The dominant role of solvent in the sequestering of bile salts by hydrophobic cationic polymeric resins

The binding behavior of sodium cholate, a trihydroxy hydrophobic bile salt, by a polyacrylamide resin with N,N,N-trimethylammonium dodecyl chloride (QPDA12) pendant group was determined with varying buffer conditions and in the presence of 1,2-propanediol as a solvent perturbant. Binding constants extracted from the fit of the binding isotherms to the Langmuir equation were obtained at several temperatures. The temperature dependence of the binding behavior indicated that binding, in comparison with that of the dihydroxy chenodeoxycholate, was weaker due to a smaller positive entropic change, despite a lowered enthalpic barrier. Enthalpy-entropy compensation with a compensation temperature of 285-290 K, characteristic of processes involving water, was found to encompass both the cholate and chenodeoxycholate data under a wide variety of conditions. Previous studies with sodium chenodeoxycholate determined that loss of hydrophobic hydration within the resin dominated the thermodynamics of the binding process, but the observations with sodium cholate revealed that solvent liberation about the bile salt is also a contributor.     

FUNCTIONALIZED β-CYCLODEXTRIN POLYMERS FOR THE SORPTION OF BILE SALTS

Poly(β-cyclodextrin) (PCD) resins were prepared by a crosslinking reaction of β-cyclodextrin with different amounts of epichlorhydrin. Some hydroxyl groups of these polymers were functionalized with alkyl quaternary ammonium groups. The polymers were tested for their ability to bind several bile salts (including the sodium salts of cholic acid, glycocholic acid, and chenodeoxycholic acid), individually and competitively, from phosphate buffer solutions. In all cases, the aminated PCD resin had a higher binding capacity for bile salts. The binding of chenodeoxycholate by the resins was much more effective than that of cholate and its conjugate, which indicates the importance of the host cavity size relative to that of the guest molecules. The degree of hydrophobicity of bile acids also plays a role in their binding by PCD resins. The variable temperature studies indicate that the electrostatic interactions become weaker at higher temperatures while the hydrophobic interactions are not as much affected.     

Polymeric Sorbents for Bile Acids: II. Oligopeptide-Containing Resins with Quaternary Amine Groups

Abstract

Polymeric sorbents for bile acids have been prepared by attaching lysine-containing peptide sequences onto a water-swellable polyamide resin, by solid phase peptide synthesis, and then attaching a terminal N,N-dimethyl glycine residue that was subsequently quaternized. The resins with relatively longer peptide sequences demonstrated a higher binding capacity, on a per active site basis, for bile acids in pH 7.4 aqueous buffer solutions at 20°C than cholestyramine and colestipol when tested under the same in vitro conditions. In solutions of low ionic strength, they also have a degree of specificity for bile acid anions. The resins have a higher binding affinity for cholic acid than for glycocholic acid, which indicates the importance of the hydrophobic interactions in the binding.     

β-Cyclodextrin dimers linked through their secondary faces with rigid spacer arms as hosts for bile salts

A convenient synthesis of β-cyclodextrin dimers in which the two cyclodextrin units are linked by rigid tethers of relatively short length through their secondary sides is reported. Compounds hexa-2,4-diynediyl- and 1,4-phenylenediethyne-briged β-cyclodextrin dimers are obtained in good yields from mono-2-O-propargyl-β-cyclodextrin through Pd-mediated oxidative homo- and heterocoupling reactions. Isothermal titration calorimetry and NMR spectroscopy (PGSE and 2D-ROESY) are used to determine the thermodynamic parameters (K, ΔH, and TΔS°) for the complexation of such β-cyclodextrin dimers with sodium cholate, deoxycholate, and chenodeoxycholate as well as to estimate the size of the supramolecular structures. The binding of bile salts is enhanced relative to that of native β-cyclodextrin. Although chenodeoxycholate salt binds in a 1:1 fashion, cholate and deoxycholate salts bind in a 1:2 sequential mode.     

Separation of Monosulfated Bile Acids by High-Performance Liquid Chromatography

Abstract

Separation of 3-, 7- and 12-monosulfates of cholate, chenodeoxycholate, deoxycholate, lithocholate and ursodeoxycholate and their glyco- and tauro-conjugates by high-performance liquid chromatography on a reversed phase column has been carried out. Effects of pH and salt concentration of a mobile phase on the k' value of sulfated bile acids were investigated with the μBondapak C₁₈ and ODS SC-02 columns. The 3-sulfated bile acids were efficiently separated on ODS SC-02 using three aqueous ammonium carbonate/acetonitrile systems. The chromatographic behaviors of 7-and 12-sulfated bile acids are also briefly discussed.     

Influence of planarity and size on guest binding with sodium cholate aggregates

Bile salt aggregates are supramolecular structures with two types of binding sites, called primary and secondary sites. The objective of this work was to explore how the nonplanarity and size of guests (biphenyl [BP], 1-1'-binaphthyl [BNP] and dibenz[b,f]oxepin [DBX]) affected their binding affinity and dynamics to sodium cholate (NaC) aggregates. Fluorescence and laser-flash photolysis experiments were performed to obtain information on the binding environment for the guests, the accessibility of quenchers to guests in the aggregate and the dissociation rate constants of the guests from the aggregates. All guests were bound to the more hydrophobic primary aggregate, showing that this site can accommodate nonplanar molecules. However, the structure of the guest affects the structure of the primary aggregates, leading to changes in the accessibility of anions to aggregate-bound guests and to changes for the guest dissociation rate constants from the aggregates.     

Separation and Determination of Bile Acids in Human Bile by High-Performance Liquid Chromatography

Abstract

A method for simultaneous determination of major bile acids in human bile is described. The unconjugated, glycine- and taurine-conjugated bile acids are extracted with Sep-pak C¹⁸ and separated into groups by ion-exchange chromatography on a lipophilic gel. Subsequently, resolution of each group into ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate is attained into two stages by high-performance liquid chromatography on a Radial-PAK A column. First, 0.3% ammonium phosphate (pH 7.7)/acetonitrile (19:8, v/v) is used for separation of the latter three as a mobile phase. Ursodeoxycholate and cholate are efficiently separated in 0.3% ammonium phosphate (pH 7.7)/acetonitrile (23:8, v/v). The present method is applicable to quantitation of bile acids in human bile with satisfactory accuracy and precision.     

Effect of the guest size and shape on its binding dynamics with sodium cholate aggregates

The binding dynamics of the guests acenaphthene, phenanthrene, fluorene, and acenaphthenol with sodium cholate aggregates were studied using laser flash photolysis and fluorescence. The location of the guests in the bile salt aggregate is determined by the guest's hydrophobicity, where acenaphthene, phenanthrene, and fluorene bind to the primary aggregates, while acenaphthenol binds to the secondary bile salt aggregates. The residence time of the guests in the primary aggregates and the access of ionic species from the aqueous phase to the guest in the aggregate depend on the size and the shape of the guest. These results show that bile salt aggregates are adaptable supramolecular host systems.     

Studies on adsorption characteristics of bile acids and methotrexate to a new type of anion-exchange resin, colestimide

The adsorption characteristics of various bile acids and methotrexate to a new type of anion-exchange resin, colestimide, were studied in vitro and compared with those to cholestyramine. For bile acids, colestimide was shown to have a higher capacity than cholestyramine. For example, approximately 1.4-fold higher for cholic acid and 2.0-fold for deoxycholic acid in water. In the presence of physiological anions, the degree of adsorption of cholic acid to both resins was greatly reduced, whereas adsorption of deoxycholic acid was only slightly reduced. Furthermore, the bed-volume of colestimide swelled about 6.8-fold in water, hence the anion-exchange groups of this resin are expected to be able to function effectively in adsorption of bile acids in the gut. In addition, colestimide was found to have high adsorption capacity for methotrexate, not only in water but also in media containing various physiological anions, and thus it is suggested that colestimide is a potential oral antidote to reduce possible toxicity by methotrexate through interruption of enterohepatic circulation.     

Novel permethylated beta-cyclodextrin derivatives appended with chromophores as efficient fluorescent sensors for the molecular recognition of bile salts

Two novel permethylated beta-cyclodextrin (PM-beta-CD) derivatives, i.e., 6I-O-(1-naphtholxy)-2I,31-di-O-methylhexakis(2II-VII,3II-VII,6II-VII-tri-O-methyl)-beta-cyclodextrin (1) and 6I-O-(8-hydroxyquinoline)-2I,31-di-O-methylhexakis(2II-VII,3II-VII,6II-VII- tri-O-methyl)-beta-cyclodextrin (2), were synthesized in satisfactory yields, and their inclusion modes, complex-induced fluorescent behaviors, binding ability, and selectivity for bile salts of biological relevance (cholic acid sodium salt, CA; deoxycholic acid sodium salt, DCA; glycochoic acid sodium salt, GCA; taurocholic acid sodium salt, TCA) were investigated by the circular dichroism, 2D NMR, steady-state, and time-resolved fluorescent spectra. The results obtained from induced circular dichroism and ROESY spectra show that the chromophore groups of 1 and 2 reside in the central cavity of PM-beta-CD, and are expelled to the region of narrow torus rim upon complexation with bile guests, which presents the binding mode of cooperative inclusion. The transfer of the chromophore groups from the central cavity to the more hydrophobic torus rim leads to the remarkable increase of fluorescent intensities and longer fluorescent lifetimes of hosts 1 and 2 upon gradual addition of bile salts, which is importantly distinct from the molecular recognition of the chromophore-modified beta-CD species with bile salts. Interestingly, hosts 1 and 2 present much stronger binding ability for bile guests than PM-beta-CD. Differing from native beta-CD, all the PM-beta-CDs are more prone to include bile salts with longer tails, such as GCA and TCA. Their corresponding binding ability and molecular selectivity are closely discussed from the viewpoints of difference of cavity size/shape between beta-CD and PM-beta-CD, effect of substituent groups, and structures of bile guests, respectively.     

Effects of Hofmeister anions on the phase transition temperature of elastin-like polypeptides

The modulation of the lower critical solution temperature (LCST) of two elastin-like polypeptides (ELPs) was investigated in the presence of 11 sodium salts that span the Hofmeister series for anions. It was found that the hydrophobic collapse/aggregation of these ELPs generally followed the series. Specifically, kosmotropic anions decreased the LCST by polarizing interfacial water molecules involved in hydrating amide groups on the ELPs. On the other hand, chaotropic anions lowered the LCST through a surface tension effect. Additionally, chaotropic anions showed salting-in properties at low salt concentrations that were related to the saturation binding of anions with the biopolymers. These overall mechanistic effects were similar to those previously found for the hydrophobic collapse and aggregation of poly(N-isopropylacrylamide), PNIPAM. There is, however, a crucial difference between PNIPAM and ELPs. Namely, PNIPAM undergoes a two-step collapse process as a function of temperature in the presence of sufficient concentrations of kosmotropic salts. By contrast, ELPs undergo collapse in a single step in all cases studied herein. This suggests that the removal of water molecules from around the amide moieties triggers the removal of hydrophobic hydration waters in a highly coupled process. There are also some key differences between the LCST behavior of the two ELPs. Specifically, the more hydrophilic ELP V5A2G(3)-120 construct displays collapse/aggregation behavior that is consistent with a higher concentration of anions partitioning to polymer/aqueous interface as compared to the more hydrophobic ELP V(5)-120. It was also found that larger anions could bind with ELP V5A2G(3)-120 more readily in comparison with ELP V(5)-120. These latter results were interpreted in terms of relative binding site accessibility of the anion for the ELP.     

Cyclodextrin dimers as receptor molecules for steroid sensors

The dansyl-modified dimer 9 complexes strongly with the steroidal bile salts. Relative to native beta-cyclodextrin, the binding of cholate (1a) and deoxycholate (1b) salts is especially enhanced. These steroids bind exclusively in a 1:1 fashion. For other bile salts (1c-1e) both 1:1 and 1:2 complexes were observed with stabilities similar to those of native beta-cyclodextrin. This indicates that only one cavity is used, with a small contribution from the second. The difference is attributed to the absence of a 12-hydroxy group in the second group of steroids. Comparison with a dimer that lacks the dansyl moiety (6) shows that this group especially hinders the cooperative binding of la and 1b. The smaller interference in the binding of the other steroids indicates that self-inclusion of the dansyl moiety hardly occurs. This weak self-inclusion is supported by fluorescence studies. The dansyl fluorescence of dimer 9 is less blue-shifted than that of other known dansyl-appended cyclodextrin derivatives; this is indicative of a more polar micro-environment. Addition of guests causes a change in fluorescence intensity.     

Binding of methyl orange and its homologs by polyion complexes consisting of a piperidinium cationic polymer and various polyanions in aqueous solution

A study was made of the formation of polyion complexes between a piperidinium cationic polymer and polyanions and of the binding of azo-dye anions (methyl, ethyl, propyl, and butyl orange) by these complexes. Sodium poly(acrylate), poly(styrenesulfonate), dextran sulfate, and carboxy-methylcellulose were used as polyanions. The resultant polyion complexes (insoluble in aqueous solutions) were compared for their ability to bind the small organic molecules in aqueous solutions, for example, of urea and an inorganic electrolyte (KCI), and exhibited a strong binding affinity toward these small anions. Polyion complexes that consisted of sodium poly(acrylate), dextran sulfate, and carboxymethylcellulose as polyanions cooperated in the binding, whereas the polyion complex of sodium poly(styrenesulfonate) did not. It was suggested that small organic anions interact with the polyion complexes primarily through electrostatic and hydrophobic forces.     

Fluorescence turn on by cholate aggregates

Bile salts, including sodium cholate (NaCh), are amphiphilic molecules with a concave hydrophilic side and a convex hydrophobic side. By forming aggregates in aqueous solution, these natural surfactants fulfill vital biological roles in the solubilization of cholesterol, lipids, and fat-soluble vitamins and thus are involved in the transport and absorption of important biological molecules. Following our success with the encapsulation of fluorescent protein chromophore (FP) analogs by synthetic hydrophobic and hydrophilic hosts, based upon substitution patterns, we now report the binding and turn on of other analogs by bile salt aggregates, observations which may lead to new tools for studying trafficking in these important systems.     

FHA domain-ligand interactions: importance of integrating chemical and biological approaches

Combinatorial library screens based on binding affinity may preferentially select ligands with ability for ionic interactions and miss the biologically relevant ligands that bind more weakly with predominantly hydrophobic interactions.     

Studies on steroids. CXXVIII. Separation and determination of bile acids by high-performance liquid chromatography

A method is described for the simultaneous determination of major bile acids by high-performance liquid chromatography without prior hydrolysis. A mixture of bile acids is divided into the free, glyco- and tauro-conjugate groups by thin-layer chromatography. Separation of each group into cholate, ursodeoxycholate, chenodeoxycholate, deoxycholate and lithocholate is attained in two stages on a muBondapak C18 column; first, 0.3% ammonium carbonate-acetonitrile (9:4) is used as a mobile phase for the separation of the last three compounds. Subsequently cholate and ursodeoxycholate are resolved by chromatography in 0.3% ammonium carbonate-acetonitrile (11:4).     

Sodium dodecyl sulfate promoting a cooperative association process of sodium cholate with bovine serum albumin

Sodium cholate (NaC) was used as a representative bile salt in the process of cooperative binding to bovine serum albumin (BSA) in a mixture with sodium dodecyl sulfate (SDS). The experiments were performed in 0.02 M Tris-HCl buffer solution (pH 7.50), in the presence of 0.1% BSA and at 25 degrees C. The aim of this study is to provide information on the performance of the BSA in the promotion of cooperative binding of sodium cholate promoted by the presence of SDS. The method used to monitor the binding was based on the analysis of the effect of SDS and NaC concentrations and their mixtures upon the fluorescence intensity of the BSA tryptophan residues. Plots of the fluorescence emission bands in terms of the A0/A ratio vs surfactant concentrations, where A0 and A represent the areas of emission bands in the presence and absence of the surfactants, respectively, were drawn in order to investigate the surfactant interaction with the protein. An alternative methodology, the specific conductivity vs surfactant concentration plots, was used, which involves mixtures of SDS and NaC to investigate the association processes, through the determination of the critical aggregation concentration (cac, when in the presence of protein) and the critical micellar concentration (cmc). The results led to a general conclusion that as the mixed micellar aggregates become richer in the bile salt monomer, the tendency to lose the reactivity with the protein increases. According to our results, a clear evidence of the predomination of BSA-SDS-NaC complexes is found only for the SDS molar fraction above approximately 0.6, and below this fraction a tendency toward free mixed micelles starts to predominate.     

Salt effect on polymer solutions

The salt effect was investigated by measurements of viscosity, cloud point, and solubility of aqueous solutions of poly(vinyl alcohol—acetate) copolymers, as these copolymers in water are sensitive in various ways to addition of various electrolytes. The major role in the salt effect is played by the anions, and water-structure breaking anions produce salting-in of the copolymers. Tetraalkylammonium halides (bromides and iodides) cause salting-in of the copolymers more effectively with increase of size of the hydrophobic cations. The Setschenow equation does not hold for the polymers except for very dilute polymer concentration. In salts of monoalkyl-substituted anions (R? COONa and R? SO₄Na) and cations (R? NH₃Cl and Br), so long as the alkyl chain is short, the salt effect is also dominated by the anions. With increase of the alkyl chain length, sodium salts of the monoalkyl-substituted anions exert a stronger salting-in effect upon the polymers than chlorides of similar long-chain cations. The significance of the counteranions is suggested for the interaction of nonionic polymers and the long-chain cations. The action of the salts on the copolymers is discussed in terms of medium effects (change of the water structure) and of the binding effect of the single ions to the polymers.     

Multiple anion binding by a zinc-containing tetratopic cyclen-resorcinarene

The synthesis and binding properties of a new tetratopic anion receptor are reported. The resorcinarene ligand bearing four cyclen moieties is able to bind four Zn²⁺ ions and subsequently bind anions. NMR titrations show proton shifts during the binding of the first one or two anions. Isothermal titration calorimetry (ITC) titrations show that two or more anions bind to one tetramer. The tetratopic receptor in methanol has high affinity for dihydrogen phosphate, acetate, and halide ions and weak affinity for nitrate and perchlorate.     

Tunable inhibition and denaturation of alpha-chymotrypsin with amino acid-functionalized gold nanoparticles

Water-soluble gold nanoparticles bearing diverse l-amino acid terminals have been fabricated to probe the effect of receptor surface on protein surface binding. The interaction of these nanoparticles with alpha-chymotrypsin (ChT) was investigated by activity assay, gel electrophoresis, zeta-potential, circular dichroism, and fluorescence spectroscopy. The results show that both electrostatic and hydrophobic interactions between the hydrophobic patches of receptors and the protein contribute to the stability of the complex. The microscopic binding constants for these receptor-protein systems are 10(6)-10(7) M(-1), with the capacity of the nanoparticle receptors to bind proteins determined by both their surface area and their surface charge density. Furthermore, it is found that the hydrophilic side chains destabilize the ChT structure through either competitive hydrogen bonding or breakage of salt bridges, whereas denaturation was much slower with hydrophobic amino acid side chains. Significantly, correlation between the hydrophobicity index of amino acid side chains and the binding affinity and denaturation rates was observed.