Mutation detection in the drug-resistant hepatitis B virus polymerase gene using nanostructured reverse micelles.

The emergence of drug-resistant hepatitis B virus (HBV) has been reported in patients with prolonged administration of lamivudine, which is a potent drug for the prevention of HBV infection. Lamivudine-resistant HBV has several types of mutations at the YMDD motif of its DNA polymerase. We successfully demonstrated that monitoring the hybridization behavior in nanostructured reverse micelles enables us to detect single nucleotide polymorphisms (SNPs). With the aid of reverse micelles, a model 40-mer oligonucleotide containing a single-base substitution was clearly distinguished from the normal, complementary oligonucleotide. In addition, we extended this technique to a high-throughput analysis. The results obtained with a 96-well micro-plate reader indicated the possibility of SNPs detection toward multiple samples of patients.     

Preparation and characterization of modified 3-aminopropylsilyl silica phases with 1,8-naphthalic anhydrides in HPLC

Modified 3-aminopropylsilyl silica stationary phases with 1,8-naphthalic anhydrides [3-(1,8-naphthalimido)propyl (NAIP), 3-(4-amino-1,8-naphthalimido)propyl (4A-NAIP), 3-(3-nitro-1,8-naphthalimido)propyl (3N-NAIP), and 3-(4-nitro-1,8-naphthalimido)propyl modified silyl silica gel (4N-NAIP)] are prepared in a single reaction. Chromatographic properties of these phases are characterized using various solutes including monosubstituted benzenes, alkyl benzenes, and polycyclic aromatic hydrocarbons (PAHs) in high-performance liquid chromatography. Even though the retention in these NAIPs is mainly based on the reversed-phase mode, 3N- and 4N-NAIP show a remarkable recognition toward aromatic hydrocarbons, which is attributed to their strong pi-pi interaction. Furthermore, the recognition ability is according to the molecular planarity and non-linearity of PAHs. Subsequently, 3N- and 4N-NAIP has a distinct selectivity from octadecyl silica and, therefore, will be a valuable alternative for a separation or a solid-phase extraction of aromatic compounds.     

Use of microcoil probehead for determination of the structure of oriented silk fibers by solid-state NMR

A microcoil probehead for solid-state NMR was developed with a two-channel radio-frequency circuit, and 13C observation with a proton-decoupling probehead was performed to obtain information on the distribution of the orientation of silk fibroin molecules in the fiber. The coil (1 mm (diameter) x 5 mm (length)) of the probehead was placed at the angles 90 degrees and 30 degrees , relative to the static magnetic field. Only 70 mug of [1-13C]Gly silk fibroin fiber was used in a magnet of 9.4 T (400 MHz for proton channel).     

Enantioselective total synthesis of antiangeogenic pentaketide dimers, epoxyquinols A and B, through an asymmetric aldol approach to their common monomeric precursor

A new enantioselective total synthesis of epoxyquinols A and B possessing unique pentaketide dimeric structures and potent antiangeogenic activity was achieved by oxidative dimerization of a monomeric pentaketide precursor obtained by the Evans asymmetric aldol reaction and a series of operationally simple transformations.     

Efficient copper-catalyzed coupling of aryl iodides and thiobenzoic acid

A highly efficient copper-catalyzed coupling reaction of aryl iodides and thiobenzoic acid, using 10 mol % of copper iodide, 20 mol % of 1,10-phenanthroline, and ⁱPr₂NEt in toluene, was developed. This methodology is applicable to a variety of aryl iodides.     

Determination of bisphenol A in human breast milk by HPLC with column-switching and fluorescence detection

A highly sensitive HPLC method was developed for the determination of xenoestrogenic compound, bisphenol A (BPA) in human breast milk samples. After a two-step liquid-liquid extraction, BPA was derivatized with fluorescent labeling reagent, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The excess fluorescent reagent could be removed effectively using a column-switching system. The separation of DIB-BPA from endogenous materials in milk was carried out on two C(18) columns and fluorescence intensity was monitored at 475 nm with the excitation of 350 nm. A good linearity (r = 0.994) was observed of BPA in the concentration range of 0.2-5.0 ng mL(-1) in breast milk, and the detection limit was 0.11 ng mL(-1) at a signal-to-noise ratio of 3. Intra- and inter-day precision (RSD, %) were less than 8.7 and 10.4, respectively. Twenty-three breast milk samples of healthy lactating women were analyzed for the BPA concentration; the mean value was 0.61 +/- 0.20 ng mL(-1), with no correlation to the lipid content of milk samples.     

Enantioselective addition of ketene silyl acetals to nitrones catalyzed by chiral titanium complexes. Synthesis of optically active beta-amino acids

A chiral titanium complex, Ti(O-i-Pr)(4)/BINOL/tert-butylcatechol, catalyzes enantioselective addition reaction of ketene silyl acetals to nitrones to give optically active beta-amino acid derivatives which are biologically active compounds and useful synthetic intermediates of natural products and pharmaceuticals such as beta-lactam antibiotics. The combined process of catalytic oxidation of secondary amines and enantioselective carbon-carbon bond formation of nitrones thus obtained with ketene silyl acetals provides a useful two-step method for the synthesis of optically active beta-amino acid derivatives and related nitrogen compounds.     

Room-temperature phosphorimetric determination of biogenic indole compounds adsorbed on a thin-layer chromatographic plate

A simple and sensitive method is described for the determination of biogenic indole compounds adsorbed on cellulose or alumina plates for thin-layer chromatography by room-temperature phosphorimetry. The optimum conditions were investigated for 5-hydroxyindole-3-acetic acid and indole-3-acetic acid. The compounds are spotted on the plates, which are then sprayed successively with sodium citrate or sodium acetate, and sodiu iodide solutions. The plates are dried completely under a stream of dry nitrogen, and immediately dipped in molten paraffin. The phosphorescence is stable for at least 3 h even in moist air. The limits of detection for nine biogenic indole compounds tested are between 2 and 300 pmol per sample spot.     

Practical synthesis of a neuropeptide Y antagonist via stereoselective addition to a ketene

[Reaction: see text]. The synthesis of neuropeptide Y antagonist 1, currently under clinical investigation for the treatment of obesity, is described. The convergent synthesis from trans-spirolactone carboxylic acid intermediate 2a and aminopyrazole 3 is predicated on a stereoselective route to the former. The coupling reaction of ethyl 4-oxocyclohexanecarboxylate (10a) with lithiated isonicotinamide 11 was investigated in detail, but even optimized conditions only provided a 45:55 ratio of trans:cis isomers (12a:12b). While selective crystallization schemes were developed to isolate the thermodynamically less stable trans isomer 2a, improved stereocontrol was subsequentially achieved by the application of ketene chemistry. The ketene formation and quench was investigated under a variety of conditions aimed at maximizing the trans:cis ratio. Reacting a mixture of carboxylic acids 2a and 2b with POCl3 in THF, followed by concomitant addition of tert-butyl alcohol in the presence of TMEDA at 35 degrees C provided a 4:1 ratio of trans:cis tert-butyl esters (18a:18b) via in situ ketene formation. Ester hydrolysis, followed by selective crystallization of undesired 2b as the HCl salt, led to isolation of 2a in 47% overall yield. Aminopyrazole intermediate 3 was synthesized via the condensation reaction of 2-fluorophenylhydrazine hydrochloride (4a) with acrylonitrile derivative 5 in 65-70% yield. Coupling of advanced intermediates 2a and 3b via activation with thionyl chloride gave a 92% yield of 1.