Synthesis and characterization of a new gemini surfactant derived from 3 α , 7 α , 12 α-trihydroxy-5 β-cholan-24-amine ( steroid residue ) and ethylenediamintetraacetic acid ( spacer )

A new gemini steroid surfactant derived from 3α,7α,12α-trihydroxy-5β-cholan24-amine (steroid residue) and ethylenediamintetraacetic acid (spacer) was synthesized and characterized in aqueous solution by surface tension measurements and fluorescence intensity of pyrene. These techniques evidence the existence of a threshold concentration, cac, below which a three layers film is formed at the air-water interface. At high concentrations, the intensity ratio of the vibronic peaks of pyrene, I1/I3, (= 0.81) is very close to published values for sodium cholate micelles, indicating that the probe is located in a region with a very low polarity and far from water. Introduction During the past few years, an increasing number of papers have been published on the surface and micellar properties of gemini surfactants. This is mainly due to their better efficiency in decreasing both the surface tension of water and the critical micelle concentration (cmc) in comparison to their corresponding monomeric analogs. Most of them contain two hydrophobic long alkyl chains and two hydrophilic groups which are linked through a flexible or rigid spacer. Although bile salts are very well known surfactants and good solubilizers of hydrophobic compounds (including drugs and cholesterol), little attention has been paid to their potential use as amphiphile residues to design new gemini surfactants. Only a few examples of gemini surfactants formed by two bile acid residues have been published. Here we have designed, synthesized and characterized a dicarboxylic gemini steroid surfactant derived from 3α,7α,12α-trihydroxy-5β-cholan-24-amine (i. e., a 24-amino derivative of cholic acid), as surfactant residue, and ethylenediamintetraacetic acid, as spacer (Figure 1).


Introduction
During the past few years, an increasing number of papers have been published on the surface and micellar properties of gemini surfactants. 1,1,2This is mainly due to their better efficiency in decreasing both the surface tension of water and the critical micelle concentration (cmc) in comparison to their corresponding monomeric analogs.

Synthesis.
The synthesis of the cholic gemini was carried out by following schemes 1 and 2. Cholic acid 24-cholanamide 24-cholanamine Scheme 1: Synthesis path of 24-cholanamine. 12e 24-cholanamide and 24-cholanamine were characterized by NMR (Figure 2and 3 respectively).To remove the methyl groups of the ester in the spacer, the compound was refluxed with KOH 1M in methanol for one hour at 80 ºC.The solvent was evaporated and the solid redissolved in water (200 mL) and acidified with HCl (pH≈ 1).When the solution is cooled, the compound precipitates in its diacid form.The precipitate was filtered and dried in a vaccum oven.The disodium salt was obtained by adding the stoichiometric amount of NaOH.Both the diacid and the disodium salts were repeatedly crystallized to guarantee the purity of the gemini compound.

Results and Discussion
In Figure 5 surface tension data, γ, are plotted against log C for 24-cholanamine (C 24 NH2) and g-2C 24 -EDTA.The absence of a minimum in the surface tension versus concentration curves of both compounds (see Fig. 5) must be noticed.This indicates the absence of any strong surface-active trace impurity in the medium. 13,14The surfactants were purified by repeated crystallization until no impurities could be detected by thin layer chromatography, by NMR-spectroscopy or FAB-MS.
For the C 24 NH2, below (c 1 =0.4 mM), A S is ~102 Å 2 /molecule, and from the straight line between c 1 and c 2 , A S is ~89 Å 2 /molecule.Both values are very close to the theoretical surface value per molecule calculated from a spacefilling model (Figure 6).
This suggests that the bile ions are lying flat at the water interface with a tighter packing of the molecules above c 1 .In this case c 2 (1.8 mM) would correspond to the concentration above which aggregates are formed.This value is one order of magnitude lower than the one published by Fini et al. 12 The analysis of the surface tension vs concentration for the g-2C 24 -EDTA evidences some noticeable differences.In agreement with the literature on gemini surfactants, 16  The first value is identical to the one published for the similar gemini g-2DC 24 -EDTA in which the starting bile residue is deoxycholic acid, 11 and was interpreted as corresponding to a film structure at the air-water interface with three layers.The length of the steroid side chain plus the EDTA bridge (∼11.7 Å), which is almost twice the length of the steroid nucleus, would allow the formation of the multilayer without preventing the interaction of the ionic groups of upper layers with water.Rosen et al 17 Tsubone et al 18 have also proposed the formation of multilayer structures to explain the aberrant behavior of some gemini surfactants.Fifty years ago Ekwall and Ekholm 19 suggested that lithocholic acid forms a single bulk phase made up of a trilayer of bile acid molecules.
Since above c 1 the slope diminishes, each molecule has more space at the interface since A S increases.This behaviour has been associated with the existence and growth of premicellar aggregates, 20 and in fact premicellization seems to be a rather general effect in gemini surfactant solutions. 3,21,22Therefore the increase of A S suggests that the three layers film is broken and molecules from the film incorporate into aggregates which start to form in the bulk solution because of the increment of the surfactant concentration above c 1 .
Figure 7 shows the pyrene I 1 /I 3 ratio plots for g-2C 24  from surface tension measurements.The gradual decrease in I 1 /I 3 has been observed for other surfactants showing premicellar association. 20It contrasts with sharp drops at a particular concentration observed for typical surfactants as SDS.Above of ~1 mM I 1 /I 3 reaches a plateau equal to 0.81.This value is close to published values for pyrene included in sodium cholate micelles 23 and reflect a very apolar micro-environment for the fluorescent probe.Fitting the experimental data to a Boltzmann type equation 24 gives values of 1.3 µM and 1.2 mM for the two threshold concentrations.

Figure 5 .-
Figure 5.-Plots of surface tension vs log[bile salt] concentration for [•] 24cholanamine in HCl solution at pH=3.1 and [¤] g-2C 24 -EDTA in 0.15M bicarbonate/carbonate buffer, pH=10.1.T = 25.0±0.5ºCProsserand Franses15 have reviewed the application of the Gibbs adsorption isotherm to surface tension of ionic surfactants at the air-water interface.For a strong ionic surfactant of ν + free positive ions and ν -free negative ions of charges z + and z -,

Figure 6 .-
Figure 6.-Representation of the surface configuration of: (a) C 24 NH2 molecule lying flat.(b) g-2C24 -EDTA lying flat (maximum angle between cholate backbones).(c) g-2C24 -EDTA in upright orientation (ionic carboxylic groups oriented towards the water and steroid moities oriented towards the aerial phase).The area occupied by a molecule depends on the angle formed by the two branches of the gemini.
Figure 7 shows the pyrene I 1 /I 3 ratio plots for g-2C 24 -EDTA at 25ºC.It can be noticed that I 1 /I 3 decreases gradually with increasing concentration of the gemini over a wide range of concentration, from log C=-5.7 (C=1.9 µM; blue line in the Figure) to log C=-3 (C=1 mM; red line in the Figure).These values are close to c 1 and c 2 determined