Yukiharu Sato

ISOMERIZATION AND PEROXIDIZING PHYTOTOXICITY OF THIADIAZOLIDINE-THIONE COMPOUNDS

Eight 5-arylimino-3,4-tetramethylene-1,3,4-thiadiazolidine-2-thiones and eight 4-aryl-1,2- tetramethylene-1,2,4-triazolidine-3,5-dithiones were synthesized and their phytotoxic activities were investigated using sawa millet (Echinochloa utilis), green microalgae (Scenedesmus acutus) and protoporphyrinogen-IX oxidase isolated from etiolated corn (Zea mays) seedlings. 5-Arylimino-3,4-tetramethylene-1,3,4-thiadiazolidine-2-thiones showed strong phytotoxic activities and the same herbicidal mode of action as known for peroxidizing herbicides. 5-Arylimino-3,4-tetramethylene-1,3,4-thiadiazolidine-2-thiones were not or very little converted into 4-aryl-1,2-tetram ethylene-1,2,4-triazolidine-3,5-dithiones either with E. utilis seedlings present for 7 days, with S. acutus cells, or using glutathione 5-transferase (GST) and glutathione (GSH). The phytotoxic activities of 4-aryl-1,2-tetram ethylene-1,2,4-triazolidine- 3,5-dithiones were stronger than those of 5-arylimino-3,4-tetram ethylene-1,3,4-thiadiazolidine- 2-thiones [cf. Sato, Y., et al., Z. Naturforsch. 49c, 49-56 (1994)].

Peroxidizing Phytotoxic Activity of 1, 3, 4-Thiadiazolidine-2-thiones and 1, 2, 4-Triazolidine-3, 5-dithiones

Using autotrophic Scenedesmus acutus cells incubated in the light and Echinochloa utilis culture, a series of isomeric compounds, namely 5-arylimino-3, 4-tetramethylene-1, 3, 4-thiadiazolidine-2-thiones (thiadiazolidine-thiones) and 4-aryl-l, 2-tetramethylene-1, 2, 4-triazolidine3, 5-dithiones (triazolidine-dithiones), were assayed with respect to growth inhibition, decrease of chlorophyll contents, protoporphyrin-IX accumulation and light-induced ethane formation level. The both types of phytotoxic compounds decreased chlorophyll contents, caused protoporphyrin-IX accumulation and ethane evolution, and inhibited growth of . Scenedesmus cells. They inhibited also protoporphyrinogen-IX oxidase, which led to rapid accumulation of protoporphyrin-IX, an intermediate of chlorophyll biosynthesis, just like peroxidizing herbicides such as p-nitrodiphenyl ethers and cyclic imides. Our comparative data on different sets of the aforementioned parameters suggest that both the thiadiazolidinethiones and triazolidine-dithiones are grouped as peroxidizing herbicides, affecting a crucial enzyme in the chlorophyll biosynthesis and inducing ethane formation by cell membrane destruction.

Very-long-chain fatty acid biosynthesis is inhibited by cafenstrole, N,N-diethyl-3-mesitylsulfonyl-1H-1,2,4-triazole-1-carboxamide and its analogs.

Abstract The rice herbicide cafenstrole and its analogs inhibited the incorporation of [1-14C]-oleate and [2-14C]-malonate into very-long-chain fatty acids (VLCFAs), using Scenedesmus cells and leek microsomes from Allium porrum. Although the precise mode of interaction of cafenstrole at the molecular level is not completely clarified by the present study, it is concluded that cafenstrole acts as a specific inhibitor of the microsomal elongase enzyme involved in the biosynthesis of fatty acids with alkyl chains longer than C18. For a strong VLCFA biosynthesis inhibition an -SO2- linkage of the 1,2,4-triazole-1-carboxamides was required. Furthermore, N,N-dialkyl substitution of the carbamoyl nitrogen and electron-donating groups such as methyl at the benzene ring of 1,2,4-triazole-1-carboxamides produced a strong inhibition of VLCFA formation. A correlation was found between the phytotoxic effect against barnyardgrass (Echinochloa oryzicola) and impaired VLCFA formation.

Isomerization of Peroxidizing Thiadiazolidine Herbicides Is Catalyzed by Glutathione S-Transferase

Abstract Thiadiazolidine-converting activity (isomerase), detected in a 45-75% ammonium sulfate precipitate from corn seedlings extracts, was purified by chromatography on hydroxyapatite and by anion exchange on Mono Q Sepharose. Two fractions 1 and 2 with isomerase activity were separated on Mono Q by combination of a stepwise elution and continuous salt gradient; fraction 2 eluting at higher salt concentrations was found the most active. Total activity could be enhanced by treatment of seedlings with naphthalic anhydride. Both fractions containing isomerase activity were further purified by glutathione-(GSH) agarose affinity chromatography and characterized by their specificity for different thiadiazolidines. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration revealed that the isomerase of fraction 2 consists either of a homodimer or a heterodimer of two proteins with apparent molecular weights of 28 and 31 kDa, respectively. The protein pattern as well as the strict dependence of activity on thiol groups (GSH or dithiothreitol) suggested a glutathione Stransferase (GST) catalyzing the thiadiazolidine conversion. Further evidence was obtained by measuring reactions specific for GSTs in both purified fractions, namely the conjugating activity for l-chloro-2,4-dinitrobenzene (CDNB ). atrazine and metazachlor. While no atrazine turnover was found, metazachlor and CDNB conjugation occurred rapidly. Both fractions differed in their activities to several GST substrates with fraction 2 being more effective in metazachlor but less active in C DN B conjugation. Inhibitors specific for GST-catalyzed reactions also inhibited thiadiazolidine conversion confirming that isomerizing activity is attributed to a GST form. We conclude that GST isoforms with different affinities towards thiadiazolidines have been isolated. CDNB activity, molecular weight, the protein pattern on SDS-PAGE as well as the amino acid sequence of one of its polypeptides suggest that fraction 1, less active in thiadiazolidine isomerization, is identical to GST I. The second peptide of this fraction was resistant to Edman degradation probably due to N-terminal blockage. The properties of the high isomerase activity found in fraction 2 are in agreement with characteristics of a GST previously termed as isoform II.

Susceptibility of ammonia-oxidizing bacteria to nitrification inhibitors.

Activity of nitrification inhibitors to several typical ammonia-oxidizing bacteria isolated recently, i. e. Nitrosococcus, Nitrosolobus, Nitrosomonas, Nitrosospira and Nitrosovibrio species was assayed using 2-amino-4-methyl-6-trichloromethyl-1,3,5-triazine (MAST), 2-amino- 4-tribromomethyl-6-trichloromethyl-1,3,5-triazine (Br-MAST), 2-chloro-6-trichloromethylpyridine (nitrapyrin) and others, and compared to confirm the adequate control of ammoniaoxidizing bacteria by the inhibitors. The order of activity of the inhibitors to 13 species of ammonia-oxidizing bacteria examined was approximately summarized as Br-MAST ≥ nitrapyrin ≥ MAST > other inhibitors. Two Nitrosomonas strains, N. europaea ATCC25978 and N. sp. B2, were extremely susceptible to Br-MAST, exhibiting a pI50 ≥ 6.40. These values are the position logarithms of the molar half-inhibition concentration. The 16S rRNA gene sequence similarity for the highly susceptible 4 strains of genus Nitrosomonas was 94% to 100% of Nitrosomonas europaea, although those of the less susceptible 3 strains of ammoniaoxidizing bacteria, Nitrosococcus oceanus C-107 ATCC19707, Nitrosolobus sp. PJA1 and Nitrosolobus multiformis ATCC25196, were 77.85, 91.53 and 90.29, respectively. However, no clear correlation has been found yet between pI50-values and percent similarity of 16S rRNA gene sequence among ammonia-oxidizing bacteria.

Inhibition of very-long-chain fatty acid formation by indanofan, 2-[2-(3-chlorophenyl)oxiran-2-ylmethyl]-2-ethylindan-1,3-dione, and its relatives.

Indanofan and its analogs inhibited the elongation of stearoyl- or arachidoyl-CoA by [2-14C]-malonyl-CoA in leek microsomes from Allium porrum. Although the precise mode of interaction of indanofan at the molecular level is not completely clarified by the present study, it is concluded that indanofan and analogs act as inhibitor of the elongase enzyme involved in de novo biosynthesis of fatty acids with an alkyl chain longer than C18, called very-long-chain fatty acids (VLCFAs). For a strong inhibition of VLCFA formation chloro substituents at the benzene ring and the oxirane group were necessary. Furthermore, the greenhouse test showed strong activity for indanofan and its analogs, and the scores coincided with cell-free elongation inhibition. The cell-free assay, however, failed to indicate any activity for an analog having a methylene instead of the oxirane group, while both Digitaria ciliaris and Echinochloa oryzicola were killed with 1 kg a.i./ha. This finding cannot be discussed because the applied use rate of 1 kg a.i./ha is too high to allow for a score differentiation. For high concentrations of this compound additional unknown inhibitory effects may be involved besides fatty acid elongation.

ENZYME-MODIFIED PHYTOTOXIC STRUCTURE OF THIADIAZOLIDINE COMPOUNDS

Abstract A simple model thiadiazolidine, 5-(4-methoxycarbonylmethylthio-phenylimino)-3,4-tetra-methylene-1,3,4-thiadiazolidin-2-one, was synthesized and its structural modification investigated using glutathione S-transferase (GST) and an esterase preparation isolated from Echinochloa utilis. The objective is a better understanding of the metabolic activation of peroxidizing thiadiazolidine compounds. The model thiadiazolidine with an ester group (thiadiazolidine ester) was isomerized by GST to a more phytotoxic triazolidine structure (triazolidine ester). Both the thiadiazolidine and the more active triazolidine ester were hydrolyzed by Echinochloa esterase to less active free acid compounds. 5-(4-carboxymethylthiophenylimino)-3,4-tetramethylene-1,3,4-thiadiazolidin-2-one (thiadiazolidine acid) and 4-(4-carboxy-methylthiophenyl)-1,2-tetramethylene-1,2,4-triazolidin-3-one-5-thione (triazolidine acid), respectively. The thiadiazolidine acid, however, was only slightly converted into the triazolidine acid in the presence of GST. It is concluded that the thiadiazolidine ester was isomerized in Echinochloa to give the triazolidine acid through the triazolidine ester. Since the triazolidine ester exhibited the highest phytotoxic peroxidizing activity GST is considered as an activating enzyme for phytotoxicity and esterase as a detoxifying enzyme to reduce phytotoxic activity. Accordingly, phytotoxic thiadiazolidine-ester type herbicides may be produced by an interplay of isomerizing GST and esterase activity contributing to herbicide selectivity among plant species.

New peroxidizing herbicides : activity compared with X-ray structure

The crystal structure of 3-(4-chloro-5-cyclopentyloxy-2-fluorophenyl)-5-isopropylideneoxazolidine- 2,4-dione (BW -4), chlorophthalim and oxyfluorfen were determined by X -ray analysis. The molecular size of these compounds was almost the same, but the angle between the oxazolidine and the phenyl ring (BW -4), the imide and phenyl ring (chlorophthalim ) and between the two phenyl rings (oxyfluorfen) differed. The phytotoxic activities, determined by growth inhibition of Scenedesmus cells, loss of chlorophyll, and peroxidative destruction of photosynthetic pigments, were different among these compounds. The angle and length of BW-3, BW-4 and oxyfluorfen showing best phytotoxic activity were found approximately 65 -95 ° and 11 - 13 Å, respectively. These preliminary findings indicate that both angle and length of the molecule have some bearing on peroxidative activity.

Metabolic Modification of Isoimide Type Peroxidizing Compounds Catalyzed by An Isoenzyme of Glutathione S-Transferase

Peroxidizing herbicides cause membrane destruction in plants by inhibiting the membrane-bound protoporphyrinogen oxidase (protox, EC 1.3.3.4) competitively (Nicolaus et al. 1995). This inhibition induces the accumulation of protoporphyrin IX (Lydon and Duke 1988; Matringe and Scalla 1988; Sandmann and Boger 1988; Wittkowski and Hailing 1988) which is sensitized by light with subsequent radical formation leading to degradation of cellular constituents with evolution of ethane (Boger and Sandmann 1990; Boger and Wakabayashi 1995).

Chemical catalysis of the isomerisation of peroxidising herbicidal thiadiazolidines

Model reactions with various -SH, -OH and -NH nucleophiles in an aprotic solvent were used to characterise the nature of the enzymatic conversion of thiadiazolidines to triazolidines. This conversion results in greater inhibition of protoporphrinogen oxidase (protox). It is inferred that GST-isoforms probably promote formation of one of the intermediates.