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Structure property relationships in the sodium muricholate derivatives (bile salts) micellization: the effect of conformation of the steroid skeleton on the hydrophobicity and micelle formation pattern recognition and potential membranoprotective properties Mihalj Poša, and Kosta Popovi# Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00375 • Publication Date (Web): 01 Sep 2017 Downloaded from http://pubs.acs.org on September 3, 2017

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Molecular Pharmaceutics

Structure property relationships in the sodium muricholate derivatives (bile salts) micellization: the effect of conformation of the steroid skeleton on the hydrophobicity and micelle formation - pattern recognition

and

potential

membranoprotective

properties Mihalj Poša*, Kosta Popović University of Novi Sad, Faculty of Medicine, Department of Pharmacy, Hajduk Veljkova 3, 21000 Novi Sad, Serbia

*To whom correspondence should be addressed: Prof. Mihalj Poša, University of Novi Sad, Faculty of Medicine, Department of Pharmacy, Hajduk Veljkova 3, 21000 Novi Sad, Serbia Tel: +381 6311 400 15 Tax: +381 21 422 760 E-mail adress: [email protected] and [email protected] (M.P.)

ACS Paragon Plus Environment

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KEYWORDS: aggregation number, steroid skeleton, conformational analysis, hydrophobic linear congeneric groups, hydrophobic drug

ABSTRACT: It is known that β-muricholic acid anions prevent membrane toxicity of hydrophobic bile acids which are being used in therapy for solubilization of the cholesterol type bile stone. Better knowledge of these derivatives' micelles is very important for understanding their physiological and pharmacological effects. β axial (a) oriented hydroxyl group from the steroid skeleton decreases the hydrophobic surface of the convex side of the steroid skeleton. Therfore, the critical micellization concetration (CMC) for steroid surfactants with β-a-OH group should increase, but in case of OH groups of different orientations forming H-bonds in the hydrophobic phase of the micelle it has the oposite effect, the CMC decreses – aggregation is more favorized. The set of muricholic acids (MCs) is composed by α-MC, β-MC, γ-MC and ωMC where α-MC and β-MC have β-axial-OH groups. The aggregation numbers (n) are determined using Moroi-Matsuoka-Sugioka thermodynamic method. CMC, enthalpy of demicellization and ∆Cp are determined by isothermal titration calorimetry (ITC). This report pioneers in the study of MC derivatives micellization. Micelles of β-MC and γ-MC belong to the linear congeneric group (LCG) and their micelles above 85 mM have constant aggregation numbers n = 4-5. Micelles of α-MC and ω-MC are outliers in relation to the LCG, their aggregation number constantly increases; at 85 mM n = 6.8 (α-MC) and 6.5 (ω-MC). In micelles of derivatives β-MC and γ-MC there is a low probability for the existence of hydrogen bonds. Micelle of α-MC probably has hydrogen bonds in its hydrophobic domain.


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1. INTRODUCTION Bile acids anions (bile salts, BSs) are steroid biosurfactants which in mammals synthesize in hepatocytes, starting from cholesterol.1,2 In humans, cholic and chenodeoxycholic acids – primary bile acids – synthesize in the liver, where they conjugate predominantly with glycine.3 In mice, primary bile acids are cholic and β-muricholic acids.4 Secondary bile acids originate from primary bile acids by deconjugation, dehydrogenation and dehydroxylation as the result of intestinal flora activity. In bile canaliculi, BSs and phospholipids form mixed micelles, which dissolve cholesterol.5-7 During digestion in the intestinal lumen, BSs form micelles – mixed micelles which solubilize lipid molecules, liposoluble vitamins and hydrophobic medications, and transport them to the intestinal epithelium.5-7 BSs promote the effect of the transportation of certain drugs through various biological barriers.8-12 Apart from the detergent effect (in anion form), bile acids demonstrate their biological effects via receptory mechanism as well. Bile acids modulate their own biosynthesis, as well as lipoprotein, glucose and energy metabolism, through nuclear (Farneosid X receptor, FXR) and membrane receptors (G-protein-coupled receptor, TGR5).13-16 As surfactants in water solution above a certain concentration (critical micellization concentration, CMC), BSs form association colloids – micelles with the aggregation number n (number of building units, i.e. monomers in a micelle): CMC and n are micellization parameters. The anions of bile acids differ from the typical surfactants with a hydrophobic tail (hydrocarbon chain) and polar head in the following structural and conformational features: 1. the geometry of spatial distribution of hydrophobic and hydrophilic molecular segment and 2. conformational rigidity of the hydrophobic molecular segment.1,17


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The BS steroid skeleton structure contains a convex hyper-plane (β side), which is the hydrophobic surface of the molecule, and a concave hyper-plane (α side) representing the hydrophilic surface of the steroid ring system.6,17 In contrast to the hydrocarbon chain of typical surfactants, the steroid skeleton is characterized by rigid conformation, i.e. with small intramolecular mobility. These structural differences are reflected in different values of aggregation parameters of BSs and typical surfactants. Surfactants with an aliphatic hydrocarbon chain exhibit significant changes of the physico-chemical solubility parameters in a critical micelle concentration, whereas in BSs these changes are gradual.18,19 The micelles of surfactants with a hydrocarbon chain have aggregation numbers from 60 to 200. The micelles of anions of bile acids usually contain from 2 to 14 building units.20-27 Several BS can form larger aggregates with aggregation numbers ranging from 30 to 60 in concentrations above the critical micelle concentration.1,7 According to the Small-Kawamura model, BSs form micelles when the hydrophobic surfaces of steroid skeletons (β sides) in aggregates face each other, i.e. when the main thermodynamic force in the formation of the micelle is the hydrophobic effect.7,28 Small makes a distinction between smaller BS micelles formed predominantly by the hydrophobic effect – primary micelles, and larger micelles (in some BS molecules) created by hydrogen bonds between primary models – secondary micelles.7,29 According to Small, the formation of primary micelles is based on the all-or-nothing principle, i.e. the micelle formed is monodispersive according to its aggregation number. However, the model which is now accepted is the one of stepwise association of BSs, which form micelles of varying sizes (with varying aggregation numbers). Therefore, the experimentally determined aggregation number is the average aggregation number of the micelle population of different numbers of building units at a total concentration of BS.24,30 Analysis of steady-state and time-resolved experiments in which


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3α-dansyl-5β-cholic acid was used as the fluorescent reporter confirms the existence of primary and secondary BS aggregates.31-33 Because BSs form micelles, the bile acid anions are used in pharmaceutical formulations as solubilizers of hydrophobic drugs for oral use.5 It is desirable that the BS administered in the pharmaceutical formulations has low membranotoxicity, or more specifically, a higher CMC value than that of sodium deoxycholate. However, with the increase in CMC values in bile salts, their capacity to solubilize the hydrophobic drug decreases. Between membranotoxicity and solubilization capacity there is an inverse proportionality.34 Therefore, it is necessary to find the optimal ratio of the hydrophilic and hydrophobic surface of the BS steroid skeleton. This can be achieved by substitution of OH groups from the steroid skeleton of natural bile acids with oxo (keto) groups.5 Bile acids belonging to the group of muricholic acids are derivatives which in the steroid skeleton of 5β-cholanic acid have a C3 α-equatorial OH group and OH groups on C6 and C7 carbons with different orientations (Figure 1) In mice, the α- and β-muricholic acids synthesize in the liver (together with cholic acid), where they conjugate with taurine. As the secondary bile acid (in mice), ω-muricholic acid is formed as a result of the activity of intestinal microbiotic flora on β-muricholic acid. Tauro-β-muricholic acid, similar to tauroursodeoxycholic acid, prevents the membrane toxic effect of hydrophobic bile acids (taurochenodeoxycholate), which represents their potential biomedical use. Unlike taurocholate and taurochenodeoxycholate, which are the agonists of FXR nuclear receptors, tauro α- and β- muricholates are antagonists.4,35-37


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Self-associations of sodium salts of muricholate derivatives (MC: α-MC, β-MC, γ-MC=H, ωMC, Figure 1) have not been researched so far; therefore the aim is to determine the critical micellization concentration and the aggregation number of MC micelle using noninvasive experimental techniques. The number of stereochemical variations in the orientation of OH groups of the steroid skeleton of MC derivatives makes it possible, using conformational analysis and the chemometric method (based on the experimentally determined micellization parameters),26,27 to indirectly examine if there exist hydrogen bonds between the building units of MC’s micelles, alongside the hydrophobic effect (interaction), which additionally (H-bonds) stabilize the aggregates. Therefore, the testing included MC derivatives as well as Na-salts of the following bile acids: cholic acid (C), chenodeoxycholic acid (CD), 7-oxolithocholic acid (7OxL), hyodeoxycholic acid (HD) and ursodeoxycholic acid (UD), whose micelles have already been tested and whose aggregates have been studied intensively.1,2,18-27,38-40 Another important goal of this paper is to explain β-MC anion membranoprotective properties in regards to structural thermodynamic characteristics of MC derivatives’ micelles.

2. MATERIALS AND METHODS 2.1. Materials Cholic, chenodeoxycholic, hyocholic, hyodeoxycholic, ursodeoxycholic, taurocholic and taurolithocholic acids were purchased from Sigma-Aldrich (Auckland, New Zealand, mass fraction purity > 0.990). The muricholic acid derivatives were obtained from Steraloids


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(Newport, RI, mass fraction purity > 0.990). 7-oxolithocholic acid (7-OxL) was prepared according to the procedure of Tullar from.41 All bile acids were transformed to their sodium salts by known procedure.2

2.2. Reverse Phase HPLC method Experiments were carried out according to our earlier paper.27 Trials were repeated (n = 5) for reproducibility. The relative capacity factor ( ki∗ ) was calculated the bile acids anions' capacity in relation to the taurocholic (TC) capacity factor (kTC ) :

ki∗ =

ki kTC

(1).

Heuman introduced the hydrophobicity factor representing the relation:

HFi =

ln ki∗ ∗ ln kTLC

(2),

∗ ( kTLC ) where is the relative retention factor of taurolitocholates (TLC).42

2.3. Determination of Average Aggregation Numbers by the Moroi-Matsuoka-Sugioka Method Solid bile acid was suspended in distilled water by stirring on a magnetic stirrer, and an increment of NaOH solution was added with the aid of micro syringe. In this way the total concentration of the bile acid anion (monomer) is regulated. After the 24-hour equilibriation, the


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pH of the clear solution was measured (Boeco BT600) without separation of the solid phase, and taking care not to disturb it.38-40 This gave one point on the titration curve and the procedure was repeated to obtain about 20 points, of which at least 5 in the micellar region. Measurements were performed at room temperature (20 °C).

2.4. Isothermal Titration Calorimetry, ITC Thermometric titration experiments were performed at T (10, 15, 20, 30, 40, 50) °C with thermal activity monitor (TAM) isothermal heat-flow micro calorimeter (ThermoMetric LKB 2277, Lund, Sweden) and a twin detector, supplied with a sample cell and a reference cell. Sample cell, equipped with a stirring facility and a Lund microtitrator, was loaded with 2 mL of water.43 A stirring rate of 60 rpm was applied and titrant (0.5 ml of BS solution in water at ≈ 10CMC) was injected into cell at 90 minute intervals in aliquots of 10 µL. Experiment was computercontrolled via DigiTam 4.1 software. Noise level of calorimeter baseline during measurements was within ±0.05 µJsec-1. Reproducibility of calorimetric peaks in titration experiment was on average better than 2%.

2.5. Spectrofluorimetric measurements of CMC Fluorescence measurements were carried out using Agilent Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Santa Clara, California, USA). Pyrene was used as a fluorescence probe molecule. All solutions of bile salts were prepared using pyrene saturated water. Fluorescence emission spectra of these solutions were recorded employing an excitation wavelength of 334 nm. The intensities of first (I1) and third (I3) vibration bands of pyrene


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emission spectrum were measured at 373 and 384 nm respectively. The I1/I3 ratio was monitored as a function of total bile salts concentration at 20 °C. The CMC values were obtained from the intersection of the straight lines above and below the CMC. The straight line above the CMC value was determined by linear regression starting from the highest concentration, while the straight line below the CMC value was obtained by linear regression beginning at the lowest concentration of investigated surfactant from the domain of stabile micelle formation.40 The regression diagnostic was repeatedly performed after addition of each subsequent experimental point. If an experimental point was identified as an outlier, by measuring Cook’s distance the regression would be stopped and the straight line would be formed using previously added experimental points.

3. RESULTS AND DISCUSSION 3.1. Hydrophobicity In reversed-phase liquid chromatography of high resolution (RPHPLC), where the stationary phase is hydrophobic, the anions of bile acids attach to the stationary phase through the largest hydrophobic plane of the steroid skeleton – the convex surface (β side) of the steroid ring system.17 Due to the relative diffuseness of the octadecyl chains of the stationary phase both C7 and C12 lateral sides of the steroid skeleton (Appendix A) are also in contact with the stationary phase.44 Therefore, according to the HF values, a bile acid anion will be less hydrophobic if it has a larger number of OH groups at the convex or lateral surfaces of the steroid skeleton.17 Bile acid anions with negative HF values are less hydrophobic than taurocholates, and the more negative the HF value, the less hydrophobic the bile acid anion will be.42 In terms of HF values


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(Table 1) α-MC has the least hydrophobic convex surface of the steroid skeleton among the muricholic acid anions. The hydrophobic nature of the steroid skeleton convex surface in the set of MC derivatives is determined by the spatial orientation of the OH groups, ie. the conformation of the molecule (Appendix B).45

3.2. Micellization The critical micellization concentration (CMC) of the researched BSs is determined by isothermal titration calorimetry (ITC) and by spectrofluorimetric measurements (SF) with pyrene as the guest molecule (Table 1). ITC is a non-invasive method, which does not disturb the micelle structure in determining the critical micellar concentration.18,19,46,47 Figure 2 A presents heat flows at successive increase of BS concentration in the micellar solution (the concentration is around 10 times higher than the CMC) in the reaction cell (the first increment of micellar solution is added to water in the reaction cell). Integrating heat peaks (Figure 2 A) within the function of the total BS concentration (ct) in the reaction cell results in the total change of enthalpy (∆H) during ITC, i.e.in the critical micellar concentration at T = const. (Figure 2 B). The following processes happen during titration: the dissolution of concentrated micellar solution to CMC, the decomposition of micelles (demicellization process) and the dissolution of monomers. The total change of the enthalpy during ITC is approximately equal to the demicellization enthalpy (∆H = –∆Hdemic).18,19,46,48 Nonetheless, the ∆HM = –∆Hdemic (∆HM = micellization enthalpy). The ITC experiments for MC derivatives have been conducted at the temperatures T (10, 15, 20, 30, 40, 50) °C (Table 2). The dependency of the critical micellar concentration on temperature is a polynome function of the second or third order (Figure 2 C). Namely, according to the pseudophase approximation in reaching the equilibrium in the


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demicellization process or micellization process (observing the equilibrium when the first • micelles formed), the chemical potential of monomers in micelle ( µ mM ) is equal to the chemical

potential of monomers from the bulk water solution ( µ m ( aq ) ) whose concentration corresponds to the critical micellization concentration ( µm0 ( aq ) – standard chemical potential of monomers in infinitely diluted water solution according to Henry): • µmM = µm( aq ) = µm0 ( aq ) + RT ln CMC (3).

0 According to the given equation the standard Gibbs energy of demicellization is ( ∆Gdemic ):

0 • ∆Gdemic = µm0 ( aq ) − µmM = − RT ln CMC

(4).

Since CMC is expressed in molar fractions in the expression (4), the following is true: 0 ∆Gdemic > 0 . Temperature differential of the standard Gibbs energy of demicellization gives the

following expression: 0 1  ∂ ( ∆Gdemic T )   ∂ ln CMC    = −  (5),  R ∂T ∂T  p,n    p ,n

i.e. by using the Gibbs-Helmholtz equation ∂ ( G T ) ∂T = − H T 2 the temperature coefficient of the critical micellar concentration is determined as: ∆H demic  ∂ ln CMC    = ∂T RT 2   p ,n

(6).


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Comparing the function slope CMC = f (T) (Figure 2 C) with the values ∆Hdemic in Table 2, it can be concluded that the observed function corresponds to the equation (6). The extreme point (minimum point) of the function CMC = f (T) of MC derivate is near the temperature (20±2-4 °C), at which the demicellization enthalpy equals zero (Table 2). During the transfer of a hydrophobic molecule (e.g. benzene, toluene, cyclohexane, etc.) from a clear liquid state to a water solution, the solubility slope x = f (T) (x being the molar fraction of the hydrophobic molecule in a water solution, i.e. solubility at a certain temperature) has the same form as the slope of function CMC = f (T), i.e. the temperature coefficient of solubility of a hydrophobic molecule is analogous to the equality of the temperature coefficient of critical micellar concentration (6):49,50 ∆H  ∂ ln x    = 2  ∂T  p ,n RT

(7).

This points to the significance of the hydrophobic effect in the process of MC derivatives micellization. Undoubtedly, hydrogen bonds between the building units are also possible in micelles, but the main contribution to the Gibbs energy of micelle formation is the change of Gibbs energy as a result of the hydrophobic effect. At the temperature (TH) where ∆Hdemic = 0 or ∆HM = 0 (∆G = ∆H - T∆S →∆G = - T∆S), the Gibbs energy of micellization (hydrophobic effect) is of entirely entropic origin. From the equation (6) it follows that the critical micellization concentration is lowest at the temperature TH, i.e. at the TH temperature, MC’s monomers have the greatest affinity towards forming aggregates (micelles). The hydrophobic effect in the micelle formation is also the greatest at the temperature TH. It originates from the ordered water molecules (compared to the water molecules from within the solution) in the hydration layer of the hydrophobic surface of the steroid skeleton which, during the formation of


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micelle with hydrophobic inner phase (domain) transfers into bulk solution, i.e. into a less ordered state. The hydrophobic effect decreases (CMC rises) with the further rise of temperature over TH: ∆HM < 0 (or ∆Hdemic > 0, Table 2). The formation of micelle will have increasingly stronger enthalpy character as a result of the increase of Van der Waals interactions (hydrophobic interactions) between hydrophobic surfaces of the building units of the micelle, while the entropic character will drop due to the decrease of the ordering of the water molecules in the hydration layer (with the temperature increase, water molecules from the hydration layer increase in rotational and vibrational energy, i.e. their entropy approaches the entropy of the molecule of water from the interior of the solution). The change in the heat capacity for the demicellization process:

 ∂∆H demic  ∆C p =    ∂T  p,n

(8),

is constant in the temperature range tested, i.e. the dependency of the demicellization enthalpy change with the temperature is linear (Table 2). The positive value ∆Cp means that during demicellization hydration of the hydrophobic monomer surface takes place, which is protected from hydration inside the micelle.18,19,48 Since in the MC derivatives in the thermal dependence of the critical micellar concentration the lowest value for CMC (ITC) is at the temperature of 20 °C (in the vicinity of TH), at which the hydrophobic effect is the most prominent in the process of micellization, the other tested BS (necessary for pattern recognition) the critical micellization concentrations in the ITC experiment are also determined at 20 °C (Table 1). In the tested BSs (non-conjugated bile acid anions) for anion C there is data in the literature about the critical micellization concentration determined by


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the ITC method (Table 1) – the value determined in our experiment corresponds to the value quoted in the literature within the margin of error. The literature also quotes CMC values for water solutions of specific BS (Table 1), which have been measured by tensiometry (dependency of the surface tension on the surfactant concentrations), also a non-invasive method,51 but at the temperature 25 °C. These values are somewhat higher than the ones we found in our experiments, but the differences of CMC and the values of various BS exhibit the same tendency. Tensiometric CMC values are probably higher due to the differences in the measurement temperature. Function of the ratio of intensities of pyrene vibration bands (I1 / I3) in relation to the total concentration of bile salts (ct) in the set of MC derivatives below CMC is stretched the least in αMC derivative (Figure 3). Namely, the more (I1 / I3) = f (ct) is stretched before the critical micellization concentration, process of self-association is in the greater extent more successive i.e. gradual - stepwise association.25 There are slightly lower values for the CMC obtained by the SF method compared to the CMC values obtained by the ITC method. This was likely due to delivery of the guest molecule (pyrene) to the BS micelles. Statistically, CMC determined by both methods are not mutually different (Table 1). Accordingly, the self-association of α-MC derivative is more favorable than the self-association of the hydrophobic β-MC stereoisomer (C7-epimer) (Appendix B). On the basis of the hydrophobicity of the steroid skeleton of α-MC derivative (the least hydrophobic in the set of MC derivatives) it would be expected that it’s CMC is greater than the CMC value of β-MC and ω-MC derivative (Table 1). Also change in (I1 / I3) = f (ct) below the CMC should be sharper with the β-MC and ω-MC derivatives than with αMC. The different behavior of α-MC derivatives than it would be expected from its hydrophobicity can be explained if it is assumed that for the formation of micelles, in addition to


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the hydrophobic effect as the main factor of the association (the main pulling force of association), there are also hydrogen bonds already at CMC values. Based on the dependency of the aggregation number on the total concentration of surfactants (Figure 4), MC derivatives can be divided into two groups of molecules. One group consists of derivatives γ-MC and β-MC, which have constant values of the aggregation number (with the fluctuation of 0.3) over 85 mM. The second group is made up of α-MC and ω-MC, whose micelles have an increase of the aggregation number over 85 mM. Similar behavior has been observed in our earlier experiments in cholic acid anions and 7-oxodeoxicholic acids which have hydrogen bonds in the micelle at concentrations significantly over the CMC, i.e. they probably build the secondary micelles as well.24 Table 1 is shown for the comparison with the values from the literature for aggregation numbers calculated using the Moroi-Matsuoka-Sugioka method.38,40

3.3. Pattern recognition and micelle structure The standard Gibbs energy of micelle formation of bile acid anions ( ∆GM0 ) at room temperature is of entropic origin (driven), i.e. the micelles are formed due to the existence of the hydrophobic effect.17,18,49 The binding of bile acid anions from the polar mobile phase to the non-polar stationary phase (RPHPLC) is also the result of hydrophobic effect,52 meaning that the driving force of this process is of entropic origin. Therefore, in the set of structurally similar molecules {1,2, … i, … n} – linearly congeneric group of amphiphilic molecules ( LCG − j ) – the standard Gibbs energy of micelle formation of each bile acid anion ( ∆GM0 (i ) LCG − j ) can be presented as the product of the coefficient ( bi ) (coefficient of vector decomposition in a one-dimensional vector


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space in relation to the ort vector) and the standard Gibbs energy of adsorption on hydrophobic stationary phase of the referent bile acid anion ( ∆GA0 ( R) LCG − j ) . Generally, a referent molecule can be any molecule from the LCG − j . Similarly, the hydrophobic surface ( Ahf (i ) LCG − j ) of any bile acid anion from the LCG − j can be presented as the product of the coefficient ( ri ) and the hydrophobic surface of the referent molecule ( Ahf ( R ) LCG − j ) . However, the coefficients bi and ri are not random, but such that there is a linear dependency between them ( b = ξ LCG − j r + β LCG − j ) which defines the hydrophobic linear congeneric group LCG − j (relation-equation (9) from Scheme 1). Taking into consideration the relation (9) from Scheme 1 and the thermodynamical condition for the equilibrium in the formation of bile acid micelle (BS) with the average aggregation number K

n : nBS

( BS)n , the following equations characterizing hydrophobic linear congeneric groups

in planes n - ln k and ln CMC - ln k are obtained (Appendix C):27 ln k = c1 + tan α1LCG n (10), ln k = c2 − tan α 2LCG ln CMC (11),

or, if instead of ln k one introduces the Heuman parameter of hydrophobicity (HF) (2): HF = c3 − tan α 3LCG ln CMC

(12),

HF = c4 + tan α 4LCG n (13),

where c1 − c4 are constants, and the tan α1LCG − tan α 4LCG are the corresponding slopes.


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In the plane ln CMC - HF the tested muricholic acid anions β-MC, γ-MC and ω-MC are in the linear congeneric group LCG − j (Figure 5 A): HF = 1.49(±0.19) − 0.56( ±0.06) ln CMC (14)

N = 8; R = 0.965; F = 82.16; sd = 0.10 This means that at the critical micellization concentration the standard Gibbs energy of micelle formation of β-MC, γ-MC and ω-MC anions originates from the hydrophobic effect. At the critical micellization concentration dimeric micelles are predominantly formed (or have a crucial role in further association),52 where the convex hydrophobic surfaces (β sides – the main hydrophobic surface of the molecule) of the steroid skeletons of micellar building units are facing each other.25,26 According to Figure 5 A α-muricholic acid anion (α-MC) is an outlier in relation to the LCG − j group (relation-equation (15) from Scheme 1). This means that the standard Gibbs energy of micelle formation of α-MC anion, apart from the Gibbs energy of hydrophobic effect, contains excess Gibbs energy (GE), which probably originates from the formation of a hydrogen bond between the building units of the micelle. Therefore, the relation (15) in the case of the α-MC = k anion is the relation (16) from Scheme 1, where

( bˆ ∆G ( R) k

0 A

LCG − j

) represents the contribution of Gibbs energy of the hydrophobic effect to the

Gibbs energy of micellization α-MC = k, and which is obtained on the basis of the hydrophobic surface of the steroid skeleton α-MC bˆk = ξ LCG − j rk + β LCG − j

obtained

in

(A

hf

( k ) = rk Ahf ( R ) LCG − j ) , i.e. the linear dependency:

the

hydrophobic

linear

congeneric

group

LCG − j : ( ∀i ∈ LCG − j ∧ ∃k ∉ LCG − j ) . In the dimeric micelle α-MC, formed in the vicinity


17


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Page 18 of 46

of the critical micellar concentration,52 the formation of a hydrogen bond is possible since the C6 β OH group has axial orientation (Figure 1). A dimeric α-MC micelle, in which the steroid skeletons of the building units are associated through the convex sides of the molecule, the C6 OH groups are facing each other, i.e. C6-OH sigma bonds of two steroid skeletons of α-MC are parallel and the distance between them is ≈3Å, which enables the hydrogen bond to be formed in the hydrophobic domain of the micelle α-MC (Figure 6 A). The bile acid β-MC anion also contains an axial (a) C6 β OH group. Therefore, one might expect a hydrogen bond to be created in the dimeric micelle β-MC between C6 β (a)OH groups of the steroid skeleton (Figure 6 B-D). However, unlike the α-MC micelle, the β-MC anion micelle enters the hydrophobic linear congeneric group at the critical micellar concentration (Figure 5 A). This can be explained by the spatial orientation of the vicinal OH groups of β-MC. In β-MC the OH group with C7 has β equatorial (e) orientation, i.e. the OH groups with C6 and C7 have β orientation and are in a synclinal (sc) position to each other (Figure 1). They can therefore bind a water molecule from the hydration layer through hydrogen bonds (cooperative bonding), which thus sterically hinders the C6 β (a)-OH group in the micelle (Figure 6 E). In α-MC the vicinal OH groups have the antiperiplanar (ap) orientation to each other (Figure 1), and, thus, these OH groups cannot cooperatively bond with the water molecule from the hydration cage. At the critical micellization concentration ∆Cp for the demicellization process the α-MC anion micelle has the highest value (250 Jmol-1K-1) compared to the other MC derivate micelles (Table 2), even though α-MC is the least hydrophobic (Table 1). A high value of ∆Cp of α-MC is probably a consequence of the fact that the hydrophobic convex surfaces of the steroid skeleton of the building units are more efficiently associated with one another due to hydrogen bonds,53 i.e. the convex surfaces of the building units are mostly parallel with one


18

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another and are thus more protected from the hydration than in other MC derivatives (Figure 5 A). The demicellization of the anion β-MC micelle for ∆Cp has the lowest value (180 Jmol-1K-1) in the MC group of bile salts. As a result of attaching water molecules through hydrogen bonds with the vicinal β OH anion groups, the convex hydrophobic surfaces of the steroid skeletons βMC in the micelle are no longer parallel (Figure 6 E) and the hydrophobic surfaces are thus exposed to hydration. In the plane HF – n (the aggregation number refers to the concentration of 85 mM) the β-MC and γ-MC anion micelles (hyocholic acid) form a linear congeneric group ( LCG − j ) with micelles of the bile acid anions (chenodeoxycholic acid, ursodeoxycholic acid and 7-oxolithocholic acid) which, as is known from previous studies, form the hydrophobic linear congeneric group,24,27 i.e. they do not form Small’s type of secondary micelles (Figure 5 B): HF = 1.74( ±0.28) + 0.27(±0.05) n

(17).

N = 5; R = 0.955; F = 31; sd = 0.15 The micelles of anions α-MC and ω-MC form a special group with the micelles of anions HD and C which have been known to form secondary micelles:24,27 HF = 1.40 + 0.13n (N = 4; R = 0.8688) (18).

By excluding the α-MC anion micelle, a linear congeneric LCG − e group is obtained (Figure 5 B): HF = 1.08 + 0.105n (N = 3; R = 0.9878) (19),


19


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Page 20 of 46

i.e. α-MC is an outlier in relation to the linear congeneric group LCG − e . Since HD and C can build secondary micelles,24,40 then ω-MC can also form secondary micelles. For two primary micelles to form a secondary micelle it is necessary for two hydrogen bonds to be created between them.24,25 Therefore, one can assume that the secondary micelles of anions of the

LCG − e group there is the same number of hydrogen bonds, i.e. their standard Gibbs energy of micelle formation has the same value as the excess Gibbs energy ( G E ) in relation to Gibbs

(

)

energy of hydrophobic effect bˆk ∆GA0 ( R )G E -LCG − j : relation-equation (20) from Scheme 1. The fact that micelles of anions ω-MC, HD and C form the linear congeneric group LCG − e is a consequence of the linear change of the Gibbs energy of hydrophobic effect

( bˆ ∆G (R) k

0 A

G E -LCG − j

)

– from the standard Gibbs energy of micelle formation – with the

(

)

hydrophobic surface of the molecule rk Ahf ( R )G E -LCG − j . The difference of the steepness value of the slope of the linear congeneric group LCG − j from the steepness value of the slope LCG − e (Figure 5 B) is the result of the inequality: ξ LCG − j ≠ ξG E -LCG − j . This is probably a consequence of the fact that in secondary micelles, i.e. micelles at the total concentration of surfactants considerably higher than the critical concentration, the hydrophobic surface of monomers exposed to hydration is changed in comparison to the hydrophobic surface of monomers in primary micelles. The relation (20) yields the equation of the slope of the linear congeneric group LCG − e which satisfies the equation (19):

HF =

GE + c4 + tan α 4LCG n c5

(21).


20

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If in micelles ω-MC, HD and C the change of Gibbs hydrophobic effect energy were insignificant compared to Gibbs hydrogen bond formation energy, then micelles ω-MC, HD and C in the plane HF – n would not form a straight line with a slope but would group around the point: G E c5 (21). The fact that the α-MC micelle anion is an outlier in relation to LCG − e is probably the consequence of the fact that this micelle is an outlier even at the critical micellization concentration (Figure 5 A), i.e. even the primary α-MC anion micelles contain hydrogen bonds. In α-MC C7 the α (a)-OH group is in synclinal position with the C14 methine group, i.e. with one part of the D ring.17,24 Therefore, this OH group, due to steric hindrance, cannot participate in the bonding of monomers through hydrogen bonds during the formation of secondary micelles (Appendix D). In the existing literature, it is shown that 7,12-bis-glucoside derivatives of certain bile acids are significantly less hydrophobic than the starting bile acids, but their critical micellization concentrations are not altered or are even lower than the CMC values of the starting derivatives. This is also explained by the presence of hydrogen bonds between building blocks of micellar aggregates. Hyocholic acid H (γ-muricholic acid = γ-MC), as well as HD, contains α equatorial OH group on the C6 carbon of the steroid skeleton which is not sterically hindered with the D ring.17,24 However, in contrast to the HD,24,40 γ-MC does not form secondary micelles, i.e. its micelle forms a hydrophobic linear secondary group. In the case of HD, a secondary micelle (the association of primary micelles) is formed by linking (through hydrogen bonds) the monomers from two primary micelles, as a result of the monomers (from different primary micelles) facing each other through the concave surface of the steroid skeleton (α side of the molecule). This spatial arrangement of monomers sterically allows the formation of a hydrogen bond between the C6 OH group of one HD anion and the C3 OH group of another HD anion, i.e. the formation of a


21


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Page 22 of 46

hydrogen bond between carboxylate function of one HD anion and the C3 OH group of other HD anion (Figure 7). Although the γ-MC has the same structural element as the HD (C3 α (e)-OH and C6 α (e)-OH group) the presence of the C7 α axial OH group which is synclinal with the C6 α (e)-OH group sterically prevents the formation of a secondary micelle (Appendix E). In anion ω-MC C7 OH group has β equatorial orientation, which means that the spatial environment around C6 α (e)-OH group on the α side of the steroid skeleton is not sterically hindered, which makes it accessible for the C3 α (e)-OH group of other monomer, i.e. the ω-MC can form a secondary micelle analogous to the anion HD (Figure 7). Tauro-conjugated β-MC derivative reduces membranotoxicity of sodium chenodeoxycholate (CD), which can be explained by micellization.4 β-MC at concentrations exceeding CMC enters a hydrophobic linear congeneric group, which means that the presence of hydrogen bonds in the β-MC micelles is not significant in comparison with α- and ω-MC micelles. This means that the β-MC micelle is dominated by hydrophobic interactions that distort unsignificantly with incorporation of chenodeoxycholic acid anions, meaning that the formation of binary mixed micelles between β-MC and chenodeoxycholic acid anions prevents micelles of CD anions to extract or remove phospholipids from cell membranes. Incorporation of CD in α- and ω-MC micelles is thermodynamically unfavorable, since there would be a disruption of hydrogen bonds (in micellar α- and ω-MC). This means that a hypothetical binary mixed micelle of CD and αMC or CD and ω-MC would be thermodynamically less stable than the starting monocomponent micelles. MC derivatives have a significantly higher critical micellization concentration than sodium deoxycholate, therefore, considering the difference in membranotoxicity, they could be used in pharmaceutical formulations as solubilizers of hydrophobic drugs. MC derivatives which do not have hydrogen bonds between the β sides of the steroid skeletons of the two building


22

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blocks in the micelle are especially significant because the hydrophobic drug incorporated in the micelle does not result of the hydrogen bond disruption, i.e. destabilization of the micelle. 4. CONCLUSIONS MC derivatives can be divided into two groups of compounds. One group contains anions β-MC and γ-MC whose micelles in the planes ln CMC– ln k and HF – n enter linear congeneric groups, i.e. these micelles have low probability of forming hydrogen bonds. The aggregation number of micelles β-MC and γ-MC above the surfactant concentration of 85 mM is constant, with the values from 4 to 5 building units per aggregate. The second group contains anion micelles α-MC and ω-MC which do not enter into hydrophobic linear congeneric groups. Hydrogen bonds are possible in micelles of both MC derivatives (α-MC and ω-MC), although a conformational analysis shows that their micelles differ. In the micelle ω-MC it is possible to attach two primary ω-MC micelles through hydrogen bonds, while in micelle α-MC this is not the case. The hydrogen bonds in micelle α-MC are in the hydrophobic domain of the micelle even at the critical micellization concentration, and the aggregation number of α-MC continually increases. High values of heat capacity change in the demicellization process of α-MC micelles points to a compact packaging of building units of this micelle and to the fact that between MC derivate micelles in α-MC micelle the hydrophobic surfaces of monomers are the most protected from hydration. If there is a β axial OH group present in the steroid skeleton, and at the same time there is no vicinal β equatorial OH group present, then the evaluated steroid surfactants have a decreased CMC value, and a higher aggregation number than would be expected based on hydrophobicity of the β convex side of the steroid skeleton.


23


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Page 24 of 46

Acknowledgments This study was financially supported by the Domus Hungarian Scholarship (Hungarian Academy of Sciences, No. 5697/30/2015/HTMT). The Ministry of Science and Technological Development of the Republic of Serbia (Project No. 172021) is acknowledged as well.

Appendix A-E. Supplementary information. Appendix A: lateral side of the steroid skeleton of bile acid; Appendix B: stereochemical analysis and hydrophobicity of MC derivatives; Appendix C: Hydrophobic linear congeneric group and outliner with excess Gibbs energy – theory; Appendix D: micelle α-MC with n = 6 and Appendix E: stereochemical analysis of possibility for formation of hydrogen bonds between steroid OH groups of hyocycholic acids H = γ-MC

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Bowe, C.; Mokhtarzadeh, L.; Venkatesen, P.; Babu, S.; Axelrod, H.; Sofia, M. J.; Kakarla, R.; Chan, T. Y.; Kim, J. S.; Lee, H. J.; Amidon, G. L.; Choe, S. Y.; Walker, S.; Kahne, D. Desing of compounds that increase the absorption of polar molecules. Proc. Natl. Acad. Sci. USA 1997, 94, 12218-12223.


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Table 1. Hydrophobicity (HF), critical micellar concentration (CMC) and aggregation number (n) (85 mM) measured at the temperature 20 °C (T= tauro derivate, ITC = isothermal titration calorimetry, ST = surface tension, SF = spectrofluorimetric) Comp. CD 7-OxL C α-MC β-MC γ-MC=H ω-MC HD

HFexp

HFlit 42

+0.51

+0.59

-0.28

CMCexp /mM ITC SF 6.0 7.0 23.5

CMClit /mM

nexp

9 (ST 25 °C)2

8.0

22.0

+0.11

+0.13

12.0

11.0

-0.71 -0.65 -0.30 -0.44

-0.84 (T) -0.78 (T) -0.45 (T)

32.0 50.0 20.0 35.0

30.0 47.5 18.0 31.5 15.0

12.4 (ITC 20 °C)44, 13 (ST 25 °C)13

nlit

6.3

7.5 (60 mM 25 °C)39 6.5 (85 mM 25 °C)24

11.5

12 (60 mM 25 °C)39

6.8 4.2 5.0 6.5

14 (ST 25 7.3 6.7 (60 mM 35 °C)40 °C)2 UD 20.5 19 (ST 25 -0.33 21.5 5.2 5.1 (60 mM 25 °C)39 2 °C) Standard uncertainties (u) are: u(HF) ≤ ±0.03, u(CMC) ≤ ±0.5 mM. u(n) ≤ ±0.3 -0.23

16.5


33


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Table 2.The change of demicellization enthalpy (∆Hdemic), critical micellar concentration (CMC) and the change of thermal capacity for demicellization (∆Cp) for MC derivatives obtained by ITC method compound α-MC

β-MC

γ-MC=H

ω-MC

T/°C

∆Hdemic /kJmol-1

CMC /mM

∆Cp / Jmol-1K-1

10

-4.25

33.0

250

15

-3.19

32.5

20

-1.65

32.0

30

0.9

32.5

40

3.25

33.5

50

5.85

34.0

10

-2.71

51.0

15

-1.19

50.5

20

-1.00

50.0

30

0.95

51.0

40

2.69

53.0

50

4.25

55.0

10

-3.35

21.0

15

-2.40

20.5

20

-1.07

20.0

30

1.45

20.5

40

3.58

22

50

5.82

23

10

-3.36

35.5

15

-2.35

35

20

-1.32

35

30

0.80

35.5

40

3.12

36

180

230

210


34

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50

5.11

37

Standard uncertainties (u) are: u(T) = ±0.1 °C, u(∆Hdemic) ≤ ±0.1 kJ mol-1 T (10, 15) °C, u(∆Hdemic) ≤ ±0.3 kJ mol-1 T (20), u(∆Hdemic) ≤ ±0.1 kJ mol-1 T (10, 40, 50) °C, u(CMC) ≤ ±0.2 mM T (10, 15) °C, u(CMC) ≤ ±0.5 mM T (20) °C, u(CMC) ≤ ±0.2 mM T (30, 40, 50) °C, u(∆Cp) ≤ ±7Jmol-1K-1. .


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Figure captions

Figure 1. Muricholic acid: molecular and conformational formulae, i.e. Newman projection formula of the environment of C6-C7 vicinal OH groups Figure 2. A: Titration of 400 mM α-MC in water into 2 mL water at 10°C - calorimetric traces: 40 injections a 10 μL aliquots; B: reaction enthalpy (-Q) vs. the total detergent concentration in the reaction cell (∆HM = –∆Hdemic); C: dependency of the critical micellar concentration on temperature (example: α-MC) Figure 3. The change in the (I1/I3) ratio depending on the total concentration of the bile acid salt (filled points: α-MC; empty points: β-MC) Figure 4. The dependency of the aggregation number on the total concentration of MC derivate anions Figure 5. A: Linear dependence between HF and ln CMC, molecule α-MC (outlier) has lower value of CMC than it would be expected from its hydrophobicity, B: Linear dependence between HF and n, molecule α-MC is outlier compared to LCGj and LCGe Figure 6. A: Formation of hydrogen bond between C6 β axial OH groups in the dimeric micelle α-MC; B: stereochemically in the dimeric micelle of β-MC formation of hydrogen bond is also possible between C6 β (e)-OH groups with two steroid skeletons, but it is not possible to form another H-bond due to inadequate angles of ω axes (C) or due to great distance (D), E: the association of water molecules with β vicinal synclinal OH groups prevents the formation of hydrogen bonds between building units Figure 7. Stereochemical analysis of possibility for formation of hydrogen bonds between steroid OH groups of hyodeoxycholic acids HD, A: conformational molecular formulas and B: projection formulas of molecular graph type (red point = carbons with OH groups, black points = carbons with angular methyl groups, grey points = methylene carbons sc to C6OH group, m = monomers, PM = primary micelles, SM = secondary micelles) Scheme 1. Collection of equations for the pattern recognition


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Figure 1. Muricholic acid: molecular and conformational formulae, i.e. Newman projection formula of the environment of C6-C7 vicinal OH groups



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Figure 2. A: Titration of 400 mM α-MC in water into 2 mL water at 10°C - calorimetric traces: 40 injections a 10 μL aliquots; B: reaction enthalpy (-Q) vs. the total detergent concentration in the reaction cell (∆HM = –∆Hdemic); C: dependency of the critical micellar concentration on temperature (example: α-MC)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. The change in the (I1/I3) ratio depending on the total concentration of the bile acid salt (filled points: α-MC; empty points: β-MC)


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10 9 8 7

α-MC

6

β-MC

n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


γ-MC 5

ω-MC

4 3 70

80

90

100

110

ct /mM

Figure 4. The dependency of the aggregation number on the total concentration of MC derivate anions



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. A: Linear dependence between HF and ln CMC, molecule α-MC (outlier) has lower value of CMC than it would be expected from its hydrophobicity, B: Linear dependence between HF and n, molecule α-MC is outlier compared to LCGj and LCGe


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Figure 6. A: Formation of hydrogen bond between C6 β axial OH groups in the dimeric micelle α-MC; B: stereochemically in the dimeric micelle of β-MC formation of hydrogen bond is also possible between C6 β (e)-OH groups with two steroid skeletons, but it is not possible to form another H-bond due to inadequate angles of ω axes (C) or due to great distance (D), E: the association of water molecules with β vicinal synclinal OH groups prevents the formation of hydrogen bonds between building units



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 7. Stereochemical analysis of possibility for formation of hydrogen bonds between steroid OH groups of hyodeoxycholic acids HD, A: conformational molecular formulas and B: projection formulas of molecular graph type (red point = carbons with OH groups, black points = carbons with angular methyl groups, grey points = methylene carbons sc to C6OH group, m = monomers, PM = primary micelles, SM = secondary micelles)


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  

GM0 (i) LCG- j bi GA0 (R) LCG- j   bi LCG- j ri LCG- j : i  LC G j (9) Ahf (i)LCG- j ri Ahf (R) LCG- j  



   

   

GM0 (k) bk G0A(R) LCG- j   bk LCG- j rk LCG- j (15) Ahf (k)LCG- j rk Ahf (R) LCG- j  

E GM0 (k) bˆk G A0 (R) LCG- j  G    LCG- j rk  LCG- j (16)  bˆk Ahf (k)LCG- j rk Ahf (R)LCG- j 

E  GM0 (k) G E LCG-e bˆk G A0 (R) G E LCG-e  G  ˆ bk G LCG-e rk G LCG-e  (20) E  E  E    G const. k LCG e Ahf (k) GE LCG-e rk Ahf (R) G E LCG-e 

   

Scheme 1. Collection of equations for the pattern recognition



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Graphical abstract


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