Effect of hofmeister series anions on the thermotropic phase behavior of bioactive O -acylcholines

Article abstract of DOI:10.1021/jp403964k

O-Acylcholines (OACs), which are true cationic lipids due to the quaternary ammonium functionality in the headgroup, exhibit interesting biological activities and medicinal properties. In the present study, a homologous series of OACs with even chain lengths (n = 12-20) have been synthesized, and their thermotropic and chaotropic phase transitions have been characterized. The role of various anions (Cl^-, Br^-, I^-, NO ₃^-, SO₄^2-, ClO₃ ^-, ClO₄^-) on the phase behavior of O-stearoylcholine was investigated by calorimetric, spectroscopic, and turbidimetric approaches. The results obtained revealed that in aqueous dispersion O-stearoylcholine undergoes a cooperative phase transition from a gel phase to a micellar structure and that the transition temperature increases when the counterions are changed in the Hofmeister series. Single-crystal X-ray diffraction studies showed that O-stearoylcholine iodide forms an interdigitated bilayer structure, with the polymethylene chain adopting an all-trans conformation. The Hofmeister effect and phase behavior were explained using the concepts of matching water affinities, water penetration into the bilayer, and electrostatic repulsion. It was also observed that one counterion per molecule is sufficient to strongly modulate the phase properties of the lipid/surfactant.

Full text of DOI:10.1021/jp403964k

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Article
Effect of Hofmeister Series Anions on the Thermotropic
Phase Behavior of Bioactive O-Acylcholines
Pradip Kumar Tarafdar, S. Thirupathi Reddy, and Musti Joginadha Swamy
J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp403964k • Publication Date (Web): 02 Aug 2013
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Effect of Hofmeister Series Anions on the Thermotropic Phase Behavior of
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Bioactive OꢀAcylcholines
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Pradip K. Tarafdar, S. Thirupathi Reddy and Musti J. Swamy*
School of Chemistry, University of Hyderabad, Hyderabad ꢀ 500 046, India
Running Title: Phase behavior of Oꢀacylcholines

*Corresponding author:
Prof. Musti J. Swamy
School of Chemistry
University of Hyderabad
Hyderabad ꢀ 500 046, India
Tel: +91ꢀ40ꢀ2313ꢀ4807
Fax: +91ꢀ40ꢀ2301ꢀ2460
Eꢀmail: mjssc@uohyd.ernet.in, mjswamy1@gmail.com
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ABSTRACT
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OꢀAcylcholines (OACs), which are true cationic lipids due to the quaternary ammonium
functionality in the head group, exhibit interesting biological activities and medicinal
properties. In the present study, a homologous series of OACs with even chainlengths (n =
12ꢀ20) have been synthesized and their thermotropic and chaotropic phase transitions have
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ꢀ
2ꢀ
ꢀ
ꢀ
been characterized. The role of various anions (Clꢀ, Brꢀ, Iꢀ, NO3 , SO4 , ClO3 , ClO4 ) on the
phase behavior of Oꢀstearoylcholine was investigated by calorimetric, spectroscopic and
turbidimetric approaches. The results obtained revealed that in aqueous dispersion Oꢀ
stearoylcholine undergoes a cooperative phase transition from a gel phase to a micellar
structure and that the transition temperature increases when the counterions are changed in
the Hofmeister series. Singleꢀcrystal Xꢀray diffraction studies showed that Oꢀstearoylcholine
iodide forms an interdigitated bilayer structure, with the polymethylene chain adopting an
allꢀtrans conformation. The Hofmeister effect and phase behavior were explained using the
concepts of matching water affinities, water penetration into the bilayer and electrostatic
repulsion. It was also observed that one counterion per molecule is sufficient to strongly
modulate the phase properties of the lipid/surfactant.
Keywords: Differential scanning calorimetry; Xꢀray diffraction; electron spin resonance;
Krafft temperature; surfactant
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INTRODUCTION
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An important characteristic of surfactants is their tendency to precipitate from aqueous
solutions, which can inhibit their activity and hence their use in many applications. The
precipitation of surfactants occurs below a characteristic temperature – known as Krafft
temperature – where surfactants are largely separated from the bulk medium and their surface
activities are significantly decreased.1,2 Above Krafft temperature the solubility of
surfactants increases dramatically in aqueous systems, and is interpreted as the melting
temperature of a hydrated solid surfactant.1,3 Since below Krafft temperature surfactants
separate out from the system, to keep the surfactants in their active form, it is desirable to
lower the Krafft temperature. It has been shown that Krafft temperature is influenced by the
ions present in solution.1,4 The influence of ions on the phase behavior of lipids/surfactants
has been studied extensively.5ꢀ19 In alkyltrimethylammonium surfactants the ability of
counterions to induce the sphereꢀtoꢀrod transition follows the Hofmeister sequence20,21 and in
dodecyltrimethylammonium micelles, the aggregation numbers have been shown to increase
along the Hofmeister sequence of the counterions.22,23 Recently, the influence of acyl chain
length on the phase behavior of surfactants were investigated.24ꢀ26 However, the influence of
Hofmeister series of ions on the modulation of Krafft temperature in a homologous series and
associated thermodynamics have not been investigated in detail. It is known that chaotropic
ions in the Hofmeister series make insoluble ion pairs with cationic surfactants,21,27 whereas
kosmotropic ions likely remain hydrated depending on the surfactant headgroup.28
Mechanistic interpretation of how ions modulate the Krafft temperature from a standard
model, for example, gelꢀmicelle transition model is important to understand the role of ions
on the phase properties of ionic lipids/surfactants. In view of this, in the present study tried
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to address the following questions. 1) How do ions modulate the Krafft temperature of a
homologous series of surfactants and what are the thermodynamics associated with it? 2)
How many ions per each surfactant molecule are required for this modulation? 3) What is
the 3ꢀdimentional structure of solid precipitate appearing below Krafft temperature? 4) Is the
modulation of the activity of surfactants/lipids by chaotropic ions different from the way they
affect the activity of nucleic acids and proteins? To address these questions we chose a
homologous series of Oꢀacylcholines, which are bioactive surfactants about which very little
is known with regard to their supramolecular structure and interactions with ions.
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OꢀAcylcholines (OACs) are fatty acid esters of choline, which are true cationic lipids
due to the quaternary ammonium functionality in the head group. OACs with different fatty
acyl chains exhibit hemolytic activity, which is comparable to that of acylcarnitines and
lysolecithins.29,30 OꢀPalmitoylcholine, isolated from the skin secretions of smooth trunk fish
(Lactophrus triqueter Linn.), exhibits toxicity against mosquitofish at low (ppm)
concentration.31 Several longꢀchain acylcholines were reported to inhibit wheat embryo Ca2+ꢀ
dependent protein kinase.32 Beside these biological properties, OACs attract interest due to
their medicinal properties. For example, OACs with various fatty acyl chains modulate blood
pressure,33 act as skin penetration enhancers for the transdermal delivery of drugs such as
acyclovir, 17βꢀestradiol, hydrocortisone etc.,34 and exhibit antimicrobial activity against a
wide range of microorganisms.35 As the OACs are enzymatically hydrolyzed to ecoꢀfriendly
components, they can potentially replace the more toxic, stable quaternary ammonium
compounds.35 Although, as indicated above, the biological and pharmacological properties of
OACs have been investigated in considerable detail, very little is known regarding their
supramolecular structure, thermotropic phase transitions and interaction with anions. In view
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of this lacuna, in the present study we have synthesized a homologous series of Oꢀ
acylcholine iodides (OACs) with even chains and characterized their thermotropic phase
transitions using DSC. The phase behavior of one of the OACs, namely Oꢀstearoylcholine
iodide (OSC) has been examined in the presence of sodium salts of various anions belonging
to the Hofmeister series, viz., NaCl, Na2SO4, NaNO3, NaBr, NaClO3, NaClO4 and NaI using
differential scanning calorimetry (DSC), electron spin resonance (ESR) spectroscopy,
turbidimetry and fluorescence spectroscopy. The results obtained indicate that counterions
modulate the phase behavior of OSC and changing the counterion can induce a transition
from a gel phase to a liquid crystalline phase, which we refer to as chaotropic phase
transition and predict that other OACs would behave similarly. Finally, the 3ꢀdimentional
structure and packing of OSC was determined by single crystal Xꢀray diffraction, which
revealed that the acyl chains are packed in an interdigitated manner.
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MATERIALS AND METHODS
Materials. Fatty acids, stearic acid spin labeled on the 5th Cꢀatom (5ꢀSASL), N, N’ꢀ
dicyclohexylcarbodiimide (DCC), 4ꢀdimethylaminopyridine (DMAP), methyl iodide, sodium
chloride, sodium bromide, sodium iodide, sodium nitrate, sodium sulfate, sodium chlorate
and sodium perchlorate were purchased from SigmaꢀAldrich (Milwaukee, WI). N, N’ꢀ
Dimethylethanolamine, SERALITE SRAꢀ400 anion exchange resin and solvents were
purchased from Sisco Research laboratory (Mumbai, India).
Synthesis of OꢀAcylcholines. OACs with even acyl chainlengths (n =12ꢀ20) were
synthesized in two steps. In the first step the fatty acid was condensed with N, N’ꢀ
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dimethylethanolamine using dicyclohexylcarbodiimide (DCC) as the coupling agent and 4ꢀ
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dimethylaminopyridine (DMAP) as the catalyst.
Fatty acid (1.15 eq), N, N’ꢀ
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dimethylethanolamine (1.0 eq) and DMAP (0.5 eq) were taken in a round bottom flask and
the mixture was azeotroped thrice with dry benzene. The mixture were dried under high
vacuum and dissolved in dry dichloromethane. DCC (1.4 eq) was added to the reaction
mixture at 0°C. The reaction mixture was stirred overnight raising the temperature from 0°C
to room temperature in an inert atmosphere. The condensation product (fatty acid ester of N,
N’ꢀdimethylethanolamine) was extracted with ethyl acetate and purified by column
chromatography. The purified and dried ester in dichloromethane was treated with 1.3 eq of
methyl iodide at 0 °C for quaternization. The product obtained (Oꢀacylcholine iodide) was
washed thrice with dry ether and dried under vacuum. The purified OACs were
characterized by IR and 1HꢀNMR spectroscopy.
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Conversion of OꢀStearoylcholine Iodide to OꢀStearoylcholine Chloride.
Oꢀ
Stearoylcholine iodide was converted to the corresponding chloride salt by ion exchange
using SERALITE SRAꢀ400 anion exchange resin. In order to get complete exchange of the
counterion to chloride, the procedure was carried out twice. The purity of the product was
characterized by 1H NMR and IR spectroscopy.
Differential Scanning Calorimetry. DSC studies were performed using a VPꢀDSC
equipment from MicroCal (MicroCal LLC, Northampton, MA, USA). Accurately weighed
lipid samples (~1 mg) were dissolved in dry chloroform and the solvent was removed under a
stream of dry nitrogen gas. The resulting lipid film was vacuum desiccated for 5ꢀ6 hrs in
order to remove residual traces of the solvent. The lipid was hydrated thoroughly with
Millipore water or with 150 mM concentration of the appropriate salt
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(Na2SO4/NaCl/NaBr/NaNO3/NaClO3/NaI/NaClO4) before performing the heating scans. In
all cases the second heating scan was considered for data analysis. Transition enthalpies
were determined by integrating peak areas under the transition curves. Transition entropies
were determined from the transition enthalpies assuming a first order transition according to
the expression:36,37
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ꢀHt = Tt.ꢀSt
(1)
where Tt is the transition temperature and ꢀHt values are taken at this temperature in
order to calculate the corresponding ꢀSt values.
Turbidimetry. Samples for turbidimetric measurements were prepared by dispersing
OSC in water, or 150 mM NaCl, or 150 mM NaI and heating to about 65ꢀ70 °C in a warm
water bath with intermittent vortexing, followed by at least three cycles of freeze thawing,
using liquid nitrogen and hot water (ca. 70 °C). After the last incubation in hot water, the
sample tube was equilibrated to room temperature and transferred to a spectrometer cuvette.
Turbidimetric measurements were performed at various temperatures between 20 and 70 °C
using a Cary 100 UVꢀVisible spectrophotometer equipped with a Peltier thermostat supplied
by the manufacturer. Turbidity was measured by recording the absorption spectrum between
350 and 450 nm and turbidity at 400 mm was considered for further analysis.
ESR Spectroscopy. Spinꢀlabeled dispersions of OSC containing probe amounts of 5ꢀ
SASL were prepared as follows. Measured volumes of OSC stocks (dissolved in
chloroform) and the spinꢀlabel stock were dispensed into glass sample tubes using a
Hamilton syringe to yield 0.5 mol% of the spin label in the lipid and the solvent was
evaporated by gently blowing dry nitrogen gas. Final traces of the solvent were removed by
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vacuum desiccation for at least three hours. The samples were then hydrated with water or
with 150 mM NaCl and kept in the dark at room temperature for 3 hrs. Then the samples
were centrifuged, and the pellets were transferred to 1 mm diameter glass capillaries. ESR
spectra were recorded on a JEOL JESꢀFA 200 ESR spectrometer equipped with an airꢀflow
temperature regulation system. Samples in 1 mm ID glass capillaries were placed in a
standard quartz ESR tube containing light silicone oil for thermal stability. Temperature was
measured with a fineꢀwire thermocouple positioned near the ESR tube.
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Crystallization, Xꢀray Diffraction and Structure Solution. Thin needle type, colorless
crystals of Oꢀstearoylcholine iodide were grown at room temperature from a mixture of
chloroform, methanol and water. A crystal of 0.28 × 0.16 × 0.04 mm size was used for data
collection in the present study. Xꢀray diffraction measurements were carried out at room
temperature (ca. 25 °C) with a Bruker SMART APEX CCD area detector system using a
graphite monochromator and MoꢀKα (λ = 0.71073 Å) radiation obtained from a fineꢀfocus
sealed tube.
Data reduction was done using Bruker SAINTPLUS program. Structure solution was
carried out in the triclinic space group. Absorption correction was applied using SADABS
program. The structure was solved successfully by direct methods in the space group Pꢀ1
and refinement was done by full matrix leastꢀsquares procedure using the SHELXLꢀ97
program.38 The refinement converged into a final R1 = 0.186, wR2 = 0.466 and goodness of
fit = 1.54. The somewhat high R1 and wR2 may be due to difficulty in growing good quality
single crystals. In this context, it may be noted that in general lipids do not yield good
quality single crystals. In addition, the diffraction patterns of lipids are dominated by strong
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subcell reflections arising from the regular hydrocarbon chain matrix, which complicate or
sometimes hinder the structure solution.39
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Crystal Parameters of OꢀStearoylcholine Iodide. Molecular formula: C23H48INO2.
Molecular formula weight: 497.5; Crystals were thinꢀplate type and colorless. Crystal
system, triclinic; Space group, Sg = Pꢀ1; Ambient temperature, T = 298(2) K; Radiation
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wavelength (λ) = 0.71073 Å; Minimum resolution = 0.84 Å; Radiation type, MoꢀKα;
Radiation source, Fineꢀfocus sealed tube; Radiation monochromator, graphite; Number of
reflections collected, 24156; Unique reflections, 9303; Reflection with I > 2σ(I), 5930;
Number of parameters, 505.
Unit cell dimensions (with standard deviation in parentheses): a = 7.992(3), b =
9.634(4), c = 35.413(14) Å; α = 86.398(7), β = 84.785(8), γ = 89.961(7)º; Volume of the cell,
V = 2709.9(19) Ǻ3; Number of molecules in the unit cell, Z = 4; Angle of tilt of Oꢀacyl
chains, θ = 1.7°; F(000) = 1064; Absorption Coefficient,
ꢁ
=0.175 mm ꢀ1; T = 298(2) K.
RESULTS
Differential Scanning Calorimetry of OꢀAcylcholines. OꢀAcylcholine iodides yielded
turbid suspensions upon hydration, which settled down over a period of time. The turbid
suspensions become optically clear at higher temperatures, suggesting that the amphiphile
undergoes a transition from a phase of high turbidity (possibly lamellar gel phase) to a phase
where aggregates are small (such as micelles), which do not scatter visible light. In order to
characterize the thermotropic phase transitions, DSC studies were carried out on hydrated Oꢀ
acylcholines (OACs) of even acyl chains (n = 12ꢀ20). OACs with different acyl chains
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exhibit a single, broad transition, except Oꢀarachidoylcholine which showed a minor
transition before the main transition. Representative heating thermograms of OACs
dispersed in water are shown in Figure 1A. Transition temperatures (Tt), widths at half
maximum of the transition (T1/2), transition enthalpies (ꢀHt) and transition entropies (ꢀSt)
obtained from the heating thermograms are presented in Table 1. It is seen from this table
that the thermodynamic parameters such as Tt, ꢀHt and ꢀSt increase with increase in the
chainlength. As discussed in more detail below, a linear chainlength dependence was
observed for ꢀHt and ꢀSt for the OACs of even chainlengths, indicating that structures of the
phases formed by all OACs in aqueous dispersions are likely to be very similar. Therefore, a
detailed characterization of the phase structure and phase transitions exhibited by one of the
molecules in the series is likely to give a fairly good idea of the phase structures adopted by
the entire homologous series. In view of this, further studies regarding the phase structure
and phase transition were carried out with Oꢀstearoylcholine iodide.
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Calorimetric Studies in the Presence of Hofmeister Ions. Further studies with OSC were
performed in the presence of 150 mM NaCl (physiological salt concentration), or 150 mM
NaI in order to investigate the role of chloride and iodide on the phase behavior.
Representative heating thermograms in water, in 150 mM NaCl and in 150 mM NaI are
shown in Figure 1B. It was observed that in the presence of 150 mM NaCl the phase
transition temperature (Tt) decreases from 61.4 to 37.7 °C, whereas it increases to 64.6 ºC in
the presence of 150 mM NaI (Figure 1B). DSC studies were also performed with OSC at
different concentrations of NaCl (25 mM to 1M) and the thermodynamic parameters
estimated from the thermograms are listed in Table S1.
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To investigate the phase behavior of OSC in the presence of other anions, DSC studies
were performed with samples containing Na2SO4, NaNO3, NaBr, NaClO3 or NaClO4 at 150
mM concentration. It may be noted that while Na2SO4 is a kosmotrope, the remaining salts
are all chaotropes. Thermograms obtained from these studies are presented in Figure 2 and
thermodynamic parameters estimated from them are summarized in Table 2. It is seen from
Figure 2 and Table 2 that the transition temperature increases along the Hofmeister series:
Na2SO4 < NaCl < NaBr < NaNO3 < NaClO3 < NaI < NaClO4.
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Turbidimetric Studies on OSC. The thermotropic phase transition of OSC was further
characterized by tubidimetric studies, monitoring the turbidity at 400 nm as a function of
temperature using a spectrophotometer. Figure 3 depicts the temperature dependence of
turbidity of OSC in water, in 150 mM NaCl and in 150 mM NaI. In all three environments
the turbidity at low temperature was relatively high and remains constant with increase in
temperature initially but exhibits a sharp decrease over a narrow temperature range and
thereafter remains at a constant level. The midpoint of the steeply declining region was taken
as the phase transition temperature (Tt). The Tt values obtained from the turbidimetric studies
were 57.5, 34.3 and 62.0 °C, respectively, for measurements performed in water, in 150 mM
NaCl and in 150 mM NaI. These values are slightly lower than the values obtained from the
DSC measurements, but are consistent with the DSC results in that the phase transition
temperature decreases by over 20 degrees in the presence of 150 mM NaCl and slightly
increases in presence of 150 mM NaI. From Figure 3 it can be clearly seen that turbidity of
the sample before phase transition in water is much higher as compared to the turbidity of the
sample prepared in 150 mM NaCl. Visual inspection also indicated that the sample (0.05
wt%) prepared in water is highly turbid as compared to the sample of the same concentration
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prepared in 150 mM NaCl (Figure 4A, B). This suggests that ions can modulate the phase
structure, and consequently, phase transition temperature (Krafft temperature) of OSC. Both
samples were optically clear above Tt, indicating that the aggregates are relatively small,
which most likely correspond to micelles.
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In order to investigate the modulation of phase structure by salt, turbidimetric studies
were performed with hydrated dispersions of OSC. In these experiments, small aliquots of
NaCl from a 2 M stock solution were added to a suspension of OSC in water. Results of
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such titrations, performed at 30 C (below the phase transition temperature of OSC hydrated
with water as well as 150 mM NaCl) and at 45 oC (which is above Tt when hydrated with 150
mM NaCl, but below Tt when hydrated with water alone) are shown in Figure 4C. The data
presented in this Figure show that at 45oC increase in NaCl concentration results in a steep
decrease in the turbidity of the OSC suspension in a narrow window of NaCl concentration
(ca. 0ꢀ25 mM) and yields a nearly optically clear solution. The sample remains clear when
the NaCl concentration is increased further (up to 200 mM). On the other hand, titrating an
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OSC suspension with NaCl at 30 C (which is below the phase transition temperature of the
sample hydrated by water or 150 mM NaCl) did not lead to any sharp changes in the
turbidity. Only a steady decrease in the turbidity was observed with increase in the chloride
concentration. Since the transition observed at 45oC is induced by the addition of ions, the
phenomenon is basically an ionꢀinduced phase transition and we refer to this as chaotropic
phase transition. As OACs with different chainlengths are isostructural, it is expected that
the other OACs will also undergo similar ionꢀinduced phase transitions.
SpinꢀLabel ESR Spectroscopy. Spinꢀlabel ESR spectroscopy enables the study of
specific environments within lipid aggregates and membranes. The outer hyperfine splitting
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(2Amax), which gives a measure of anisotropic motion of the probe, provides information
regarding the structure and dynamics of lipid aggregates. The ESR spectra of 5ꢀdoxyl stearic
acid (5ꢀSASL) incorporated at probe concentration in OSC, dispersed in water and in 150
mM NaCl recorded at different temperatures are shown in Figures 5A & 5B, respectively. At
25 °C the spectra recorded in water and in 150 mM NaCl exhibit strong spinꢀspin
broadening, clearly indicating that in this phase the acyl chains of OSC are tightly packed.
The 2Amax value of 5ꢀSASL in OSC at 25 oC was found to be 64.1 G and 61.7 G, respectively
in water and in 150 mM NaCl. The value of the 2Amax obtained in water at room temperature
is close to the value exhibited by the gel or crystalline phase of lipids and suggests that OSC
may adopt a gel or crystalline phase. The 2Amax value observed in 150 mM NaCl is
somewhat smaller and therefore most likely corresponds to the gel phase but not the
crystalline phase. The 2Amax values decrease with increase in temperature with a steep
decrease being seen near the phase transition temperature in both the environments. By
monitoring the temperature dependence of 2Amax the phase transition temperature was
determined as 58 and 36 °C, respectively, for measurements performed in water and 150 mM
NaCl (Figure 5C). These observations are also consistent with a lowering of the phase
transition temperature in the presence of 150 mM NaCl, as seen from the results of DSC and
turbidimetric studies presented above. In both environments the spectra become more
isotropic above the phase transition temperature. Qualitatively similar spectra have been
obtained for 5ꢀSASL incorporated in octadecylꢀtrimethylammonium bromide (C18TAB)
micelles40 and other micelles. This observation, together with the results of the turbidimetric
studies presented above, strongly suggests that in water and in 150 mM NaCl OSC exists in a
micellar state above the phase transition temperature.
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DSC of Oꢀstearoylcholine chloride.
Results of DSC measurements with Oꢀ
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stearoylcholine chloride (counterion is chloride) dispersed in water are shown in Figure 6.
From this figure it can be seen that OSC chloride gives an endothermic transition centered at
18.9 oC, whereas OSC with iodide counterion gives a transition around 61.4 oC (see Figure 1
and Table 1). These results demonstrate that counterion plays a definite role in modulating
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the transition temperature. The transition temperature increases to 32.9 C at 150 mM NaCl,
which could be due to the screening of the electrostatic repulsion between the charged head
groups, resulting in a tightening of the acyl chain packing41 (Figure 6).
Structure and Packing. Since spectroscopic studies have shown that preferred
conformations of acyl chains observed in solidꢀstate by Xꢀray analyses are predominant also
in the gel phase of lipids,39 determination of the 3ꢀdimentional structure of lipids in the solid
state by single crystal Xꢀray diffraction and analysing the intermolecular interactions will be
of immense value in understanding their structure and organization in the hydrated state.
Therefore, in the present study, we have determined the 3ꢀdimensional structure of Oꢀ
stearoylcholine iodide (OSC) and analyzed the molecular packing and intermolecular
interaction in the crystal lattice. An ORTEP of the molecular structure of Oꢀstearoylcholine
iodide is shown in Figure S1 and a packing diagram is given in Figure 7. Selected bond
distances, bond angles and torsion angles are given in Table S2. The hydrocarbon portion of
the acyl chains of OSC adopts an allꢀtrans conformation in the solid state, with all the torsion
angles in the acyl chain region being close to 180°. The iodide ions are close to the
positively charged trimethyl ammonium group with the nitrogenꢀiodide distance being 4.93
Å. The packing diagram (Figure 7) shows that the OSC molecules are packed in a headꢀtoꢀ
tail manner with a common hydrocarbon chain matrix that is confined on both sides by a
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boundary layer of polar groups. This type of chain packing, known as interdigitation of acyl
chains, accommodates two hydrocarbon chains per head group. In addition to the iodide
counter ion, for two OSC molecules, a water molecule is also accommodated in the crystal
lattice. The iodide and bulky choline moiety in the polar head group increase the observed
molecular area to 38.5 Å2, which is approximately twice that of hydrocarbon chain cross
section (19ꢀ20 Å2).42 This is consistent with the formation of the interdigitated bilayer
structure. The layer thickness (carbonyl carbonꢀcarbonyl carbon distance) of OSC is 21.8 Å
and the allꢀtrans acyl chains are nearly perpendicular to the bilayer plane, with a virtually
negligible tilt angle of 1.7° with respect to the bilayer normal.
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DISCUSSION
Although, as indicated in the introduction, Oꢀacylcholines with saturated acyl chains
exhibit interesting biological properties and have potential therapeutic applications,29ꢀ35 the
phase behavior and structureꢀactivity relationships of OACs are not yet understood. In order
gain better understanding about the structure–function relationships for these amphiphiles we
have characterized their phase behavior and thermodynamics of phase transitions in aqueous
dispersion and investigated the effect of counterions on them. The results obtained are
discussed below.
Chainlength Dependence of Transition Enthalpy and Transition Entropy. The
chainlength dependence of transition enthalpy and transition entropy for the aqueous phase
transitions of OACs of even chainlengths are given in Figures 8A and 8B, respectively. The
enthalpy and entropy data for OACs of even chainlengths could be fit well to expressions (2)
and (3), given below:36,43
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ꢀHt = ꢀHo + (nꢀ2) ꢀHinc
ꢀSt = ꢀSo + (nꢀ2) ꢀSinc
(2)
(3)
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where n is the number of Cꢀatoms in the acyl chains and ꢀHo and ꢀSo are the end
contributions to ꢀHt and ꢀSt, respectively, arising from the terminal methyl group and the
polar region of the OAC molecule. ꢀHinc and ꢀSinc are the incremental values of ꢀHt and ꢀSt
contributed by each CH2 group. Linear least squares analysis of the chainlengthꢀdependent
values of ꢀHt and ꢀSt for the even chainlengths of OACs yielded the incremental values
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(ꢀHinc, ꢀSinc) and end contributions (ꢀHo, ꢀSo). These values are listed in Table S3. For the
sake of comparison corresponding values obtained with longꢀchain Nꢀacylethanolamines
(NAEs) and fatty acids are also given in this Table. The ꢀHinc value of 1.82 kcal/mol/CH2
observed here for the OACs with even chainlengths indicates that the incremental enthalpy
contributed by each CH2 unit in the OACs is higher than that observed with the hydrated
NAEs and dry fatty acids.37 Similarly, the ꢀSinc values obtained here for the OACs with even
acyl chains are also considerably higher than the ꢀSinc values obtained for the NAEs and fatty
acids (Table S3). These observations suggest that the acyl chains in OACs are packed more
tightly than the acyl chains in NAEs and fatty acids.
Chainlength dependence of transition temperatures. Although the data presented in
Table 1 show that the transition temperatures increase with increase in acyl chainlength, the
Tt values do not increase in linear proportion to the acyl chainlength. In order to understand
this better the transition temperatures have been plotted as a function of acyl chainlength
(Figure 8C). From this figure it is seen that the Tt values increase in a smooth progression
but with decreasing increments as the chainlength is increased. As the acyl chainlength
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increases, the total contribution from the polymethylene portion towards the total enthalpy
and entropy of the phase transition will be sufficiently large that the end contributions are
negligible in comparison. Therefore, at infinite acyl chainlength, equations (2) and (3) can
be reduced to:36
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ꢀHt = (nꢀ2)ꢀHinc
ꢀSt = (nꢀ2)ꢀSinc
(4)
(5)
Then the transition temperature for infinite chainlength, Tt∞, will be given by:
Tt∞ = ꢀHinc/ꢀSinc
(6)
From the incremental enthalpy and entropy, the Tt∞ value for the OAC of even acyl
chainlength has been estimated as 360.4 K. The chainlength dependence of the transition
temperatures of OACs of even acyl chainlengths was fitted to eq. 7, predicted from the linear
dependence of transition enthalpy and transition entropy given in equations 1 and 2:44,45
Tt = ꢀHt/ꢀSt = (ꢀHinc/ꢀSinc) [1 – (no – no’)/(n – no’)]
(7)
where no (= – ꢀHo/ꢀHinc) and no’ (= – ꢀSo/ꢀSinc) are the chainlengths at which the
transition enthalpy and transition entropy, respectively, extrapolate to zero. From the
nonlinear least squares fit shown in Figure 8C, it is evident that the transition temperatures of
OACs with even chainlengths are described quite accurately by equation 7. In addition, the
fitting parameters yielded the transition temperature at infinite chainlength (Tt∞) for the
OACs of even acyl chains as 390.7 K with χ2 of 0.27.
Role of ions on phase behavior. Results obtained from DSC, turbidimetric and ESR
spectroscopic studies suggest that the turbid suspensions of OACs, which settled down over a
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period of time become optically clear above the transition temperature. As the OACs tend to
form precipitates near physiological temperature, their solubility might be very low in this
temperature range. Now if their solubility in water is too low and they tend to settle down
from the water then the question that rises is, how they are biologically active?
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The tendency of surfactants to precipitate from aqueous solutions inhibits their activity
and the hydrated solid precipitate can be termed ‘inactive state’ in view of their lack of
activity in this state. As the hydrated solid precipitate transforms to micelles above Krafft
temperature, it is desirable to find ways to decrease the Krafft temperature, which would
result in an increase in the temperature range over which the surfactant would be active. In
the present studies we have found that 150 mM NaCl induces significant changes in the
phase properties of OSC and leads to a large decrease in the phase transition temperature
(here, Krafft temperature). On the other hand in presence of 150 mM NaI, the phase
transition temperature increases slightly as compared to the sample dispersed in water alone
(see Figures 1B and 3). In water, in 150 mM NaCl and 150 mM NaI (all the environments)
the phase above transition temperature is most likely micellar, as the lipid dispersions were
optically clear and have very low optical density as revealed from turbidimetric
measurements (Figures 3, 4). The ESR spectra recorded above the phase transition
temperature in aqueous dispersions were more isotropic, which also support the micellar
structure above the phase transition (Figure 5). Lowering of the transition temperature in the
presence of 150 mM NaCl suggest that OACs with chloride counterion exhibit much lower
phase transition temperature than when the counterion is iodide. This indicates that ions
significantly modulate the phase behavior of OACs. To gain further information regarding
the number of ions required in the modulation, OSC with chloride counterion was
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synthesized and the DSC studies were performed with an aqueous dispersion of the sample.
The results obtained show that OSC with chloride counterion exhibits a much lower phase
transition temperature than OSC with iodide counterion (Figure 6). Our result suggest that
one ion per lipid molecule in the solution is sufficient to modulate the phase properties of
OACs.
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The effect of electrostatic interactions on the gelꢀtoꢀfluid transition of charged
phospholipid bilayer membranes have been investigated in detail.46ꢀ48 In such systems, by
modulating salt concentration and changing the pH, it was concluded that electrostatic
repulsion between the headgroups significanty affects the gelꢀtoꢀfluid transition,46ꢀ48 although
free energy of interaction between the hydrophilic segment and water is also important.49
While the electrostatic repulsion among charged lipid headgroups favors the fluid phase with
disordered acyl chains, the van der Waals attractions favor the chainꢀordered gel phase.
Thus, the Coulombic interaction competes with van der Waals interaction and below the
phase transition temperature the counterions would be expected to be strongly associated
with the gel phase thus making the area per headgroup small, whereas above transition
temperature counterions would be released into the surrounding aqueous solution (due to
entropic reason) leads to the fluid phase. In case of amphiphiles, the release of counterions
into aqueous solution makes the lamellar fluid phase very unstable due to electrostatic
repulsion and the gel phase transforms directly to micellar phase. We, therefore expect that
the electrostatic interactions and counterions could well modulate Krafft transition of ionic
surfactants, where the gel(or crystalline) phaseꢀtoꢀmicelle transition remains an important
parameter for surfactant solubility.
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The modulation of Krafft temperature of OSC by ions follows the Hofmeister series,
where kosmotropic ions lower the Krafft temperature and chaotropic ions increases the Krafft
temperature. The Hofmeister effect for macromolecules (protein, nucleic acids) in aqueous
solution are ubiquitous, although precise origin of action of the ions in the series has not yet
been understood.16,18 However, in case of surfactants a fairly good understanding about the
role of Hofmeister ions was obtained with the ionꢀpair formation vs hydration
arguments28,50,51. It was based on law of “matching water affinities” and attributes that
chaotropes can form direct ionꢀpairs with other chaotropes, while kosmotropes would pair
only with other kosmotropes, but chaotropes do not come close contact with
kosmotropes28,50,51. In other words, ions with equal water affinities will associate with each
other and tend to form contact ionꢀpairs in solution, whereas those with differing water
affinities will tend to separate.51 Qualitatively, small cations were found to prefer small
anions or anionic groups (e.g., acetate), lead to kosmotropeꢀkosmotrope interaction, while
larger cations (potassium) are attracted to larger anions or anionic groups (e.g.,
methylsulfate).28 Since choline group was regarded earlier as a chaotrope28, it will associate
with chaotropic ions and forms contact ion pairs with ions such as iodide, perchlorate,
chlorate etc. In such ion pairs, the screening of the charge will be effective and they are
likely to be much less hydrated than the individual ion and headgroup. The 3ꢀdimentional
structure of OSC iodide ionꢀpair shows that molecules in the crystal pack in an interdigitated
manner that allows both close packing of the alkyl chains (leading to strong van der Waals
interactions) and also significant reduction in headgroup repulsion.52 The moderately
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ꢀ
chaotropic Clꢀ, Brꢀ, NO3 ions are expected to be weakly associated with the choline group
and because of the repulsion between the bulky headgroups and hydration they most likely
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adopt a tilted bilayer gel phase below Krafft temperature. It would be expected that due to
hydration and electrostatic repulsion of the choline headgroups, in the presence of
moderately chaotropic and kosmotropic ions the area of headgroup per acyl chain will be
more than the ionꢀpair, formed in the case of strongly chaotropic ions, e.g., iodide.
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The insolubilization of the surfactants below Krafft temperature is usually accompanied
with the crystallization of alkyl chains (tightly packed bilayer/interdigitated bilayer gel
phase) of the surfactant molecules. Above Krafft temperature the system will lose enthalpic
stabilization (enthalpy of transition) but will gain more entropy, which leads to the transition
from a tightly packed gel to micellar phase. The enthalpy of transition in presence of
chaotropic ions is higher than the corresponding enthalpy in presence of weakly chaotropic
and kosmotropic ions (Table 2). Mechanistically, the transition from a tightly packed bilayer
phase to a micellar phase involves changes in the topology of lipid structures and thus must
occur through intermediates involving significant hydrocarbonꢀwater contact. In presence of
moderately chaotropic and kosmotropic ions the choline headgroup as well as ions will
remain hydrated. This will lead to relatively large headgroup area, which is responsible for
loose packing of acyl chains and more water penetration into the bilayer. On the other hand,
in the presence of more chaotropic ions the ionꢀpair formation will lead to screening of
charge, resulting in a decrease in the penetration of water into the bilayer. Since the
intermediate structure formed during the transition to a micellar phase involves significant
water hydrocarbon interaction, the extent of water penetration into the bilayer may also
account for the modulation of Krafft temperature. Alternatively, release of counterions into
the surrounding aqueous solution above the transition temperature could well explain the
observed Hofmeister effect. Strongly associated ions (here chaotropic ions) will be more
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reluctant to separate from the ionꢀpair in the gel or crystalline phase. The removal of
counterions from the surface triggers strong electrostatic repulsion and hydration of
headgroups, necessary for the formation of micellar phase.
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Our present results show that chaotropic ions increase the Krafft temperature of OSC
and therefore at physiological temperature these chaotropic ions (such as iodide and
perchlorate) induce precipitation and the system behaves as if it is in a virtually inactive state
(crystalline phase). On the other hand it exists in an active state (micelle) in presence of
chloride counterion at physiological temperature. The Krafft temperature of OSC increases
when the ions are changed along the Hofmeister series, suggesting that at a particular
temperature the probability of formation of inactive crystalline form increases along the
series. This fact will have an important consequence since physiological fluids contain the
chloride salts (KCl, NaCl), which may help to keep the lipids and surfactants in their active
state at physiological temperature. Similar effect of different ions on proteins has been
examined and it was shown that these ions play a definite role in saltingꢀin or saltingꢀout.
However, in case of proteins the chaotropic ions induce unfolding and greater solubility,
hence saltingꢀin ions and in case of OACs the chaotropes form insoluble ion pairs which lead
to precipitation.
SUMMARY AND CONCLUSIONS
Thermotropic phase transitions of a homologous series of Oꢀacylcholines, which are
biologically relevant lipids, have been characterized by differential scanning calorimetry. A
linear dependence has been observed in the thermodynamic parameters, ꢀHt and ꢀSt
associated with the chainꢀmelting phase transitions. The first crystal structure in this lipid
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series, that of Oꢀstearoylcholine iodide has been solved by single crystal Xꢀray diffraction.
The structure demonstrates that the OSC molecules adopt an interdigitated bilayerꢀtype
arrangement, with the polymethylene portion of the acyl chains adopting an allꢀtrans
conformation. DSC, spectroscopic and turbidimetric studies indicate that in aqueous
dispersion Oꢀstearoylcholine undergoes a cooperative phase transition from a gel phase to a
micellar structure and that the transition temperature increases when the counterions are
changed in the Hofmeister series. The OACs in chloride salt environment have a lower
Krafft temperature and they will be in active form (micelle) near body temperature, whereas
in iodide environment they exist in an inactive state (crystalline precipitate).
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Supporting Information Available: Parts of Methods and Results, including three tables
(S1 – S3) and two figures (S1, S2) are given in Supporting Information. This material is
available free of charge via the internet at http://pubs.acs.org.
ACKNOWLEDGEMENT
This work was supported by a research project from the Department of Science and
Technology (India) to MJS. PKT and STR thank the Council of Scientific and Industrial
Research (India) for Senior Research Fellowships. We thank the University Grants
Commission (India) for their support through the UPE (to University of Hyderabad) and
CAS (to School of Chemistry) programs. The authors are grateful to Prof. Samudranil Pal
and Dr. Abhik Mukhopadhyay of this School for advice in Xꢀray data analysis.
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REFERENCES
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(1) Tsujii, K.; Mino, J. Krafft Point Depression of Some Zwitterionic Surfactants by
Inorganic Salts. J. Phys. Chem. 1978, 82, 1610–1614.
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