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Abstract. In order to increase the tissue level of tetrahydrobiopterin (BH 4 ), supplementation with 6R-tetrahydrobiopterin (6RBH 4 ) has been widely employed. In this work, the effectiveness of 6RBH 4 was compared with 7,8-dihydrobiopterin (7,8BH 2 ) and sepiapterin by administration to mice. Administration of 6RBH 4 was the least effective in elevating tissue BH 4 levels in mice while sepiapterin was the best. In all three cases, a dihydrobiopterin surge appeared in the blood. The appearance of the dihydrobiopterin surge after BH 4 treatment suggested that systemic oxidation of the administered BH 4 had occurred before accumulation of BH 4 in the tissues. This idea was supported by the following evidences: 1) An increase in tissue BH 4 was effectively inhibited by methotrexate, an inhibitor of dihydrofolate reductase which reduces 7,8BH 2 to BH 4 . 2) When the unnatural diastereomer 6SBH 4 was administered to mice, a large proportion of the recovered BH 4 was in the form of the 6R-diastereomer, suggesting that this BH 4 was the product of a dihydrofolate reductase process by which 7,8BH 2 converts to 6RBH 4 . These results indicated that the exogenous BH 4 was oxidized and the resultant 7,8BH 2 circulated through the tissues, and then it was incorporated by various other tissues and organs through a pathway shared by the exogenous sepiapterin and 7,8BH 2 in their uptake. It was demonstrated that maintaining endogenous tetrahydrobiopterin in tissues under ordinary conditions was also largely dependent on an methotrexate-sensitive process, suggesting that cellular tetrahydrobiopterin was maintained both by de novo synthesis and by salvage of extracellular dihydrobiopterin.

A novel drug efflux gene (named sepA, staphylococcal efflux pump gene) is cloned from antiseptic-resistant mutants of Staphylococcus aureus into Escherichia coli. The sepA gene conferred the reduction of susceptibility to acriflavine and the acceleration of ethidium bromide efflux from the E. coli cells. The sepA (474 bp) encoded the protein that has four predicted transmembrane segments. These results indicate that sepA gene is a multidrugresistant gene and encodes a drug efflux protein.

Abstract:We have developed an efficient method for the esterification or thioesterification of equimolar amounts of carboxylic acids and alcohols or thiols using a novel reagent, p-toluenesulfonyl chloride (TsCl) together with N-methylimidazole. The present method is simple, mild, and reactive, uses readily available and economical reagents. The choice of amine is critical for the present method. The amine, N-methylimidazole, has two roles: (i) as an HCl scavenger for the initial smooth generation of mixed anhydrides between carboxylic acids and TsCl and (ii) successive formation of highly reactive ammonium intermediates from mixed anhydrides. This method could be applied to various types of carboxylic acids, alcohols, and thiols: a) several functionalities were tolerated; b) two N-Cbz amino acids were smoothly esterified without racemization; and c) the labile 1b-methylcarbapenem key intermediate and a pyrethroid insecticide, prallethrin, were successfully prepared. The related amide formation between carboxylic acids and primary or secondary amines was also performed. The proposed reaction mechanism involves a novel method for producing the reactive acylammonium intermediates. The production of these intermediates was rationally supported by a careful 1 H NMR monitoring study.

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