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The antioxidant (AOX) activities of alanyl tyrosine dipeptide-type surfactants with several chain lengths were investigated. The critical micelle concentration decreased exponentially with the carbon number of the hydrophobic chain of the surfactant. The antioxidative property was investigated using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS) assay and the oxygen radical absorbance capacity (ORAC) assay. The AOX activity was found to be strongly dependent on the chain length in the monomer solution. Therefore, an increase in the molecular size negatively influenced the AOX ability of the tyrosine residue. However, it was almost independent of the chain length of the surfactant in the micellar solution. The micelle particles acted as a catalyst for the reduction of the radicals in the ORAC assay.

Thioester surfactants, [C 12 H 25 N(CH 3 ) 2 (CH 2 ) m SCOCH 3 ] Br (C 12 mSAc, m = 4, 11, 12), yielded thiol surfactants via thiol-thioester exchange upon addition of dithiothreitol in aqueous solution. The thiolthioester exchange reaction was enhanced in a micellar system owing to the concentration effect. The enhanced aggregation upon deprotection of the thioacetate group was observed by dynamic light scattering measurements. The thiol surfactants yielded disulfide-linked gemini surfactants upon air oxidation and incubation with hydrogen peroxide. In contrast, the thiol surfactants yielded thioester double-tailed products by esterolytic reactions with p-nitrophenyl hexanoate. The introduction of an alkyl chain to the second side chain significantly decreased the critical micelle concentration and induced the micellar growth.

Water-insoluble genistein was solubilized in aqueous medium by using phospholipid vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycerophosphocholine (DOPC) with 0-30% cholesterol. For each vesicle, the maximum solubilization amount of genistein was investigated by X-ray scattering measurement. In addition, the antioxidant capacity of the solubilized genistein was evaluated by the ABTS assay. Genistein was found to be solubilized by 10-20% and 40-50% of the vesicle concentrations of pure DPPC and DOPC respectively. The maximum solubilization amount of genistein decreased to 0-10% and 20-30% when 30% of cholesterol is present in the respective vesicles. Cholesterol is solubilized in a hydrophobic core whereas genistein is solubilized in the polar head region or in the polar-apolar interface. The overlapping of solubilizing sites affected the solubilization of genistein when cholesterol was present in the vesicles. Moreover, the lamellar interval was largely affected by cholesterol in compared to the little impact of genistein because the later can indirectly affect the acyl chains. Genistein solubilized in DOPC showed the same degree of antioxidant capacity as that of vesicle-free genistein system. On the other hand, genistein solubilized in DPPC had lower antioxidant activity than the former systems. The distinction of antioxidant activity at different systems probably related to the difference of accessibility of ABTS radical cation to solubilized genistein through different vesicles. Finally, cholesterol-free DOPC vesicles were found to be the best solubilizer for genistein among the investigated systems.

A series of cationic surfactants containing the thioacetate group, [C n H 2n+1 N(CH 3 ) 2 (CH 2 ) 3 SCOCH 3 ] Cl (C n 3SAc, n = 12, 14, 16), were prepared and their properties in aqueous solution were investigated by conductivity, fluorescence, and dynamic light scattering measurements. The critical micelle concentrations (CMCs) of C n 3SAc decreased to about half the value of the corresponding alkyltrimethylammonium chloride. Thioacetate was eliminated with the addition of dithiothreitol (DTT) as well as NaOH. HPLC (high performance liquid chromatography) analysis and NMR (nuclear magnetic resonance) spectroscopy showed that thiol surfactants, [C n H 2n+1 N(CH 3 ) 2 (CH 2 ) 3 SH]Cl (C n 3SH), were generated upon the addition of DTT in aqueous solution via thiol-thioester exchange, whereas gemini surfactants, [C n H 2n+1 N(CH 3 ) 2 (CH 2 ) 3 SS(CH 2 ) 3 N(CH 3 ) 2 C n H 2n+1 ]2Cl (2C n 3SS), were generated upon incubation in alkaline solution via hydrolysis and air oxidation.

pH responsive surfactants, [C 12 H 25 N(CH 3 ) 2 (CH 2 ) n SCOCH 3 ]Br (C 12 nSAc, n = 4, 11, 12), were prepared, and their properties in aqueous solution were examined. The critical micelle concentration (cmc) and critical vesicle concentration (cvc) were determined based on changes in conductivity, as well as by fluorescence measurements, and light scattering methods. A significant increase in the light scattering intensities of the C 12 nSAc (n=11, 12) systems suggested that the growth of aggregates was accompanied by considerable counterion binding with increasing surfactant concentration. The diameter of C 12 11SAc, recorded by the dynamic light scattering measurements, was about 9.6 ±1.0 nm, which was slightly smaller than that for didodecyldimethylammonium bromide (DDAB) vesicles. The thioester group was easily hydrolyzed upon the addition of NaOH, while it was hardly hydrolyzed with the addition of HCl. The time course of alkaline hydrolysis was examined by the conductivity measurements and high-performance liquid chromatography analysis. [C 12 H 25 N(CH 3 ) 2 (CH 2 ) 11 SS(CH 2 ) 11 N(CH 3 ) 2 C 12 H 25 ]2Br (2C 12 11SS) was generated in the C 12 11SAc alkaline solution because of air oxidation. The C 12 11SAc alkaline solution gradually became an opaque blue color with increasing light scattering at 346 nm, indicating the remarkable growth of vesicles. The chemical structure of 2C 12 11SS was consistent with that of a disulfide linked double tailed surfactant, similar to DDAB. The disulfide linkage between the double tailed surfactants will contribute to the stabilization and growth of vesicles.

Disulfide linked gemini surfactant having a long spacer chain, [C 10 H 21 N(CH 3 ) 2 (CH 2 ) 11 SS(CH 2 ) 11 N(CH 3 ) 2 C 10 H 21 ]2Br (2C 10 11SS), was prepared by the hydrolysis and oxidation process of thioester group for [C 10 H 21 N(CH 3 ) 2 (CH 2 ) 11 SCOCH 3 ]Br. The critical vesicle concentrations of double tailed surfactants such as dialkyldimethylammonium bromide were observed by the conductivity and light scattering methods. The disulfide bonds of gemini surfactant, [C 12 H 25 N(CH 3 ) 2 CH 2 CH 2 SSCH 2 CH 2 N(CH 3 ) 2 C 12 H 25 ]2Br (2C 12 SS), were rapidly cleaved by the addition of water-soluble dithiothreitol. However, it took long time to cleave the disulfide bonds of so-called double tailed surfactants 2C 10 11SS due to vesicle formation. The dynamic light scattering method showed that the diameters of 2C 12 SS micelles were increased with the cleavage of disulfide bonds, whereas those of 2C 10 11SS aggregates remained almost constant at 17.6 ±1.3 nm in similar size with dialkyldimethylammonium bromide vesicles. The time course of disulfide cleavage was examined by the conductivity and HPLC analysis. The produced thiol surfactants were returned to their original gemini surfactants by the addition of H 2 O 2 .

We examined the "micelle-vesicle transition" through the mixing effect of single-tailed thiol surfactants produced by the cleavage of gemini surfactants, [C12H25N(CH3)2CH2CH2SSCH2CH2N(CH3)2C12H25] 2Cl (C12SSC12), which have a disulfide bond in the spacer chain. Phase diagrams of C12H25N(CH3)2CH2CH2SHCl-didodecyldimethylammonium chloride (C12SH-DDAC) and C12SSC12-DDAC were determined by conductivity and pyrene fluorescence probe methods. The aggregate diameters were evaluated by dynamic light scattering (DLS). The critical vesicle concentration (CVC) was confirmed by the abrupt increase in the intensity of light scattering with excitation at 335 nm. Vesicle formation was confined to the DDAC-rich region of the C12SSC12-DDAC system, while the vesicle formation region for the C12SH-DDAC system spread out with the addition of dithiothreitol (DTT) to C12SSC12-DDAC. This implies that single-tailed surfactants can induce a more favorable environment for molecular packing of the vesicular surface. The micelle-vesicle transition occurs with disulfide spacer chain cleavage of gemini surfactants at a particular specific concentration range.

This study investigates the aggregation behavior of caseinate and the solubilization of genistein in aqueous caseinate solution. The critical aggregation concentration (CAC) of caseinate was obtained from the fluorescence intensity of 8-anilino-1-naphthalenesulfonic acid (ANS), which was enhanced by ANS-protein interactions and the hydrophobicity of caseinate. The increasing solubility of genistein in caseinate was confirmed by HPLC measurements; above and below the CAC, the genistein/caseinate molar ratio is 1:1 and 10:1, respectively. The latter ratio indicates that more caseinate molecules surround genistein below the CAC. However, the solubility of genistein in caseinate is unaffected by calcium ions. Atomic force microscopy (AFM) shows that casein sub-micelles are similarly structured in the presence and absence of genistein. In AFM phase images, the caseinate sub-micelle is brightened in the presence of genistein, implying that the particle becomes more rigid, probably because genistein attaches to the surface or to the narrow part of the sub-micelle. The diameter of sub-micelle aggregates is two times that of caseinate alone (24 nm versus 12 nm). These results were confirmed by cryo-TEM observations.

Hybrid surfactants were generated through the simple mixing of fluorocarbon and hydrocarbon gemini surfactants in aqueous solutions at 25°C. Disulfide exchange between the disulfide in the spacer chain occurred in the mixed micelles. However, the generation of hybrid gemini surfactants was particularly inhibited by the addition of salt. The suppression of the electrostatic repulsion between the hydrophilic headgroups led to the close packing of the hydrophobic chains in the micelles, resulting in the increased immiscibility of the fluorocarbon and hydrocarbon surfactants. On the other hand, when fluorocarbon-fluorocarbon or hydrocarbon-hydrocarbon surfactants were mixed, equilibrium with a 1:2 ratio of symmetric and dissymmetric gemini surfactants was attained after incubation for 24 h.

We prepared a fluorinated gemini surfactant containing a disulfide bond in its spacer chain, [C₈F₁₇(CH₂)₃N(CH₃)₂CH₂CH₂SSCH₂CH₂N(CH₃)₂(CH₂)₃C₈F₁₇]2Cl, and its analogue with a hexamethylene spacer. Monomeric thiol surfactant, [C₈F₁₇(CH₂)₃N(CH₃)₂CH₂CH₂SH]Cl, was readily produced by the cleavage of the gemini surfactant using dithiothreitol in water. The critical micelle concentration was determined using surface tension, conductivity, and fluorescence probe methods. The critical micelle concentration of the monomeric surfactant was significantly larger than that of the gemini surfactant. The surface tension of aqueous solution for the cleaved monomeric thiol surfactant returned gradually to the original value through the formation of the disulfide bond via air oxidation.

Cationic surfactants containing a thioether group in the hydrophobic chain were prepared by the reaction of alkanethiol and bromocholine bromide. The aqueous solution properties of the thio-surfactants were investigated and compared with conventional cationic surfactants. The Krafft temperatures of the thio-surfactants were lower than that of alkyltrimethylammonium bromide. The critical micelle concentrations were determined by the conductivity method, and pyrene and SPQ fluorescence probe methods. A linear relation was observed for the plots of cmc vs. chain length. However, the cmc of the thio-surfactants depended on the position of the thioether group in the hydrophobic alkyl chain. It was found that the thioether group exhibited quenching ability toward SPQ fluorescence. The variation of the surfactant monomer concentration in equilibrium with micelles can be estimated from Stern-Volmer plots.

We found the generation of dissymmetric gemini surfactants from symmetric ones in aqueous micelle solutions of gemini surfactants with a spacer containing disulfide linkage. The equilibrium was attained during 5 h incubation at 25 degrees C with 1:2 ratio of symmetric to dissymmetric ones when the total concentrations were sufficiently high compared with monomer concentrations. The generation of dissymmetric form is attributed to the disulfide exchange between the spacer chains, which occurs in the mixed micelles of gemini surfactants with different hydrocarbon chains. The pseudo-phase separation model for micellization was successfully used to predict not only the cmc of binary and ternary mixtures composed of symmetric and dissymmetric gemini surfactants but also the monomer concentrations after the equilibrium. Reversibility of the disulfide exchange was ascertained by the generation of symmetric gemini surfactants from dissymmetric ones.

The incorporation behaviors of some N-dodecanoyl amino acid-type surfactants into L-alpha-dipalmitoylphosphatidylcholine (DPPC) vesicles in aqueous solution were investigated. From the leakage measurements of a vesicle-entrapped fluorescence probe, it was found that these surfactants did not affect the DPPC vesicle so much at a very low concentration less than a one-tenth of the critical micelle concentration (CMC), but caused a significant release of the probe form the vesicles even at just below the CMC. The leakage induced by amino acid-type surfactants was promoted by increasing hydrophobicity of the amino acid. However the polarization of DPH embedded in the DPPC membrane was almost unchanged by incorporation of the surfactants at the concentration below the CMC except for the N-dodecanoylphenylalanine system. The binding enthalpy and the binding constant of the surfactant to DPPC vesicles were estimated by isothermal titration calorimetry. While the binding enthalpy was independent of the kind of surfactant, the binding constant increases with increasing hydrophobicity of the amino acid species in the surfactants. The molar ratio of bound surfactant to DPPC molecule in the saturated state was much greater than that for sodium dodecyl sulfate system. Further the ratio was not influenced by the structure of the amino acid side chain.

The aggregation behaviors of three stereoisomers of tetramethylammonium N-dodecanoyl phenylalanylphenylalaninate in dilute aqueous solution were investigated. From surface tension, fluorescence intensity using probes, and heat of dilution measurements, it was suggested that the critical aggregation concentration was the same between the enantiomers, but was obviously different between the diastereomers. It was also found that these surfactants formed large aggregates at lower concentrations. These large aggregates were then transformed to micelles at higher concentrations similarly to the potassium N-acyl phenylalaninate system. Furthermore, the fluorescence intensity of auramine increased strikingly in the N-dodecanoyl-L-phenylalanyl-L-phenylalanine (homo-chiral dipeptide-type surfactant) system. The fluorescence intensity of auramine in the aggregate of homo-chiral dipeptide-type surfactant was 20 times larger than that in the hetero-chiral dipeptide-type surfactant.

Monomeric thiol surfactants, [C(n)H(2n+1)N(CH(3))(2)CH(2)CH(2)SH]Br, were produced by the cleavage of gemini surfactant containing a disulfide bond in the spacer chain, [C(n)H(2n+1)N(CH(3))(2)CH(2)CH(2)SSCH(2)CH(2)N(CH(3))(2)C(n)H(2n+1)]2Br. The disulfide bond was completely reduced by the addition of four times moles of dithiothreitol in water at room temperature. The critical micelle concentrations of monomeric surfactants were significantly increased in comparison with original gemini surfactants. The monomeric thiol surfactants were stable in the presence of dithiothreitol, whereas they returned gradually to their original gemini surfactants within several days due to air oxidation in water without dithiothreitol. The micelle formation induced by the disulfide linkage formation was suggested by the fluorescence intensity ratio of pyrene. The time course of decrease in thiol concentration associated with the recovery of gemini surfactants was confirmed by the absorption spectra utilizing the reactions with 4,4'-dithiopyridine.

We developed new benzofurazan (NBD) labeled probes for fluorocarbon surfactant systems. The fluorescence behavior depended on the solubilization site of the fluorescent probes in the surfactant aggregates. The NBD-labeled probes suffered virtually complete reduction in the presence of Na(2)S(2)O(4) owing to the solubilization at the surface of 2-hydroxy-1,1,2,3,3-pentahydroperfluoroundecyldiethylammonium bromide (FC(8)DAB) aggregates. On the other hand, N-(3-sulfopropyl)acridinium (SPA) in FC(8)DAB aggregates showed residual fluorescence in spite of NaBH(4) addition. The large vesicles of FC(8)DAB were confirmed by DLS measurements. These facts suggest that SPA is solubilized in an inner water phase of the vesicles. The NBD labeled fluorescence probe is quite effective for the study of the aggregation behavior of fluorocarbon surfactants.