José Vázquez Tato

Complexation of Bile Salts by Natural Cyclodextrins

The complexation of three trihydroxy sodium bile salts--cholate, glycocholate, and taurocholate--and their three related sodium dihydroxy bile salts--deoxycholate, glycodeoxycholate, and taurodeoxycholate--by f -, g - and n -cyclodextrins ( f -CD, g -CD and n -CD), has been studied by using 1D and 2D-NMR techniques. Trihydroxy bile salts form 1:1 complexes with g -CD and n -CD, while dihydroxy bile salts form 1:2 (bile salt:cyclodextrin) complexes with g -CD and 1:1 complexes with n -CD. ROESY experiments stated that the side chain, ring D, and part of ring C of the steroid body of the bile salts, are included into the cavities of g -CD and n -CD, in 1:1 complexes. The A ring of the steroid body is included into the cavity of the second g -CD in the 1:2 complexes. The only structural difference between related bile salts is the existence or not of a hydroxyl group at C7. The bigger cavity of n -CD allows this region of bile salts to be located inside the cyclodextrin cavity and therefore n -CD does not discriminate between both types of bile salts. However, in trihydroxy bile salts, this region clearly remains outside the g -CD cavity. The absence of the C7 hydroxyl group enlarges the hydrophobic region of the steroid body, allowing a favourable interaction with the hydrophobic cavity of a second g -CD. It is concluded that g -CD molecularly recognises bile salts, distinguishing di- from trihydroxy ones, while n -CD does not. The steroid body of bile salts is too big to enter into the f -CD cavity and only an interaction between their side chain and f -CD is observed.

Catanionic Tubules with Tunable Charge

Thethree-dimensionalstructureswithnanoscopicdimensionsthat are yielded by the self-assembly of lipids and surfactantsare of particular interest for their applications in nano-technology.Intheseapplications,thepossibilityofcontrollingthe charge of the particles allows the regulation of funda-mental aspects, such as the ability of the particles to loadmolecules (drugs, DNA, proteins, etc.), to aggregate, and topenetrate membranes. Within the possible surfactant supra-molecular architectures, tubular structures have recentlydrawn much research interest.

Synthesis and Characterization of a New Gemini Surfactant Derived from 3α,12α-Dihydroxy-5β-cholan-24-amine (Steroid Residue) and Ethylenediamintetraacetic Acid (Spacer)

A new gemini steroid surfactant derived from 3alpha,12alpha-dihydroxy-5beta-cholan-24-amine (steroid residue) and ethylenediamintetraacetic acid (spacer) was synthesized and characterized in aqueous solution by surface tension, fluorescence intensity of pyrene, and light scattering (static and dynamic) measurements. These techniques evidence the existence of a threshold concentration (cac), below which a three layers film is formed at the air-water interface. Above the cac, two types of aggregates--micelles and vesicle-like aggregates--coexist in a metastable state. Filtration of a solution with a starting concentration of 2.6 mM (buffer 150 mM, pH 10) allows isolation of the micelles, which have an average aggregation number of 12, their density being 0.28 g cm(-3). Under conditions where only the vesicle-like aggregates are detected by dynamic light scattering, a value of 5.5 x 10(4) was obtained for their aggregation number at 30 microM, their density being 6.8 x 10(-4) g cm(-3). At high concentrations, the intensity ratio of the vibronic peaks of pyrene, I1/I3, (=0.68) is very close to published values for deoxycholate micelles, indicating that the probe is located in a region with a very low polarity and far from water. A hypothesis to explain the observed aggregation behavior (small aggregates are favored with increasing gemini concentration) is outlined.

Kinetics of formation of supramolecular tubules of a sodium cholate derivative

We report a kinetic study of the supramolecular tubule formation of the bile salt derivative [3β,5β,7α,12α]-3-(4-t-butylbenzoilamine)-7,12-dihydroxycholan-24-oic acid sodium salt (Na-tbutPhC). At high bicarbonate buffer concentration (pH∼10) this salt shows gelator properties. Starting from gels or viscous solutions, the tubule formation is triggered by increasing the temperature beyond the critical value of 34–36 °C. For gels, when the process takes place, the transition to sols occurs. The process is easily triggered and can be followed by several techniques. We used static light scattering (SLS), circular dichroism (CD), small angle X-ray scattering (SAXS) along with transmission electron (TEM) and optical microscopies. The CD results show that fibrils with a clockwise arrangement of the bile salt derivative are present in the samples at room temperature. When the tubule formation starts, evolutions of the CD and SLS profiles are observed indicating that the formation process begins with the aggregation of the fibrils accompanied by a simultaneous peculiar reciprocal reorientation of the surfactant molecules. After that, as pointed out by the long time evolution of the curves, a slow transformation towards the final well defined tubules occurs, involving an adjustment of the molecular packing. In the meanwhile, the slow ordering of the tubule walls in well spaced layers takes place, as inferred by SAXS. The TEM images show that short disordered tubules are formed, because of the aggregation of fibrils, in the beginning. Moreover they highlight a final elongation of the tubules taking place without a further aggregation of fibrils. Optical microscopy frames, collected during the process, point out that the tubules grow singly even at quite a high concentration, thus supporting the data interpretation.

Catanionic Gels Based on Cholic Acid Derivatives

In this paper, the preparation and characterization of an anionic and a cationic surfactant obtained by chemical modifications of a natural bile acid (cholic acid) are reported. The bile acid was modified by introducing a diamine or a dicarboxylic aromatic residue on the lateral chain. The pure cationic surfactant self-assembles in a network of fibers with a cross-section gyration radius of about 15.1 Å, providing hydrogels with a pH-dependent compactness. On the other hand, the anionic molecule gives rise to prolate ellipsoid micelles. Homogeneous catanionic mixtures have also been obtained, with molar fraction of each surfactant ranging from 0.125 to 0.875. At total surfactant concentration of 0.05% (w/v), the mixtures form gels of fibrils partially arranged in secondary twisted superstructures. Comparison of this concentration with the minimum gelation concentration of the pure cationic derivative (0.16% w/v) suggests that, in the mixtures, the presence of the electrostatic component in self-assembly of the molecules allows the formation of gels starting from more dilute samples. In view of these achievements, this work suggests that catanionic mixtures can be exploited to enhance the efficiency of gelators.

Determination of second-order association constants by global analysis of 1H and 13C NMR chemical shifts.: Application to the complexation of sodium fusidate and potassium helvolate by β- and γ-cyclodextrin

The host-guest interaction between the steroid antibiotics sodium fusidate and potassium helvolate as guests and the hosts beta- and gamma-cyclodextrin was studied by 13C and 1H NMR techniques. The analysis of chemical shifts of individual nuclei leads to inconsistent values of the association constants and fails generally in the case of mixtures of 1:1 and 1:2 stoichiometries. The problem of parameter correlation is identified and the global analysis of two or more nuclei is proposed as a very effective method for the detection of complexes of higher stoichiometries and for the precise determination of the involved association constants. A matrix formulation of global analysis and the determination of confidence intervals is described. An analytical solution of the cubic equation, necessary for the description of higher order complexes, is presented in detail and its use together with commercial fitting software is compared with dedicated implementations. gamma-Cyclodextrin forms with both studied steroids, sodium fusidate and potassium helvolate, 1:1 complexes with high values of the association constants, K(1)=(60+/-24)x10(3)lmol(-1), and K(2)=(22+/-9)x10(3)lmol(-1), respectively. To the contrary, beta-cyclodextrin forms 1:1 and 1:2 (guest:host) complexes with both steroids, with moderate K(1) and low K(2) values (K(1)=(0.74+/-0.13)x10(3)lmol(-1), K(2)=(0.210+/-0.075)x10(3)lmol(-1)), and (K(1)=(2.42+/-0.87)x10(3)lmol(-1), K(2)=(0.06+/-0.09)x10(3)lmol(-1)), respectively.

Sugar-Bile Acid-Based Bolaamphiphiles: From Scrolls to Monodisperse Single-Walled Tubules.

The introduction of a mannose residue on carbon 3 of lithocholic acid gives rise to an asymmetric and rigid bolaamphiphilic molecule, which self-assembles in water to form elongated tubular aggregates with an outer diameter of about 20 nm. These tubular structures display a temporal evolution, where the average tube diameter decreases with time, which can be followed by time-resolved small-angle X-ray scattering experiments. Cryogenic transmission electron microscopy images collected as a function of time show that at short times after preparation tubular scrolls are formed via the rolling of layers, after which a complex transformation of the scrolls into single-walled tubules takes place. At long time scales, a further evolution occurs where the tubules both elongate and become narrower. The observed self-assembly confirms the tendency of bile acids and their derivatives to form supramolecular aggregates with an ordered packing of the constituent molecules. It also demonstrates that scrolls can be formed as intermediate structures in the self-assembly process of monodisperse single-walled tubules.

Kinetics and mechanism of the nitrosation of N-acetyltryptophan and of the denitrosation of N-acetyl-N1-nitrosotryptophan

Both the formation and the denitrosation of N-acetyl-N1-nitrosotryptophan have been studied kinetically in aqueous solution at 25 °C at acidities between 1 M-HClO4 and pH 4. A value of 850 l mol–1 has been obtained for the equilibrium constant for the formation of N-acetyl-N1-nitrosotryptophan. At acidities ([H+]) greater than 0.1 M the rate constants for both nitrosation and denitrosation increase linearly with the concentration of acid, and the reaction rates are unaffected by the addition of nucleophiles (Br– and SCN–). The results are consistent with a mechanism for nitrosation where the rate-limiting step is the proton transfer from the protonated N-nitroso species to the medium. For denitrosation the corresponding protonation of the nitrosamine is rate-limiting. These conclusions are confirmed by the results obtained when the reactions are carried out in heavy water. However, in the pH range 1–4 the rates of both nitrosation and denitrosation are independent of the acidity of the medium and are again unaffected by the presence of nucleophiles or buffers. It is suggested that in this region nitrosation occurs at C-3. This is followed by deprotonation and an internal NO migration from C to N which is rate-limiting. This mechanism also accounts for earlier results on the denitrosation reaction at even lower acidities (pH 4–7), where acid catalysis and nucleophilic catalysis are found. Results of experiments in heavy water are compatible with this mechanism.

Formation of tubules by p-tert-butylphenylamide derivatives of chenodeoxycholic and ursodeoxycholic acids in aqueous solution.

The formation of tubules by p-tert-butylphenylamide derivatives of chenodeoxycholic and ursodeoxycholic acids in aqueous solution is investigated. The critical aggregation concentrations of the new surfactants are much lower than those of ursodeoxycholate and chenodeoxycholate, indicating the enhanced surfactant properties resulting by the presence of the hydrophobic p-tert-butylphenyl group. The molecular areas at the air-water interface suggest the formation of monolayer films with molecules upright oriented. The shape of the aggregates was investigated by TEM. The main structure present in solution corresponds to tubules. The estimated value for the wall thickness of tubules suggests that a bilayer structure is formed. Host of positively charged latex beads by tubules suggests that their inner and outer surfaces are negatively charged. The acid form of the chenodeoxycholate derivative was recrystallized from toluene and its crystal structure analyzed.

Spectra and structure of complexes formed by sodium fusidate and potassium helvolate with β- and γ-cyclodextrin

Abstract The complexation of two steroid antibiotics of the fusidane family, sodium fusidate and potassium helvolate, by β-CD and γ-CD has been studied by using 1D and 2D-NMR techniques. Both guests form 1:1 complexes with γ-CD and 1:2 (guest:cyclodextrin) complexes with β-CD. Thus, both antibiotics behave as monotopic and ditopic guests when they are complexed by γ-CD and β-CD, respectively. Both steroids enter into the cavity of the γ-CD by the side chain, reaching the central region of the steroid (rings C and D), whereas the A and B (partially) rings remain outside. For β-CD complexes, ROESY spectra show a remarkable absence of interactions of the protons of the C and D rings, whereas clear interactions corresponding to the side chain, and A and B rings are observed. The obtained equilibrium constants (see previous paper) are discussed in terms of the structures proposed for the complexes. NMR spectra of sodium fusidate are revised, and a full assignment of the 1 H and 13 C NMR spectra is presented for potassium helvolate.