2/4-Substituted-9-fluorenones and their O-glucosides as potential immunomodulators and anti-herpes simplex virus-2 agents. Part 5.

In pursuing a research on the antiviral and immunomodulatory activity of tilorone congeners, two new series of compounds were prepared and pharmacologically explored: 9-fluorenone carboxyhydroxyesters, indicated as AG, and 9-fluorenone carboxyhydroxamides, indicated as MG. Two of them, AG17 and MG3, were used as sugar acceptors in the transglycosylation reactions performed by alpha- and beta-glucosidases extracted from the marine mollusc Aplysia fasciata providing different alpha- and beta-, mono- and oligosaccharides. Then aglycons and saccharides were assayed for cytotoxicity, for anti-herpes virus-2 properties on peripheral blood mononuclear cells (PBMC) and for their capability to trigger human cells to produce antiviral cytokines such as IFNalpha and TNFalpha. Some promising compounds were individuated whereas the utility of the biocatalytic procedures in the preparation of pure anomeric material was further focused.

Facile biocatalytic access to 9-fluorenylmethyl polyglycosides: evaluation of antiviral activity on immunocompetent cells.

The biological activities of a series of mono- and oligosaccharides (beta-xylosides and alpha-glucosides) of 9-fluorenylmethanol were investigated together with mono-beta-galactoside and beta-glucoside of this aglycone, produced by biocatalytic routes. By using marine glycoside hydrolases and inexpensive donors such as maltose or xylan, access to mono-alpha-glucoside or mono-beta-xyloside of 9-fluorenylmethanol was obtained. Additionally, interesting polyglycoside derivatives were isolated. Biological testing indicated that in vitro treatment with these carbohydrate derivatives may influence the balance of cytokines in the environment of human peripheral blood mononuclear cells (PBMC), restricting the harmful effect of herpes simplex type 2 replication. In fact, these carbohydrate derivatives tested in WISH cells did not show any significant antiviral activity.

Functional and structural characterization of ovine ornithine transcarbamoylase.

Ornithine transcarbamoylase from ovine liver has been purified to homogeneity. Like all anabolic OTCs, the ovine enzyme is a trimer, constituted by identical subunits of 34 kDa. Sequence analysis of the 54 N-terminal residues of ovine OTC shows a high degree of homology with the human enzyme. The optimum pH and the Michaelis constants for the catalytic reaction were determined. The ovine enzyme is the most thermostable one among mammals OTCs, its critical temperature being 6 degrees C higher than those measured for the other enzymes. The enzyme has been crystallised and the structure determined at 3.5 A resolution. Crystals belong to the cubic P4(3)32 space group, with a = b = c = 184.7 A and a solvent content of about 80%. There is no evidence of any ligand in the active site cavity, indicating that the crystals contain an unliganded or T state of the enzyme. The unliganded OTCase enzyme adopts a trimeric structure which, in the crystal, presents a three-fold axis coincident with the crystallographic one. The conformation of each monomer in the trimer is quite similar to that of the liganded human protein, with the exception of a few loops, directly interacting with the substrate(s), which are able to induce a rearrangement of the quaternary organisation of the trimer, that accounts for the cooperative behaviour of the enzyme following the binding of the substrates.