Enantiomer separation by reversed-phase liquid chromatography with novel hydrophobic phases composed of chiral cationic surfactants

This paper describes enantiomer separation using four kinds of chiral stationary phases (CSPs) where quaternary ammonium surfactants containing L-valine diamide moieties into long alkyl chains were bound to silicagel supports by reversed phase liquid chromatography. Our aim was to examine hydrogen bonding association of the chiral moiety in hydrophobic phase brought about by aggregation of the micelle-forming surfactants on the surface. The following CSPs were thus derived from the vinyl-terminated chiral surfactants via hydrosilylation: CSP 1 from N-[3-(10-undecenoyl-L-valylamino)propyl]-N,N,N-trimethylammonium bromide, CSP 2 from N-[6-(10-undecenoyl-L-valylamino)hexyl]-N,N,N-trimethyl-ammonium bromide, CSP 3 from N-[3-(10-undecenoyl-L-valylamino)propyl]-N-octadecanyl-N,N-dimethyl-ammonium bromide and CSP 4 from N-[6-(10-undecenoyl-L-valylamino)hexyl]-N-octadecanyl-N,N-dimethylammonium bromide. The degree of hydrophobicity in the interfacial phase was observed by measuring pyrene fluorescence in aqueous media including an organic modifier. Retention of racemic N-acylleucine isopropyl esters was highest in CSP 4, followed by 3, 2, and 1. Largest alpha values toward enantiomer separation were observed for CSP 4 where the chiral moieties were kept through a hexamethylene unit apart from the polar head groups and to which another long alkyl chain was attached, as compared with those for CSP 4. In CSP 4, the chiral moiety to interact with enantiomeric solutes should be buried into the interfacial phase deeply in more extent than CSP 3. In a similar manner, CSP 2 has more effective for enantiomer separation than CSP 1. The interfacial phase of these CSPs was easily exposed to the bulk phase because of the affinity between the bulk phase and the polar head groups as well as their electrostatic repulsion. However, degree of the enantiomer separation can be controlled by the depth of the chiral moiety in the hydrophobic interfacial phase.     

Rhodium-catalyzed reaction of thiols with polychloroalkanes in the presence of triethylamine

[reaction: see text] RhCl(PPh(3))(3) catalyzes a reaction of thiols with polychloroalkanes in the presence of triethylamine. This reaction serves as a convenient new method to produce formaldehyde dithioacetals, ethylenedithioethers, thioformates, and dithiocarbonic esters under mild conditions.     

Two new dimeric acridone alkaloids from Glycosmis citrifolia

Two new acridone dimers, glycobismines-F (1) and -G (2), were isolated from the roots of Glycosmis citrifolia collected in Taiwan. The structures of the new compounds were determined based on spectral analysis.     

Constituents of holothuroidea, 12. Isolation and structure of glucocerebrosides from the sea cucumber Holothuria pervicax

Ten glucocerebrosides, HPC-3-A-HPC-3-J, have been isolated from their obtained parent glucocerebroside molecular species HPC-3, together with other glucocerebroside molecular species HPC-1 and HPC-2, from the less polar lipid fraction of a chloroform/methanol extract of the sea cucumber Holothuria pervicax. The structures of these glucocerebrosides have been determined on the basis of chemical and spectroscopic evidence. Reversed-phase HPLC, including a recycling system, was effective in isolating these glucocerebrosides, revealing a very close resemblance in structure, though the problem due to regio-isomers remains.     

Antigen�Cantibody interactions of influenza virus hemagglutinin revealed by the fragment molecular orbital calculation

Effective interactions between amino acid residues in antigen?Cantibody complex of influenza virus hemagglutinin (HA) protein can be evaluated in terms of the inter-fragment interaction energy (IFIE) analysis with the fragment molecular orbital (FMO) method, in which each fragment contains the side chain of corresponding amino acid residue. We have carried out the FMO-MP2 (second-order Moeller?CPlesset) calculation for the complex of HA antigen and Fab antibody of influenza virus H3N2 A/Aichi/2/68 and obtained the IFIE values between each amino acid residue in HA and the whole antibody as the sums over the residues contained in the latter. Combining this IFIE data with experimental data for hemadsorption activity of HA mutants, we succeeded in theoretically explaining the mutations in HA observed after the emergence of influenza virus H3N2 A/Aichi/2/68 in an earlier study, except for those of THR83. In the present study, we employ an alternative way of fragment division in the FMO calculation at the carbonyl C site of the peptide bond instead of the C_?? site used in the previous work, which provides revised IFIE values consistent with all the historical mutation data in the antigenic region E of HA including the case of THR83 in the present prediction scheme for probable mutations in HA.     

Ribonuclease protection assay on microchip electrophoresis

We describe the potential of microchip electrophoresis with a Hitachi SV1210, which can be used to evaluate the integrity of total RNA, for the analysis of mRNA expression. The ribonuclease (RNase) protection assay was performed by using microchip electrophoresis with cyanine 5 (Cy5) labeled 248-base antisense RNA probe (riboprobe) encoding adipose-type fatty acid binding protein (A-FABP) as the riboprobe. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary strand RNA. Results were obtained in 120 s, and the same amount of Cy5-labeled antisense riboprobe as used in the conventional method can be used. Furthermore, 8 times more sensitive detection of mRNA by microchip electrophoresis could be obtained. An obvious increase in the mRNA expression of A-FABP, which is known as a differentiation marker of adipocytes, occurred during the adipocyte differentiation of 3T3-L1 cells. These results clearly indicate the potential of microchip electrophoresis for the analysis of mRNA expression in cells.     

A fluorescent single domain antibody against plumbagin expressed in silkworm larvae for fluorescence-linked immunosorbent assay (FLISA)

A fluorescent single-domain antibody (fluobody), a chimera of a green fluorescent protein (AcGFP) with a single chain variable fragment antibody (scFv), against plumbagin (5-hydorxy-2-methyl-1,4-naphthoquinone; PL) was successfully expressed in the hemolymph of silkworm larvae using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system to develop a rapid, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA). In this study, two kinds of fluobody, in which the PL-scFv was fused at the N-terminus (N-fluobody) or C-terminus of AcGFP (C-fluobody), were expressed in silkworm larvae for comparative purposes. Interestingly, both fluobodies expressed in the BmNPV bacmid DNA system retained both of their original functions as an AcGFP and a PL-scFv, although the functions of the N-fluobody were found to be inferior to those of C-fluobody when they were expressed in Escherichia coli. Moreover, an improvement in the limit of quantification for PL measurement was observed in FLISA (24 ng mL(-1)) compared with conventional ELISA (0.2 μg mL(-1)). Since both the C-fluobody and N-fluobody are useful probes for FLISA and the time-, cost-consuming refolding step required in the conventional bacterial expression system can be avoided when they are expressed in the BmNPV bacmid DNA system, the silkworm expression system is useful for expressing fluobodies when developing FLISA.     

Sequence‐Specific DNA Recognition by Cyclic Pyrrole–Imidazole Cysteine‐Derived Polyamide Dimers

Pyrrole–imidazole (PI) polyamides bind to the minor groove of the DNA duplex in a sequence‐specific manner and thus have the potential to regulate gene expression. To date, various types of PI polyamides have been designed as sequence‐specific DNA binding ligands. One of these, cysteine cyclic PI polyamides containing two β‐alanine molecules, were designed to recognize a 7 bp DNA sequence with high binding affinity. In this study, an efficient cyclization reaction between a cysteine and a chloroacetyl residue was used for dimerization in the synthesis of a unit that recognizes symmetrical DNA sequences. To evaluate specific DNA binding properties, dimeric PI polyamide binding was measured by using a surface plasmon resonance (SPR) method. Extending this molecular design, we synthesized a large dimeric PI polyamide that can recognize a 14 bp region in duplex DNA.