Characterization of DDAB/Cholesterol Vesicles and Its Comparison with Lipid/Cholesterol Vesicles.

Vesicles prepared by synthetic surfactant, DDAB (dilauryldimethylammonium bromide), were modified with cholesterol and their membrane surface properties of the vesicle were characterized through the analyses of fluorescent probes, such as Laurdan (6-lauroyl-2-dimethylaminonaphthalene) and DPH (1,6-diphenyl-1,3,5-hexatriene). The self-assembly of DDAB with cholesterol showed stable vesicle structure with a mean diameter of 127 nm through the dynamic light scattering analysis. While the DDAB vesicle showed high polarity and high fluidity, the modification of the DDAB vesicle with cholesterol lead to the formation of "heterogeneous phase" on the vesicle membrane. DDAB:cholesterol = 70:30 vesicle showed unique characteristics that represents polar environment but lower fluidity. A novel platform for the chemical process in aqueous media can be expected by using the artificial surfactant vesicles modified with cholesterol.

Fluorescent Probe Study of AOT Vesicle Membranes and Their Alteration upon Addition of Aniline or the Aniline Dimer p-Aminodiphenylamine (PADPA).

Artificial vesicles formed from sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in aqueous solution are used successfully as additives for enzymatic oligomerizations or polymerizations of aniline or the aniline dimer p-aminodiphenylamine (PADPA) under slightly acidic conditions (e.g., pH 4.3 with horseradish peroxidase and hydrogen peroxide as oxidants). In these systems, the reactions occur membrane surface-confined. Therefore, (i) the physicochemical properties of the vesicle membrane and (ii) the interaction of aniline or PADPA with the AOT membrane play crucial roles in the progress and final outcome of the reactions. For this reason, the properties of AOT vesicles with and without added aniline or PADPA were investigated by using two fluorescent membrane probes: 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6-lauroyl-2-dimethylaminonaphthalene (Laurdan). DPH and Laurdan were used as "sensors" of the membrane fluidity, surface polarity, and membrane phase state. Moreover, the effect of hexanol, alone or in combination with aniline or PADPA, as a possible modifier of the AOT membrane, was also studied with the aim of evaluating whether the membrane fluidity and surface polarity is altered significantly by hexanol, which, in turn, may have an influence on the mentioned types of reactions. The data obtained indicate that the AOT vesicle membrane at room temperature and pH 4.3 (0.1 M NaH2PO4) is more fluid and has a more polar surface than in the case of fluid phospholipid vesicle membranes formed from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Furthermore, the fluorescence measurements indicate that mixed AOT-hexanol membranes are less fluid than pure AOT membranes and that they have a lower surface polarity than pure AOT membranes. PADPA strongly binds to AOT and to mixed AOT/hexanol membranes and leads to drastic changes in the membrane properties (decrease in fluidity and surface polarity), resulting in Laurdan fluorescence spectra, which are characteristic for intramembrane phase separations (coexistence of ordered and disordered domains). This means that highly fluid AOT membranes transform upon the addition of PADPA into membranes that have ordered domains. Although the relevance of this finding for the enzymatic oligomerization of PADPA is not yet clear, it is also of interest if one likes to use heterogeneous vesicle membranes as additives for carrying out membrane surface-confined reactions that do not necessarily involve PADPA as a reactant.

Liposomes Can Achieve Enantioselective C-C Bond Formation of an α-Amino Acid Derivative in Aqueous Media.

We first report that a highly enantioselective C-C bond formation reaction was achieved with liposomes in aqueous media. Alkylation of N-(diphenylmethylene)glycine tert-butyl ester (DMGBE) with benzyl bromide was conducted in the presence of cetyltrimethylammonium bromide micelles, resulting in a high conversion of DMGBE but little enantiomeric excess (e.e.) of the product. The same reaction was then carried out in 1,2-dioleoyl-sn-glycero-3-phosphocholine liposome suspensions, where the e.e. values were high (at least 90 % (S)), indicating that the liposome membranes can behave as the promoter of the enantioselective reaction. Changing the type of lipid to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine to form a more ordered bilayer membrane lowered the reaction conversion but still maintained high e.e.% , that is, >90 (S), regardless of lipid chirality. It is indicated that multiple interactions between the DMGBE intermediate and lipid molecules promoted the migration of the intermediate into the interior of the membrane, whose bottom side (Si face) could be free for alkylation. These results suggest that liposomes can promote and regulate the alkylation of amino acid derivatives.

Enantioselective C-C Bond Formation Enhanced by Self-Assembly of Achiral Surfactants.

The use of achiral surfactant assemblies as a reaction platform for an alkylation reaction resulted in a high enantiomeric excess. Dilauryldimethylammonium bromide (DDAB) vesicles were modified with cholesterol to promote alkylation of N-(diphenylmethylene)glycine tert-butyl ester (DMGBE) with benzyl bromide, resulting in high conversion (∼90%) and high enantioselectivity (up to 80%). The R-enantiomer was formed on using the DDAB vesicles, whereas the use of phospholipid liposomes prepared from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) produced an excess of the S-enantiomer. Considering the chemical structures of the reaction substrates and amphiphiles as well as the membrane structures and properties of DDAB vesicles and DOPC liposomes, it is suggested that the enantiomeric excesses result from the location of the quaternary amine of the amphiphiles and the DMGBE at the outer surface of the membrane. We show that the enantioselective reaction at the surface of the self-assembly could be regulated by adjusting the chemical structures and resulting membrane properties of the self-assembly.

Functional implication of archaeal homologues of human RNase P protein pair Pop5 and Rpp30.

PhoPop5 and PhoRpp30 in the hyperthermophilic archaeon Pyrococcus horikoshii, homologues of human ribonuclease P (RNase P) proteins hPop5 and Rpp30, respectively, fold into a heterotetramer [PhoRpp30-(PhoPop5)2-PhoRpp30], which plays a crucial role in the activation of RNase P RNA (PhopRNA). Here, we examined the functional implication of PhoPop5 and PhoRpp30 in the tetramer. Surface plasmon resonance (SPR) analysis revealed that the tetramer strongly interacts with an oligonucleotide including the nucleotide sequence of a stem-loop SL3 in PhopRNA. In contrast, PhoPop5 had markedly reduced affinity to SL3, whereas PhoRpp30 had little affinity to SL3. SPR studies of PhoPop5 mutants further revealed that the C-terminal helix (α4) in PhoPop5 functions as a molecular recognition element for SL3. Moreover, gel filtration indicated that PhoRpp30 exists as a monomer, whereas PhoPop5 is an oligomer in solution, suggesting that PhoRpp30 assists PhoPop5 in attaining a functionally active conformation by shielding hydrophobic surfaces of PhoPop5. These results, together with available data, allow us to generate a structural and mechanistic model for the PhopRNA activation by PhoPop5 and PhoRpp30, in which the two C-terminal helices (α4) of PhoPop5 in the tetramer whose formation is assisted by PhoRpp30 act as binding elements and bridge SL3 and SL16 in PhopRNA.

Pseudo-Interphase of Liposome Promotes 1,3-Dipolar Cycloaddition Reaction of Benzonitrile Oxide and N-Ethylmaleimide in Aqueous Solution.

The hydrophobic interior of a liposome membrane was used as a platform for the organic synthesis of hydrophobic compounds in water. The 1,3-dipolar cycloaddition of benzonitrile oxide (BNO) and N-ethylmaleimide (EMI) in liposome suspensions was carried out, and an increase in the reaction rate constant was observed depending on the liposome characteristics. While the reaction rate constant in 1,4-dioxane was 1.5 times higher than that in water, the reaction rate constant in an aqueous solution of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposome was 3 times higher than in water. The amount of substrate, BNO, accumulated in the DOTAP liposome was higher than that in 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), indicating that BNO prefers to be distributed in the liposome membrane in the liquid-disordered phase. The membrane polarity, GP340, as monitored by Laurdan, varied with the presence of BNO, while EMI slightly affected the membrane properties of the liposomes. These results suggest that the pseudo-interphase afforded by the liposome membrane can promote the 1,3-dipolar cycloaddition between BNO and EMI in water.