Derivatives of thymidine containing o-carboranylalkyl groups at the N-3 position and derivatives of 2'-deoxyuridine containing o-carboranylalkylmercapto groups at the C-5 position were synthesized. The alkyl spacers consist of 4-8 methylene units. The synthesis of the former compounds required 3-4 reaction steps in up to 75% overall yield and that of the latter 9-10 reaction steps with significantly lower overall yield. Derivatives of thymidine substituted with carboranylalkyl substituents at the N-3 position and short spacers were phosphorylated by both recombinant and purified cytosolic thymidine kinase (TK1) to a relatively high degree. None of the tested 2'-deoxyuridine derivatives possessing carboranyl substituents at the C-5 position were phosphorylated by either recombinant or purified TK1. The amounts of phosphorylation product detected for some of the C-5-substituted nucleosides with recombinant mitochondrial thymidine kinase (TK2) were low but significant and decreased with increasing lengths of the alkyl spacer. The data obtained in this study do not seem to support the tether concept applied in the synthesis of the new C-5- and N-3-substituted carboranyl nucleosides intended to reduce possible steric interference in the binding of carboranyl nucleosides with deoxynucleoside kinases. Instead, it appeared that a closer proximity of the bulky carborane moiety to the nucleoside scaffold resulted in better substrate characteristics.

Cleavage of the arylglycerol b-aryl ether linkage is the most important process in the biological degradation of lignin. The bacterial b-etherase was described previously and shown to be tightly associated with the cellular membrane. In this study, we aimed to detect and isolate a new extracellular function that catalyses the b-aryl ether linkage cleavage of high-molecular lignin in the soil fungi. We screened and isolated 2BW-1 cells by using a highly sensitive fluorescence assay system. The b-aryl ether cleavage enzyme was produced by a newly isolated fungus, 2BW-1, and is secreted into the extracellular fraction. The b-aryl ether cleavage enzyme converts the guaiacylglycerol b-O-guaiacyl ether (GOG) to guaiacylglycerol and guaiacol. It requires the Ca alcohol structure and p-hydroxyl group and specifically attacks the b-aryl ether linkage of high-molecular mass lignins with addition of two water molecules at the Ca and Cb positions. Lignin-biodegradation systems in nature can be summarized as follows. Initially, basidiomycetes secrete peroxidases and/or laccases and degrade the aromatic polymer lignin [1][2][3][4][5][6][7]. The role of each enzyme in this complicated process is an active area of research and debate. Thus far, mainly white rot fungi, Phanerochaete chrysosporium and Trametes (Coriolus) versicolor, have been studied regarding these peroxidases. P. chrysosporium produces two types of peroxidases, manganese peroxidase (MnP) and lignin peroxidase (LiP) and T. versicolor generally produces laccase. Laccase reacts with polyphenols including lignin, and other lignin-derived aromatic compounds, that, in turn, can be both polymerized and depolymerized. MnP can oxidize Mn 2+ to Mn 3+ ; Mn 3+ , in turn, is able to oxidize a wide range of phenolic substrates including phenolic lignin. LiP can directly oxidize a variety of phenolic and nonphenolic aromatic compounds. These peroxidases remove an electron and a proton from phenolic hydroxyl, aromatic amino groups or other aromatic side chains to form free radicals. Although this acts to cleave Ca-Cb linkages and b-O-4 linkages in the lignin structure, the free radicals cause random depolymerization of lignin. The various lowmolecular mass lignins produced by these peroxidases and/or laccase are decomposed to carbon dioxide and water by specific bacterial enzymes, such as ring-fission enzymes [8,9]

The phytoremediation of soils contaminated with organic pollutants offers a low-cost method for removal of such pollutants. We have attempted to enhance the environmental decontamination functions of plants by introducing appropriate enzymatic activities from microorganisms. In the present study, we introduced an extracellular fungal enzyme, the laccase of Coriolus versicolor, into tobacco plants. One transgenic plant, designated FL4, produced laccase that was secreted into the rhizosphere. FL4 was able to remove 20 micromol bisphenol A or pentachlorophenol per gram dry weight. The efficiency of this removal was apparently greater than that of control lines. Our results should stimulate efforts to develop plant-based technologies for the removal of environmental pollutants from contaminated environments.

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