Taiichi Higuchi’s research while affiliated with Kyoto University and other places

The high-pressure SNAr reaction was first applied to the synthesis of new functionalized diaza-crown ethers, which are directly connected to various heteroaromatic substituents as cation binding sites. The yields of the reaction were moderate to excellent (51% to quantitative). In a CH2Cl2 liquid membrane cation-transport experiment, diaza-crown ethers having 2′-thiazoyl-, 2′-benzoxazoyl-, 2′-pyrazinyl-, and 6′-pyridazinyl groups exhibited almost perfect Ag + ion selectivity. Cation extraction and 13C-NMR titration experiments revealed that attachment of heteroaromatics to the diaza-crown ring, if in the proper position, provides excellent Ag+ ion specificity. Since the binding and transport selectivity of these crown ethers were higher than those of the known crowns, the high-pressure technique proved a useful method for synthesis odf a new type of specific crown ethers. Single crystals of the complexes of diaza-crown ethers with silver trifluoromethanesulfonate (AgO3SCF3): AgTf and the corresponding metal-free diaza-crown compounds were successfully prepared. The short Ag+…Ag+ distances (2.790 and 2.969 Å) in the Ag complexes were confirmed for these Ag complexes by X-ray crystallography.

Präzedenzlose Reaktivität zeigt der N2-Ligand im Titelkomplex (Me8[16]anS4 = 3,3,7,7,11,11,15,15-Octamethyl-1,5,9,13-tetrathiacyclohexadecan): Mit Arylhalogeniden und Benzylbromid reagiert er in Toluol bei Raumtemperatur ohne Bestrahlung zu MoII-Aryldiazenido- bzw. -Dibenzylhydrazido-Komplexen. Mit Methanol und Aceton entsteht NH3 bzw. Me2C N N CMe2; Phosphan-Analoga des Titelkomplexes gehen keine derartigen Reaktionen ein. Die Struktur des Diazenido-Komplexes trans-[MoI(N2-p-C6H4CO2Me)(Me8[16]anS4)] ist rechts gezeigt.

We determined the three-dimensional structures of aspartate aminotransferase (AspAT) from Escherichia coli and its complex with inhibitor (2-methyl-L-aspartate) at 1.8A resolution. This enzyme reversibly catalyzes the transamination reaction and is a dimer of two identical subunits. Each subunit has 396 amino acid residues and one pyridoxal 5'-phosphate as a cofactor, and is divided into two domains, one large and the other small. Upon binding of the inhibitor, the small domain rotates by 5 degrees toward the large domain to close the active site. This domain movement is caused mainly by small but important main-chain conformational changes in the residues located over the domain interface of the small domain. In chicken mitochondrial AspAT, the domain movement was larger, with a rotational angle of 13 degrees. By comparison of these two structures, the difference in the rotational angles was found to be caused by the larger opening of the domain in the open form of chicken mitochondrial AspAT. Although the overall structures of these two enzymes were almost identical, the surface area of the domain interface in the E. coli enzyme was larger than that in mitochondrial AspAT, suggesting that the structure of the domain interface is responsible for the degree of movement of the small domain.

Isomers of ursodeoxycholic acid, 3 beta,7 alpha- and 3 beta,7 beta-dihydroxy-5 beta-cholan-24-oic acids (3 beta 7 alpha U and 3 beta 7 beta U) crystallize in the orthorhombic space group P2(1)2(1)2(1) containing one molecule and in the monoclinic group P2(1) containing two independent molecules in an asymmetric unit. The cell dimensions are a = 28.032(17), b = 9.973(5), c = 8.049(6) A for 3 beta 7 alpha U, and a = 11.771(8), b = 27.999(12), c = 6.637(2) A, beta = 90.78(6) degrees for 3 beta 7 beta U, respectively. Both structures were solved by the direct method and refined to the residual values of 0.065 (3 beta 7 alpha U) and 0.059 (3 beta 7 beta U). The conformations of the D rings of each molecule are different from each other: 3 beta 7 alpha U intermediate, 3 beta 7 beta U (A and B) half-chair form. In the crystal, 3 beta 7 alpha U molecules are connected by a hydrogen bond extended nearly parallel to the bc plane and 3 beta 7 beta U molecules are connected by the helical hydrogen bond system or by the zigzag one parallel to the ac plane.

Binuclear silver (I) complexes of double armed diaza-crown ethers, 7,16-bis(2′-thiazoyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane and 7,13-bis(2′-thiazoyl)-1,4,10-trioxa-7,13-diazacyclopentadecane were prepared. In these complexes, two Ag+ cations were cooperatively coordinated by thiazole-functionalized side arm and parent diaza-crown ring, and their Ag···Ag′ separations (2.790 and 2.969 Å) were either shorter (≈0.1 Å) or slightly longer (≈0.08 Å) than the intermetallic distance (2.889 Å), respectively.

Site-directed mutagenesis of Tyr70 in the active site of Escherichia coli aspartate aminotransferase (AspAT) followed by kinetic studies has elucidated the roles of the hydroxyl group and benzene ring of Tyr70. X-ray crystallographic analysis showed that replacement of Tyr70 by Phe did not alter the active-site conformation of the enzyme. Comparison of the kinetic parameters of the four half-transamination reactions (the pyridoxal 5'-phosphate form of the enzyme with L-aspartate or L-glutamate and the pyridoxamine 5'-phosphate form with oxalacetate or 2-oxoglutarate) between the wild-type and [Tyr70----Phe]AspATs showed that the mutation increases the energy level of the transition state by 2 kcal.mol-1 for all the four substrates, suggesting some contribution of the hydroxyl group of Tyr70 to the transition state. When Phe70 was further replaced by Ser, the energy level of the transition state for L-glutamate or 2-oxoglutarate, but not for L-aspartate or oxalacetate, was further increased by 2-3 kcal.mol-1, suggesting that the presence of a benzene ring at position 70 is essential for recognizing the L-glutamate-2-oxoglutarate pair as substrates.

Tyr225 in the active site of Escherichia coli aspartate aminotransferase (AspAT) was replaced by phenylalanine or arginine by site-directed mutagenesis. X-ray crystallographic analysis of Y225F AspAT showed that the benzene ring of Phe225 was situated at the same position as the phenol ring of Tyr225 in wild-type AspAT. The mutations resulted in a great decrease in the rate of the transamination reaction, suggesting that Tyr225 is important for efficient catalysis. The kinetic analysis of half-transamination reactions of Y225F AspAT with four substrates (aspartate, glutamate, oxalacetate, and 2-oxoglutarate) and some analogues (2-methylaspartate, succinate, and glutarate) revealed a considerable increase in the affinities for all these compounds. In contrast, affinity for the amino acid substrates was decreased by mutation to arginine, but affinities for the keto acid substrates and the two dicarboxylates (succinate and glutarate) were increased. The electrostatic interaction between O(3') of the coenzyme [pyridoxal 5'-phosphate (PLP)] and the residue at position 225 affected the pKa value of the Schiff base, which is formed between the epsilon-amino group of Lys258 and the aldehyde group of PLP; based on the spectrophotometric titration the pKa values were determined to be 6.8 for wild-type AspAT, 8.5 for Y225F AspAT, and 6.1 for Y225R AspAT in the absence of substrate. The absorption spectra of the three AspATs were almost identical in the acidic pH region, but the spectrum of Y225F AspAT differed from that of wild-type or Y225R AspAT in the alkaline pH region.(ABSTRACT TRUNCATED AT 250 WORDS)