Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties

Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs. (c) 2012 Elsevier B.V. All rights reserved.

The 2:1 cycloadducts from [3+2] 1,3-dipolar cycloaddition of nitrile oxide and vinylacetic acid. Synthesis and liquid crystal behaviour

Four liquid crystals (LC) 3,7a-bis(4-alkyloxyphenyl)-7,7a-dihydro-6H-isoxazolo[2,3-d][1,2,4]oxadiazol-6-yl)acetic acid (7a-d) were synthesised and the mesomorphic behaviour reported. The LCs were characterised as 2: 1 bisadducts, which were obtained from a double [3+2] 1,3-dipolar cycloaddition. In the first step, the cycloaddition of 4-alkyloxyphenylnitrile oxide (4a-d) and vinylacetic acid (5) gave the initial unobserved 1:1 cycloadducts 2-[3-(4-alkyloxyphenyl)-4,5-dihydroisoxazol-5-yl]acetic acid (6a-d). In the second step, the addition of a second equivalent of 4 to 6 yielded the 2: 1 bisadducts 7a-d without any traces of 6. All compounds 7a-d were unstable during the transition from the mesophase to the isotropic state upon first heating as evidenced by the large peaks in the differential scanning calorimetry traces. Due to the chemical instability of the compounds upon heating, the transition temperature related to the smectic C to smectic A transitions was acquired by means of an image processing method. X-Ray diffraction experiments were also used to analyse the liquid-crystalline phases. A theoretical calculation was performed using density functional theory (DFT) methods at the PBE1PBE/6-311+G(2d,p) level (with solvent effect) in order to get information about the energetic profile of the 2: 1 cycloaddition. DFT studies revealed that the cycloaddition process is controlled by the HOMO(dipolarophile) - LUMO(1,3-dipole), and that the double [3+2] 1,3-dipolar cycloaddition reaction is quite possible.

Side-chain Modifications of Highly Functionalized 3(2H)-Furanones

A series of 3(2H)-furanones, based on side-chain modifications of a parent 3(2H)-furanone, was synthesized in good yield. The parent compound was prepared by hydrogenolysis, and subsequent acid hydrolysis, of isoxazole derivatives. The isoxazole was prepared by a [3+2] 1,3-dipolar cycloaddition reaction between 3-butyn-2-ol and nitrile oxide.