fig. S1- S5). The H
NMR spectra of NAT showed resonances at 3.44 -3.60 δ (2H, t) and 2.98-2.96 δ (2H, t)
for the methylene groups at α and β positions to the sulfonate moiety, 2.11-2.19 δ (2H, t)
and 1.46-1.61 δ (2H, bs) for the methylene group at α and β positions to the amide
carbonyl, respectively. Resonance of the terminal methyl group was seen at 0.92−0.74 δ
(3H, t), whereas the resonance of the polymethylene group was observed at 1.16−1.30 δ
1
3
(
12−28H, m). These spectral data are presented in the supporting information. The C
NMR spectra of NAT showed resonances at 13.70 δ for terminal methyl group of the acyl
8

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chain, 49.9 δ for the methylene moiety at α positions of the sulfonate group, 35.10 δ for
the methylene moiety attached to NH and C=O group, other methylene carbons are seen
at 29.3, 25.6 and 22.6 δ. Resonance corresponds to the amide carbonyl was observed at
1
1
75.9 δ. For all the synthesized compounds, number and nature of H NMR resonances
are matching with the structure of the corresponding compounds. For compounds N10T
1
3
and N12T, the numbers of C NMR resonances are well in accordance with structure.
For the compound N14T, few resonances are grouped into single peak, hence the number
of resonances observed are less in number than the expected number. For compounds
1
3
N16T and N18T, C NMR spectra were not recorded due to solubility problem. These
spectral data are presented in the supporting information (Fig. S6 to S13).
Table 1. Condensation of taurine with lauroyl acid chloride*
entry
Base
NaHCO3
K CO
Solvent
Chloroform
Chloroform
Result
1
2
3
4
2
3
No reaction and lauroyl acid was
recovered
Pyridine
Et N
Chloroform
3
NAT was obtained in trace
quantities
5
Et N
CH CN
3
3
6
7
8
Et N
NaOH
NaOH
CH CN/H O (19:1, v/v)
CH CN/H O (19:1, v/v)
3 2
NAT was obtained in 12% yield
NAT was obtained in 27% yield
NAT was obtained in 62% yield
3
3
2
THF/H O (19:1, v/v)
2
*
To a mixture of taurine (1.0 mmol) and a base (5 equiv) in specified solvent (5.0 mL), lauoryl chloride
(
1.0 mmol) was added and stirred for 12 h.
3
.2. Determination of critical micellar concentration
An amphiphilic molecule with long acyl chain fatty acids was preferred to adopt one of
the following structures in hydrated form: interdigitated gel phase, hexagonal phase and
micelles [25, 26]. NAT is composed of a polar sulfonate group and hydrophobic acyl
chain, therefore, it is expected that NAT can form micelles in aqueous solution.
Fluorophores like 8-anilinonapthalene-1-sulfonate were widely used to determine the
critical micellar concentration of an amphiphilic molecule [22]. ANS is weakly
fluorescent in aqueous solution and exhibit strong fluorescence in lipophilic environment.
9

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Figure 1: (A) Fluorescence spectra of ANS at various concentration of N10T. (B)
Fluorescence intensity of ANS is plotted as a function of N10T concentration. (C)
Fluorescence spectra of NPN at various concentration of N10T. (D) Fluorescence
intensity of NPN is plotted as a function of N10T concentration. Increasing concentration
of a 100 mM stock solution of N10T was added to 3 mL of ANS/NPN. Excitation
wavelength for ANS and NPS is 340 nm and 370 nm, respectively. Solutions were
o
prepared in 10 mM phosphate buffer, pH -7.4. Temperature = 25 C.
Fig. 1A shows the effect of N-decanoyltaurine (N10T) on the emission spectra of
ANS. The addition of N10T increases the emission intensity of ANS and induces a blue
shift of about 30 nm in the peak wavelength, indicating that the environment of ANS
becomes more hydrophobic. Fig. 1B shows the enhancement of fluorescence intensity at
4
90 nm as a function of N10T concentration. Two straight lines can be drawn through the
experimental points; their intersection (4.07 mM) is taken as the critical micellar
concentration (CMC) above which N10T aggregates to form micelles (Fig. 1B). The
CMC of N10T was also studied with another fluorescent probe, NPN [23]. Fig. 1C shows
that the addition of N10T increases the emission intensity of NPN and a blue shift of
about 70 nm in the peak wavelength. The determined CMC (4.16 mM) is comparable to
the CMC determined with ANS (Fig. 1D), suggesting that both probes are suitable for the
determination of CMC of an amphiphilic molecule. The fluorimetric method of CMC
determination was further verified by surface tension measurement. The CMC of N12T
1
0

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determined by pendant drop method using goniometer is 1.99 mM (Fig. S14). This value
is in close agreement with the reported fluorimetric method (Table 2), indicate that ANS
and NPN is a suitable probe to study the CMC of NAT. The results of fluorescence
titration of ANS with N-lauryltaurine (N12T), N-myristoyltaurine (N14T), N-
palmitoyltaurine (N16T) and N-stearoyltaurine (N18T) are shown in Fig. 2 and the CMC
values are reported in Table 2. Noteworthy that, the addition of 50 mM of sodium
chloride prevents the formation of micelles, indicate the contributions of electrostatics in
the formation of micelles (Fig. S15).
Figure 2: Fluorescence titration of ANS with (A) N12T, (B) N14T, (C) N16T and (D)
N18T. The concentration of ANS is 5 M. 80 mM, 15 mM, 5 mM and 1 mM stock
solution of N12T, N14T, N16T and N18T, respectively were used in the titration.
o
Solutions were prepared in 10 mM phosphate buffer, pH -7.4. Temperature = 25 C.
Table 2: Critical micellar concentration of various N-acyltaurines. CMC values given are
the averages obtained from three independent experiments and reported as mean with
standard error.
NAT
CMC determined with the fluorescent probe
ANS (mM)
4.005 ± 0.092
1.460 ± 0.113
0.272 ± 0.040
NPN (mM)
3.975 ± 0.262
1.660 ± 0.566
0.305 ± 0.050
N10T
N12T
N14T
1
1

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N16T
N18T
0.082 ± 0.013
0.018 ± 0.001
0.117 ± 0.037
0.023 ± 0.001
The relationship between the CMC values and the carbon number of the alkyl chain in N-
acyltaurine is shown in Fig. 3. This plot gave a good linear relationship and followed
Traube’s rule [27]. The slope of the line yields a value for G/RT and the energy of
micellar formation deduce from the slope is of -0.61 kcal/mol per methylene group of the
alkyl chain, which is close to those obtained with lysolecithin [28].
Figure 3: Logarithm of CMC as a function of carbon numbers of the alkyl chain in N-
acyltaurine. Some data point’s error bar is smaller.
3
.3. Phase transitions of NAT
Figure 4: (A) DSC calorigrams of various N-acyl taurines and (B) Chain length
dependence phase transition temperatures of NAT. Solutions were prepared in 10 mM
phosphate buffer, pH -7.4.
1
2

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Differential scanning calorimetric experiments were carried out to determine the phase
transitions exhibited by the aqueous dispersions of NAT and to determine the change in
enthalpy and change in entropy associated with the phase transitions. DSC heating scans
of fully hydrated NAT were shown in Fig. 4A. Apart from N-decanoyltaurine, all other
NATs showed broad transition with a full width at half maximum range between 2.6 to
o
3
.8 C. The transition temperatures of the NAT increases with increasing chain length
though the magnitude of change decreases as the chain length increase (Fig. 4B). This is
similar to the trends observed for the aqueous dispersion of fatty acids, N-
acylethanolamines (NAE) [16], N-acyltyramine (NATA) [29], N-acyldopamines (NADA)
[
21], N-acylglycine (NAG) [19] and other N-acyl fatty acid amides [30]. A broad
transition observed with NAT can be due to the coexistence of two phases over the
temperature range before it proceeds to the complete fluid phase at high temperature.
Similar broad transition was reported for palmitoylcarnitine, where it forms an
interdigitated bilayer structure in gel phase and hexagonal phase (H ) at high temperature
I
[
25]. However, further studies are required to delineate the exact structure of the NAT
aggregates at different temperature.
1
3

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Figure 5: Chain length dependence of (A) transition enthalpies and (B) transition
entropies of hydrated N-acyltaurines. Solid line represents linear least-squares fit of the
data.
Table 3: Transition temperature (T ), transition enthalpies (H ) and transition entropies
t
t
(
S ) of N-acyltaurines
acyl chain length
t
o
-1
-1 -1
T ( C)
H (kcal.mol )
S (cal.mol .K )
t
t
t
NAT
1
1
1
1
2
4
6
8
21.24
38.13
53.89
62.65
3.46
5.92
7.89
11.76
19.02
23.90
28.88
10.04
The change in enthalpy and change in entropy associated with chain-melting
phase transitions of various NAT are given in Table 3. Both transition enthalpy (H ) and
t
transition entropy (S ) follows a linear trend with respect to chain length (n) and the data
t
could be fitted with the following equation 2 and 3,
0

H = (n – 2) H + H
(2)
(3)
t
inc
0
St = (n – 2) S + S
inc
1
4

ACCEPTED MANUSCRIPT
0
0
where H and S are the end contributions to the transition enthalpy and transition
entropy, respectively, arising from the terminal methyl group and the polar head group
region of NAT. Hinc and Sinc are the incremental values of transition enthalpy and
transition entropy per methylene group. The linear chain length dependence of H and
t
St indicate that the structure, molecular packing and intramolecular interaction of
various NAT are likely to be very similar. From Fig. 5, the incremental values and end
contributions of H and S were obtained from the linear least square analysis and
t
t
presented in the Table 4.
Table 4: Incremental values (H , S ) of chainlength dependence and end
inc
inc
0
0
contributions (H , S ) to phase transition enthalpy and entropy of N-acyltaurines.
a
b
Thermodynamic parameters
NAE
NAT
NAG
1.15
-
1



H (kcal.mol )
0.95
1.09 ± 0.03
-7.28 ± 0.46
2.96 ± 0.16
inc
0
-1
-
2
0.52
.37
.1
-4.34
2.95
-10.06
H (kcal.mol )
-1
-1
S (cal.mol .K )
inc
0
-1 -1
3
-17.36 ± 2.11

S (cal.mol .K )
a
b
For comparison thermodynamic values are adopted from ref. [16]; For comparison thermodynamic values
are adopted from ref. [19]
As noted from Table 4, NAT displays larger negative end contributions of
0
0
enthalpy, H and entropy, S than the NAEs and NAGs. The end contributions from
0
enthalpy, H mainly arise from the taurine moiety. It was observed that upon hydration
-1
taurine moiety gains sustainable enthalpy stabilization (-7.28 ± 0.46 kcal.mol ) with the
expense of ordering of water structure as a result reduction in entropy. The enthalpy
stabilization could arise from hydrophobic and hydrogen bonding interactions. The
0
negative S is also indicative of hydrophobic effect playing a role in the packing of
NAT [21], which could arise due to the ordering of water molecules around the sulfonate
moiety of the headgroup. Hinc value of NAT was higher than NAEs [16] and
comparable to NAGs [19], while Sinc value of NAT was higher than NAEs and NAGs.
These results indicated that the hydrophobic effect plays a role in the acyl chain packing
of NAT, a similar observation was proposed for NAGs acyl chain packing. The average
incremental contribution of each CH to the transition enthalpy H and entropy, S is
2
inc
inc
higher than NAE indicating a substantial contribution for NATs acyl chain packing
comes from the repulsion between the charged head group. In addition to repulsion
between headgroup at the interaface, the hydrogen bonding between the amide N-H and
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carbonyl oxygen atoms of adjacent NAT molecules might contribute to high curvature in
the head group region and resulted in higher H and S . NAT will not form micelles
inc
inc
in the presence of NaCl, which supports the involvement of weak interaction such as H-
bond in the stabilization of micelles (Fig. S15). Based on the thermodynamic analysis, we
suggest that the hydrophobic forces govern the self-assembly of NATs acyl chains to
form a hydrophobic core and expose the polar head group to the aqueous environment, as
a result NATs prefer to adopt a micellar structure as characterized in the fluorescence
study (Table 2).
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4
. Conclusion
A facile method to synthesize homologous series of NATs in reasonable yield with good
purity was reported. The acyl chain length dependence of the CMC and phase transition
exhibited by NATs has been investigated. The results obtained from fluorimetric titration
indicate that the CMC follows a linear trend with respect to acyl chain length and obeys
Trabue’s rule. It has found that the energy of micellar formation of -0.61 kcal/mol per
methylene group of the alkyl chain. The values of H andS per CH group of NATs
inc
inc
2
is significantly higher than the NAEs, suggests the acyl chain packing differs
considerably between NAEs and NATs. Moreover the higher negative end contributions
0
0
of entropy, S and enthalpy, H observed with NATs indicate that the hydration of
head group plays an important role in the self-assembly of NATs. Further studies aimed
at the miscibility of NATs with other lipids, such as phosphatidylcholine and
phophatidylethanolamine, is expected to offer detail insight into their biological role.
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Acknowledgement
The Department of Science and Technology, Government of India (SB/FT/LS-217/2012)
is acknowledged for the financial support. DST FIST grant to SCBT, SASTRA for
establishing infrastructure facilities is gratefully acknowledged.
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Graphical abstract
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Highlights




A simple procedure to synthesis of N-acyltaurine was reported
NAT forms micelles, the CMC decreases with increasing alkyl chain length
Phase transition temperature increases with increasing alkyl chain length
Transition enthalpy and transition entropy follows linearity
2
2