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Calcineurin, which is a Ca2+/calmodulin-dependent protein phosphatase, is a key mediator in calcium signaling in diverse biological processes and of clinical importance as the target of the immunosuppressant FK506. To identify a mutant(s) in which calcineurin is activated, inhibiting cellular growth as a result, we screened for a mutant(s) whose temperature sensitivity would be suppressed by FK506 from the budding yeast non-essential gene deletion library. We found that the temperature sensitivity of cells in which the conserved Verprolin VRP1 gene had been deleted, which gene is required for actin organization and endocytosis, was suppressed by either FK506 or by cnb1 deletion. Indeed, the calcineurin activity increased significantly in the ∆vrp1 cells. Finally, we demonstrated that the ∆vrp1 strain to be useful as an indicator in a positive screening for bioactive compounds inhibiting calcineurin.

The influence of the S-enantiomer, R-enantiomer and racemate species of indanofan on the elongation of very-long-chain fatty acids (VLCFA) elongation was assayed using a leek microsomal preparation. Double-reciprocal plots of the rate of VLCFA production versus the concentration of C18 : 0-CoA demonstrated to be a competitive type of inhibition by indanofan. The I50 values of (S)-indanofan, (R)-indanofan and the (RS)-indanofan racemate for elongation of C18 : 0- and C20 : 0-acid were estimated to be about 2×10−8, >10−3 and 5×10−8 M, respectively. These findings prove that the S-enantiomer inhibited the elongation of the C18 : 0-and C20 : 0-acid ca. 2–3 times more strongly than the racemate, yielding almost no inhibition by the R-enantiomer. The elongation of C18 : 0- as well as of the C20 : 0-acid is inhibited by the S-enantiomer only. © Pesticide Science Society of Japan

Phytotoxic activity of several middle-chain fatty acids, especially pelargonic acid (C9 acid) was investigated. C9–C11 acids caused strong non-selective damage to plants such as crabgrass, cucumber, velvetleaf, and tobacco, while C6 and C14 fatty acids had almost no activity. Middle-chain fatty acids caused a strong and rapid electrolyte leakage. They reached highest conductivity in 3 h in the case of cucumber cotyledons. Middle-chain fatty acids caused a decrease of the amount of polar lipids, particularly MGDG and PG, and chlorophylls. They also caused an increase of free fatty acids in 24 h after treatment. These results suggested that middle-chain fatty acids caused severe damage to cell membranes and thylakoid membranes of treated leaves. C6 volatile compounds such as cis -3-hexenal, trans -2-hexenal, and cis -3-hexenol were generated in less than 1 h after spraying pelargonic acid to tobacco leaves. The application of pelargonic acid was thought to be the trigger for linolenic acid degradation in the thylakoid membranes.

Pelargonic acid (PA), an aliphatic 9-carbon monocarboxylic acid, is a phytotoxic burn-down compound. In the light peroxidizing activity can be measured as ethane and propane formation with cress or tobacco seedlings. This effect is strong at low pH (4–5), and saturated acids with 9–10 carbon atoms represent the optimum chain length. Methyl or ethyl esters are inactive, and safeners have no influence. In contrast to the peroxidative herbicides like acifluorfen methyl neither photosynthesis nor protoporphyrin IX is involved, although peroxidation requires light. Chlorophyll is necessary since etiolated seedlings show little peroxidation. Singlet oxygen quenchers like eugenol markedly reduce peroxidation. Membrane leakage of a similar rate is observed in light as well as in darkness. PA was described as a penetration enhancer intercalating with membranes. Our data corroborate that conclusion. Accordingly, the herbicidal mode of action of pelargonic acid is due first to membrane leakage in dark or light and second to peroxidation driven by radicals originating in the light by sensitized chlorophyll displaced from the thylakoids.

A bird's eye review was tried in Part 2 of this series, ‘Phytotoxic sites of action for molecular design of modern herbicides’, in order to select the best selection of known and some novel plant-specific targets for molecular design of modern herbicides, which affect amino acid, lipid and cell wall biosynthesis. Although amino acid biosynthesis pathways, particularly those for aromatic amino acids, ammonia assimilation and branched amino acids, have been confirmed as reasonable herbicidal target domains, the other targets affecting plant growth more markedly than inhibition of 5-enolpyruvylshikimate-3-phosphate synthase, glutamine synthetase and acetolactate synthase are discussed. In three essential enzymes involved in fatty acid biosynthesis in or in the vicinity of chloroplasts, acetyl-CoA carboxylase (ACCase), elongase(s) for very long chain fatty acids (VLCFA) and linolate monogalactosyldiacylglycerol desaturase, ACCase and elongase are more important targets for new herbicides. Although the effect of cellulose biosynthesis inhibitors is restricted to cell wall formation in growing plant cells only, there is a good chance to design the low-use rate herbicides also in this class of inhibitors. Other possible targets for new herbicides are also discussed.

A bird's eye review was tried to select the bio-rational targets from known and novel plant-specific ones for the molecular design of modern herbicides, which exhibit efficient phytotoxicity at a low-use rate and preserve a good environment in the 21st century. In phytotoxic sites in the photosynthetic electron transport (PET) system discussed in the present article (Part 1), the generally called bleaching herbicides interfering with the biosynthesis of photosynthetic pigments, chlorophylls and carotenoids, and the biosynthesis of plastoquinone, were considered to be good models for the molecular design of modern herbicides. The PET itself was still considered as an interesting target site for new herbicides, although they need to exert their action in all green leaves of weeds to achieve herbicidal efficacy. Because these herbicides never form a tight binding with D1-protein, their use-rate cannot be expected to be as low as the herbicides inhibiting chlorophyll or branched amino-acid biosynthesis. Other herbicidal targets found in chloroplasts, namely ATP and NADPH formations, have already been omitted from the worldwide biorational molecular design program of herbicides targeting the PET system.

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.

A series of replacement experiments of [14C]-triazines, [14C]-atrazine and [7-14C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine, bound to thylakoids isolated from wild-type and atrazine-resistant Chenopodium album (lambsquarters) were conducted. Replacement experiments of [14C]-triazines bound to wild-type Chenopodium thylakoids with non-labeled atrazine and 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine were carried out, to elucidate whether benzylamino-1,3,5-triazines use the same binding niche as atrazine. [14C]-Atrazine and [7-14C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine bound to wild-type thylakoids were replaced by non-labeled 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine and non-labeled atrazine, respectively. The above two replacements showed mutual competition. To clarify further whether benzylamino-1,3,5-triazines bind at the D1-protein to amino acid residue(s) different from atrazine or not, experiments to replace [7-14C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazines bound to atrazine-resistant Chenopodium thylakoids by non-labeled atrazine, 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU and DNOC were carried out. Although the bound [7-14C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine was difficult to be replaced even with high concentrations of atrazine, [14C]-labeled 1,3,5-triazine was competitively replaced by non-labeled 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU or DNOC. Thus, 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine herbicides are considered to bind to the same niche at the D1 protein as atrazine, but use amino acid residue(s) different from those involved with atrazine binding.

A series of 3(2H)-oxo-N-(substituted phenyl)-4,5,6,7-tetrahydro-1,2-benzisothiazoles (2) were obtained via four reaction steps starting from 2-chlorocyclohexene-1-carboxylic acid. The sulfur atom of 3-oxo-4,5,6,7-tetrahydro-1,2-benzisothiazoles (2) was oxidized with an equimolecular amount of 3-chloroperbenzoic acid (3-CPBA) in chloroform to give the corresponding 3-oxo-4,5,6,7-tetrahydro-1,2-benzisothiazole-1-oxides (3). Oxidation of 2 with two moles of 3-CPBA afforded 3-oxo-4,5,6,7-tetrahydro-1,2-benzisothiazole-1,1-dioxides (4). Phytotoxic activities of the compounds (2-4) synthesized were assayed by inhibition of protoporphyrinogen-IX oxidase isolated from corn as well as by growth inhibition, chlorophyll decrease and peroxidative destruction of cell membranes of the green microalga Scenedesmus acutus. Among the compounds (2-4), 4 showed the strongest activities according to all phytotoxic parameters, exhibiting phytotoxicities characteristic of peroxidizing herbicides. 3(2H)-oxo-2-[4-chloro-3-(isopropoxycarbonyl)phenyl]-4,5,6,7-tetrahydro-1,2-benzisothiazole-1,1-dioxide (4h) was the strongest of the compounds 4 tested.

The effects of Photosystem II inhibiting herbicides, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), atrazine and two novel 2-benzylamino-1,3,5-triazine compounds, on photosynthetic oxygen evolution and chlorophyll a fluorescence induction were measured in thylakoids isolated from Chenopodium album (wild type and atrazine-resistant plants) and cyanobacterial intact cells. The resistant plants have a mutation of serine for glycine at position 264 of the D1 protein. Diuron and two members of a novel class of 2-benzylamino-1,3,5-triazine compounds were almost as active in wild-type as in atrazine-resistant thylakoids, indicating that the benzylamino substitution in the novel triazines may be important for the lack of resistance in these atrazine-resistant plants. The inhibition by the herbicides of oxygen evolution in the cyanobacteria was somewhat lower than in the thylakoids of Chenopodium album wild type, probably caused by a slower uptake in the intact cells. The so-called OJIP fluorescence induction curve was measured during a one second light pulse in the absence and in the presence of high concentrations of the four herbicides. In the presence of a herbicide we observed an increase of the initial fluorescence at the origin (Fo′), a higher J level, and a decreased steady state at its P level (Fp). The increase to Fo′ and the decreased leveling Fp are discussed. After dark adaptation about 25% of the reaction centers are in the S0 state of the oxygen evolving complex with an electron on the secondary electron accepting quinone, QB. The addition of a herbicide causes a transfer of the electron on QB to the primary quinone acceptor, QA, and displacement of QB by the herbicide; the reduced QA leads to a higher Fo′. The decrease of Fp in the presence of the herbicides is suggested to be caused by inhibition of the photo-electrochemical stimulation of the fluorescence yield.

The herbicide thenylchlor and its analogs inhibited the incorporation of [1- 14 C]oleate and [2- 14 C]malonate into very-long-chain fatty acids (VLCFAs) in Scenedesmus cells and leek microsomes from Allium porrum , respectively. Although the precise mode of interaction of thenylchlor at the molecular level is not completely clarified by the present study, it is concluded that thenylchlor acts as an inhibitor of the elongase enzyme involved in biosynthesis of fatty acids with alkyl chains longer than C 18 . For a strong inhibition of VLCFA formation, an N,N-disubstituted amide moiety of chloroacetamides is required. Furthermore, 2,6-dimethyl substituents at the benzene ring and electron-donating groups such as methoxyl or methyl at the 3 position of the thiophene ring produce a strong inhibition of VLCFA formation. A correlation was found between the phytotoxic effect against barnyardgrass ( Echinochloa oryzicola ) and the decreased VLCFA formation.

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.

The bioactivation of thiadiazolidine-type peroxidizing compounds was examined with thiosemicarbazides as model intermediates. Peroxidizing activities of three sets of thiadiazolidines (5-arylimino-3,4-tetramethylene-1,3,4-thiadiazolidines), thiosemicarbazides (4-aryl-1-ethylthiocarbonyl-1,2-tetramethylenethiosemicarbazides), and triazolidines (4-aryl-1,2-tetramethylene-1,2,4-triazolidines) were assayed for protopor- phyrinogen-IX oxidase (Protox) inhibition with Protox isolated from corn etioplasts and for phytotoxic parameters of growth, chlorophyll content, and ethane evolution obtained with Scenedesmus acutus cells. Protox inhibitory activity of thiosemicarbazides was intermediate between that of thiadiazolidines and triazolidines. Phytotoxic parameters of thiosemicarbazides obtained from S. acutus cells were quite identical to those of triazolidines, although phytotoxic parameters of thiadiazolidines exhibited a slightly different pattern. Phytotoxic activities of 5-(4-bromophenyl)-3,4-tetramethylene-1,3,4-thiadiazolidin-2-one (thiadiazolidine 1 ) and 5-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4-tetramethylene-1,3,4-thiadiazolidin-2-one (thiadiazolidine 4 ) were similar to those of the corresponding triazolidines, but phytotoxicities exhibited by 5-(4-(4-chlorobenzyloxy)phenyl)-3,4-tetramethylene-1,3,4-thiadiazolidin-2-one (thiadiazolidine 7 ) were 10 times less active with S. acutus than those of the corresponding triazolidine. Three thiosemicarbazides exhibited rapid conversion into triazolidines both in buffer at pH 7 (ca. 25–50%, 5 min incubation) and in Scenedesmus cultures (ca. 75%, 5 h incubation). Thiadiazolidines 1 and 4 were converted into corresponding triazolidines (ca. 70%) in S. acutus but were converted in buffer only in the presence of glutathione S -transferase plus ethylmercaptan, although thiadiazolidine 7 was not converted in the Scnedesmus culture. Apparently, the conversion step from thiadiazolidine to the thiosemicarbazide intermediate is enzymatic and the thiosemicarbazide intermediate to triazolidine is a spontaneous, nonenzymatic step.

To investigate the mechanism by which short-chain hydrocarbons such as ethane are formed under the influence of peroxidizing herbicides, we examined oxyfluorfen-induced lipid hydroperoxide content and changes of fatty acids constituting the neutral lipid in mung bean cotyledons. Lipid hydroperoxides, detected by a chemiluminescence-HPLC system, were separated into neutral lipid, free fatty acid, and phospholipid fractions using BUND ELUT NH2. Lipid hydroperoxide is present primarily in the neutral lipid fraction. This indicates that lipid peroxidation caused by oxyfluorfen may occur in the neutral lipid including glycolipid. Next, we analyzed the change of fatty acid composition in the neutral lipid in which the most lipid hydroperoxide was observed. Polyunsaturated fatty acids (PUFAs: C18:2, C18:3) markedly decreased after a 1-h treatment with oxyfluorfen under the light, but increased during the next 5 h. However, the PUFA content decreased from 6 to 13 h after oxyfluorfen treatment. It is suggested that peroxidation occurs during the early stages after herbicide application, and antioxidants together with fatty acid synthesis are instrumental in recovering PUFAs decreased by peroxidation in plant cells. However, the defense system of a plant is overtaxed by the continuous oxidative stress, eventually leading to destruction of cell membranes. Whereas the amount of PUFAs decreased, monounsaturated (C16:1, C18:1) fatty acid content tended to increase by oxyfluorfen treatment. It is suggested that the regulatory network for the biosynthesis of different fatty acid species may be disturbed by the oxidative stress imposed.

A mode of action study of herbicidal diphenylpyrrolidinones was carried out through carotenoid analyses in intact Scenedesmus cells and by a cell-free plant-type phytoene desaturase assay using Escherichia coli transformants. A series of forty-eight diphenylpyrrolidinones decreased the carotenoid content of Scenedesmus cells in the light and inhibited phytoene desaturase. The relationship between substituents at various positions and inhibition of phytoene desaturase is discussed. Using very active bleaching diphenylpyrrolidinones, a 10−5 M concentration affected neither the ζ-carotene desaturase nor the protoporphyrinogen-IX oxidase. Although some differences in their inhibitory activity were found between the in vivo and cell-free assays, it is concluded that the compounds are essentially bleachers affecting carotenoid biosynthesis in plants. Enzyme kinetics studies with recombinant phytoene desaturase revealed a non-competitive inhibition with respect to the substrate phytoene. A competition against the inhibitor was shown by the cofactor NADP+, suggesting an interaction of pyrrolidinones at the cofactor-binding site of phytoene desaturase. © 2001 Society of Chemical Industry

Peroxidizing herbicides have a common mode of action, i. e. inhibition of chlorophyll biosynthesis. A molecular target of these herbicides is protoporphyrinogen-IX oxidase (Protox). Corn is widely used for Protox inhibitory assays by peroxidizing herbicides because the plant enzyme has high and stable activity. The Protox isolated from one corn variety, Anjou, has been used for the assay in our laboratory and several others. This variety can no longer be used however, because of its reduced tolerance against corn diseases. Therefore, we collected twelve corn varieties and compared their Protox activities and sensitivities against peroxidizing herbicides. Among the tested varieties, DK212MF exhibited the highest activity. This variety has approximately two times higher activity than Anjou. Tolerance of Anjou and DK212MF to peroxidizing herbicides was evaluated by a pot test, because the varieties have quite different Protox activities. DK212MF was less sensitive to the peroxidizing herbicides oxyfluorfen and chlorophthalim than Anjou. Furthermore, DK212MF accumulated less Proto-IX than Anjou, which was believed to be one of the causes of its lower sensitivity in vivo to the peroxidizing herbicides.

Enzyme activity of protoporphyrinogen-IX oxidase (Protox) prepared from 34 kinds of plant sources, containing 18 monocotyledons and 16 dicotyledons was evaluated. Protox(es) originated in monocotyledonous plants such as Zea mays cv. Anjou and DK212, Lolium perenne and Poa annua exhibited high enzyme activity, and Protox(es) in dicotyledonous plants such as Agrostemma githago and Arabidopsis thaliana showed a little higher enzyme activity than that of the above monocotyledonous plants. Highly active Protox(es) obtained from Zea mays cv. Anjou, Lolium perenne, Agrostemma githago and Arabidopsis thaliana were selected for the Protox inhibitory assay of peroxidizing herbicides. Echinochloa utilis was also selected for control. Protox inhibition by six peroxidizing herbicides such as oxyfluorfen, chlorophthalim, BW-91, pyraflufen-ethyl, DLH-1777 and LS-82556 was investigated using highly active Protox from the above monocotyledons and dicotyledons. As a result, six peroxidizing herbicides exhibited high inhibitory activity to Protox. Pyraflufen-ethyl showed the highest inhibition. The order of Protox inhibitory activity was pyraflufen-ethyl > oxyfluorfen > BW-91 > chlorophthalim > DLH-1777 > LS-82556, in experiment using four Protox(es) such as Zea mays cv. Anjou, Echinochloa utilis, Agrostemma githago and Arabidopsis thaliana except Lolium perenne. Oxyfluorfen exhibited the highest inhibitory activity to Protox of Lolium perenne. On the other hand, DLH-1777 showed a little less activity to Protox of Zea mays than that of other four weeds. DLH-1777 may become a model compound to find out new selective herbicides. Protox from four weeds will be used for peroxidizing herbicides assay, since the Protox showed high sensitivity to structually different peroxidizers.

The effects of nine novel 2-benzylamino-1,3,5-triazines on photosynthetic reactions were measured in thylakoids isolated from wild-type and atrazine-resistant plants of Chenopodium album. The resistant plants have a mutation of serine for glycine at position 264 of the D1 protein. The measurement of oxygen evolution and chlorophyll a fluorescence induction indicated a 2–4-fold stronger inhibition by the 6-trifluoromethyl analogues of Photosystem II-dependent electron flow than atrazine. Analogues having a 6-methyl-, 6-monofluoromethyl or 6-difluoromethyl substitution were weak inhibitors, indicating that the 6-trifluoro group is very important for strong inhibition. All the nine novel 2-benzylamino-1,3,5-triazines were almost as active in wild-type as in atrazine-resistant thylakoids, indicating that the benzylamino substitution may be important for the lack of resistance in the atrazine-resistant plants.

高いPET阻害活性を有する2-ベンジルアミノ-4-メチル-6-トリフルオロメチル-1, 3, 5-トリアジン系化合物において, フッ素原子の置換効果を調べるために, トリフルオロメチル基をジフルオロメチル基, フルオロメチル基, メチル基に変換した化合物を合成し, それら化合物のPET阻害活性を測定した. その結果, PET阻害活性は, フッ素原子数の増加とともに増大することが分かり, 全体的には活性の強さはCF3-≧CHF2->CH2F->CH3-誘導体の順であった. 高いPET阻害活性を附与させるためにはトリアジン環の置換基の1つとして2個以上のフッ素原子で置換されたメチル基すなわち, トリフルオロメチル基またはジフルオロメチル基を導入する必要性が確認された. また, ベンジルアミノ基のベンゼン環4位にハロゲン原子を導入した化合物は無置換の化合物よりも高いPET阻害活性を示した. これらの化合物の除草活性を土壌, 茎葉, 湛水処理試験によって評価したところ, PET阻害活性の高い化合物が強い除草活性を示した. 例えば, 2-(4-ブロモベンジルアミノ)-4-ジフルオロメチル-6-メチル-1, 3, 5-トリアジンは, 非常に強いPET阻害活性及び除草活性を示した.

New fluoroalkyl-substituted 1,3,5-triazine derivatives were synthesized and screened for herbicidal activity using a greenhouse pot test. Surprisingly, a series of 2-alkyl-4-fluoroalkyl-6-aralkylamino-1,3,5-triazines e.g. 6-(4-bromo-benzylamino)-2-methyl-4-trifluoromethyl-1,3,5-triazine was found to possess strong pre- and post-emergence herbicidal activities, although the conventional herbicidal 1,3,5-triazines generally should have a 2-substituted-4,6-diamino-1,3,5-triazine structure for herbicidal activity. Our compounds show strong Photosynthetic Electron Transport inhibitory activity (PI 50 c 7). Although their herbicidal effect is considered to be caused by a process similar to that for the conventional 1,3,5-triazine herbicide atrazine, they can control atrazine-resistant Chenopodium album effectively, and will thus form promising trial compounds for new triazine herbicide design.

2-アラルキルアミノ-1,3,5-トリアジン化合物のPET阻害活性を測定し, 構造と活性との相関関係について検討した. その結果, トリアジン環の4,6位にメチル基とトリフルオロメチル基を有する2-ベンジルアミノ-1,3,5-トリアジンが高いPET阻害活性を示した. また, ベンジル位に不斉炭素を有するα-メチルベンジルアミノ-1,3,5-トリアジン誘導体において, (-)-エナンチオマーは(+)-エナンチオマーより100倍高い活性を示した. 更にベンゼン環上の置換基を検討したところ, 3または4位にハロゲン原子またはトリフルオロメチル基を導入した化合物はより高い活性を示した. それらの化合物はアトラジンやシマジンやより高い活性を示した.

Protoporphyrinogen-IX oxidase inhibition and peroxidizing phytotoxicity were determined using a series of fluorinated and non-fluorinated alkyl 2-chloro-5-(3,4,5,6-tetrahydrophthalimido)benzoat and their parent benzoic acid derivatives. Protoporphyrinogen-IX oxidase from corn etioplasts was extracted, and ethane formation by intact cells of the green algae Scenedesmus acutus was measured. The benzoates with a fluorine atom exhibited stronger peroxidizing phytotoxicity (higher ethane formation) than the benzoates having no fluorine atom although protoporphyrinogen-IX oxidase inhibition by the fluorinated analogues was found to be weaker or almost equal to the non-fluorinated analogues. These data strengthen and generalize our previous findings with cyclic imides having an N-(4-chloro-2-fluoro-5-carboxyphenyl) group. The fluorinated analogues, which are not necessarily stronger protoporphy-rinogen-lX oxidase inhibitors, exhibit stronger peroxidizing phytotoxicity with intact plant cells than those without a fluorine atom.

新規な2-ベンジルアミノ-1,3,5-トリアジン系化合物を合成し, 除草活性を評価した. 構造と活性との相関関係について検討したところ, トリアジン環上の4位にメチル基, 6位にトリフルオロメチル基を導入した2-ベンジルアミノ-1,3,5-トリアジン化合物が高い除草活性を示した. 更にベンゼン環上の置換基を検討したところ, 3または4位にハロゲン原子を導入した化合物はより高い除草活性を示した. ポット試験において高い除草活性を示した2-(3-クロロベンジルアミノ)-4-メチル-6-トリフルオロメチル-1,3,5-トリアジンと2-(4-クロロベンジルアミノ)-4-メチル-6-トリフルオロメチル-1,3,5-トリアジンはシマジンやアトラジンより高いPET阻害活性を示した.

GSTは還元型GSHの存在下でN-(4-ブロモフェニル)-3,4,5,6-テトラヒドロイソフタルイミド (1) をN-(4-ブロモフェニル)-3,4,5,6-テトラヒドロフタルイミド (2) に, また, 5-(4-ブロモフェニルイミノ)-3,4-テトラメチレン-1,3,4-チアジアゾリジン-2-オン (3) を異性体であるトリアゾリジン型 (4) に異性化した. しかしながら, 5-(4-ブロモフェニルイミノ)-3,4-テトラメチレン-1,3,4-チアジアゾリジン-2-チオン (5) は異性化されにくかった. (1) と (3) は異性化後にプロトポルフィリノーゲンオキシデースをより強く阻害した. GSTは一般的に農薬の毒物学においては解毒化酵素として知られているが, 上記のような異性化による活性化はGSTとGSHの新しい機能である.

1,3,5-トリアジンのハロメチル置換基が硝化細菌の硝酸化成作用に及ぼす影響について調べることを目的として, 26種のハロメチル-1,3,5-トリアジン系化合物を用いて試験を行なった. 1,3,5-トリアジン系化合物の硝酸化成抑制効果は二つの実験方法によって測定した. 畑土壌を用いたアンモニアから硝酸 (NO3--N) への酸化と, Nitrosomonas europaea ATCC 25978の細胞懸濁液を用いたアンモニアから亜硝酸 (NO2--N) への酸化を測定した. その結果, トリクロルメチル基を有する化合物は畑土壌と菌体の両方において, 部分的に塩素化されたメチル基 (CH2ClおよびCHCl2) をもつ化合物よりも強い硝酸化成抑制効果を示すことが判明した. トリブロモメチル基をもつ1,3,5-トリアジン系化合物は N. europaea ATCC 25978の細胞懸濁液では非常に強い抑制効果を示したが, 土壌試験においては弱い抑制効果しか示さなかった. また, N. europaea ATCC 25978を用いた試験では基質として (NH4)2SO4を用いた場合には抑制効果がみられたが, NH2OH・HClを用いたときにはみられなかった. したがって, 検討したすべてのハロメチル-1,3,5-トリアジン系化合物の作用点はアンモニアからヒドロキシルアミンへの酸化過程にあると推測された.

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.

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.

Confocal fluorescence microscopic images were used to investigate the accumulation site of protoporphyrin IX (PPIX) within liverwort cells (Marchantia polymorpha L.) treated with peroxidizing herbicides as acifluorfen methyl (AFM). A high level of PPIX accumulation was observed in the cells during 12 h to 24 h after the addition of AFM . The results obtained from confocal fluorescence microscopic images gave clear evidence that the accumulation of PPIX occurred only in the chloroplasts, but was not observed in the cytosol or at the plasma membrane. The presence of PPIX in the chloroplasts strongly suggests that protoporphyrino­ gen (Protogen) accumulates by inhibition of protoporphyrinogen oxidase (Protox) which is the target enzyme for peroxidizing herbicides. The plastidic occurrence of PPIX provides evidence of either the presence of an additional herbicide-resistant Protox or of a non-enzy-matic Protogen-oxidation system in the Marchantia chloroplast.

The benzoxazine derivatives are a new chemical family of diphenyl ether herbicides, which exhibit a strong peroxidizing herbicidal activity on mono- and dicotyledonous species in preemergence and postemergence tests. Twenty derivatives were synthesized, and their herbicidal activity was determined to examine structure-activity relationships. Among the compounds investigated, it was found that the fluorine atom introduction into the trifluoromethylbenzene moiety together with an oxazine ring instead of a nitro group led to the most active herbicide.

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)].

18種の5-アリールイミノ-3,4-テトラメチレン-1,3,4-チアジアゾリジン-2-チオンと4-アリール-1,2-テトラメチレン-1,2,4-トリアゾリジン-3,5-ジチオン系化合物を合成し, それらの植物毒性を Scenedesmus acutus と Echinochloa utilis を用いて検討した. これらの化合物は, Echinochloa の根伸長阻害を示し, また光照射下で Scenedesmus 細胞中で, protoporphyrin-IXを蓄積し, ethane を発生し, chlorophyll を減少させ, 細胞の生育を阻害した. すなわち, 両者は chlorophyll 生合成経路中の protoporphyrinogen-IX oxidase を阻害し, その結果蓄積する物質が関与して生じる酸素ラジカルによって, チラコイド膜を破壊して ethane を発生する作用機構 (peroxidizing mechanism) をもつと考えられる. 両者の間では, 5-アリールイミノ-3,4-テトラメチレン-1,3,4-チアジアゾリジン-2-チオンの植物毒性が, 4-アリール-1,2-テトラメチレン-1,2,4-トリアゾリジン-3,5-ジチオンに比べて高かった.

1,2,4-Triazolo[1,5-a]-1,3,5-triazine 系化合物は, 報告例の少ない, 合成困難な化合物である. したがって, それら化合物の植物毒性等に関する報告も少ない. 本研究は1,2,4-triazolo[1,5-a]-1,3,5-triazine 系化合物の新しい合成法の開発と植物毒性の検定を目的としたものである. 新しい合成法は三段階で行なわれる. まず, 容易に入手できる benzylideneaminoguanidines (I) と N-cyanoformimidate (II) を室温下に反応させ, 鎖状中間体 1-benzylideneamino-2-cyanoiminomethyl guanidines (III) を得る. 次に, この中間体を熱メタノールで閉環させ, 1,2-dihydro-1,2,4-triazolo[1,5-a]-1,3,5-triazines (IV) を得る. この化合物 (IV) をI2で酸化すると最終目的物である1,2,4-triazolo[1,5-a]-1,3,5-triazines (V) が得られる. このようにして合成した, 1,2,4-triazolo[1,5-a]-1,3,5-triazine 誘導体 (IVおよびV), および中間体の 1-benzylideneamino-2-cyanoiminomethyl guanidines (III) の植物毒性を緑藻類 Scenedesmus acutus を用いて検定したところ除草剤等として使用するには必ずしも十分ではないものの, 7-amino-2-(4-chloro-2-fluorophenyl)-1,2,4-triazolo[1,5-a]-1,3,5-tliazine は生長阻害効果とクロロフィル減少効果を示した.

Certain 5-(arylimino)-3,4-tetramethylene-1,3,4-thiadiazolidin-2-ones (thiadiazolidines) are peroxidizing bleaching herbicides which interrupt chlorophyll biosynthesis, inhibit the activity of protoporphyrinogen oxidase, lead to accumulation of protoporphyrin IX, and induce ethane formation in the light. The same effects are caused by their isomers, the 4-aryl-1,2-tetramethylene-l,2,4-triazolidin-3-one-5-thiones (triazolidines). Couples of thiadiazolidines and corresponding triazolidine isomers were synthesized. Thiadiazolidines with a 4-bromophenylimino, 4-chlorophenylimino, 4-chloro-2-methylphenylimino, 4-chloro- 2-fluorophenylimino, 4-chloro-2-fluoro-5-propargyloxyphenylimino and 4-chloro-2-fluoro- 5-isopropoxyphenylimino moiety were converted to the corresponding triazolidines both with Echinochloa seedlings or a spinach homogenate present, depending on the 5-arylimino moiety. The 5-[4-(chlorobenzyloxy)phenylimino]-3,4-tetramethylene-1,3,4-thiadiazolidin-2- one analogue did not convert to the corresponding triazolidine under both conditions. Thiadiazolidines as well as triazolidines having a 4-chloro-2-fluoro-5-methoxycarbonylmethylthiophenyl moiety were converted to an unidentified compound whose structure is assumed to be 4-(4-chloro-2-fluoro-5-carboxymethylthiophenyl)-1,2-tetramethylene-1,2,4- triazolidin-3-one-5-thione. Apparently, the general conversion mechanism is caused by enzymatic hydrolysis of thiadiazolidines to an unstable intermediate which rapidly and spontaneously changes to the corresponding triazolidine isomer.

本研究では, 強い硝酸化成抑制作用を示す1, 3, 5-triazine 系化合物の N. europaea ATCC 25978株の無細胞抽出渡に対する影響を硝化作用に伴うと予測される溶存酸素の消費量を酸素電極を用いて測定することにより評価した. また, その結果をすでに報告されている細胞系試験の結果と比較した. 細胞の破砕はフレンチプレスを用いて行った. その他にも凍結融解法や超音波破砕法を試みたが N. europaea ATCC 25978株の細胞破砕には適さなかった. N. europaea ATCC 25978株無細胞抽出液の1, 3, 5-triazine系化合物の硝酸化成抑制効果は N. europaea ATCC 25978株細胞系に対するものと同等以上であった. さらに, その無細胞抽出液中の溶存酸素の減少はアンモニア態窒素を加えた後にすぐ開始した. 酸素消費の阻害が認められたことから, 1, 3, 5-triazine 系化合物はアンモニアモノオキシゲナーゼを阻害したと考えられる。また, 無細胞系において溶存酸素を測定することによって硝酸化成抑制活性試験が可能であり, 膜物質による影響を受けやすい化合物についても膜透過を必要としないことからより正確に硝化作用抑制活性を測定できると考えられる。

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.

トリクロロメチル-1,3,5-トリアジン類の畑土壌における硝酸化成抑制作用を調べることを目的として種々の1,3,5-トリアジン系化合物を合成し, 硝酸化成抑制効果 (pI50) を試験した. 検討したすべてのトリクロロメチル-1,3,5-トリアジン類は土壌中のアンモニア態窒素の硝酸化を遅らせた. 硝酸化過程の中間体であるヒドロキシルアミンおよび亜硝酸態窒素の蓄積が見られないことは, トリクロロメチル-1,3,5-トリアジンがヒドロキシルアミンの生成過程を主に阻害していると考えられた. pI50値と疎水性パラメータ (log P) との間には良好な関係が認められた. 硝酸化成抑制効果はトリアジン環上に置換基としてトリクロロメチルとアミノ基が共存している化合物が好ましいことがわかった.

18種のチアジアゾリジンとトリアゾリジンを合成し, それらの植物毒性を Echinochloa utilis, Scenedesmus acutus を用いて検討した. これら化合物は既知の peroxidizing 除草剤と同様に, 光照射下で Scenedesmus においてその成育を阻害し, クロロフィル含量を減少させプロトポルフィリン-IXを蓄積し, エタンを発生した. また, 光照射下で Echinochloa の根伸長を抑制した. 以上の結果から, 両化合物群は peroxidizing 除草活性を示すことが判明した. すなわち, 両者はクロロフィル生合成系の酵素活性を阻害し, その結果蓄積する物質が関与して生じる酸素ラジカルによってチラコイド膜が破壊されてエタンを発生する作用機構をもつと考えられる. また, チアジアゾリジンとトリアゾリジンの間で活性が近かったことから, チアジアゾリジンの活性は, チアジアゾリジンからトリアゾリジンへの異性化に基づくものと考えられる.

Cell suspension cultures of the liverwort, Marchantia polymorpha L. were found useful to study the influence of peroxidizing herbicides either on the greening process or on the fully green cells. The cells of both physiological stages exhibit a characteristic sensitivity to the herbicides. The sensitivity increased rapidly during the exponential phase of growth, reached a maximum during the late exponential phase, and then decreased in the stationary phase. We investigated the kinetics of accumulation of protoporphyrin IX (PPIX) in Marchantia cells treated with several peroxidizing herbicides at various stages of cell growth, and observed a correlation between accumulation of PPIX and herbicidal damage. The glutathione (GSH) content in the cell was also investigated to examine the role of GSH against herbicide treatment. In the light, GSH levels in the cells treated with AFM rose rapidly reaching a peak after 8 h, and rapidly decreased subsequently. The beginning of PPIX accumulation coincided with the decline of GSH after 8 h of treatment. Obviously, GSH plays a key role in protection against oxidative damage caused by AFM in the early treatment period. In the dark, AFM also induced an accumulation of GSH and PPIX, followed by a decline in GSH and PPIX contents during a 20 h incubation. The decline of PPIX was observed several hours after GSH starts to decrease, remaining at a constant level for the following 40 h, leading to accumulation of an other fluorescent still-unknown pigment.

Using Scenedesmus , a series of peroxidative cyclic imides was assayed with respect to degradation of chlorophyll (bleaching), light-induced ethane formation, growth inhibition, and protoporphyrin IX (proto IX) levels. The p I 50 values of the first two parameters, comprising a range of about four decimal powers of inhibitory activity, were quantitatively correlated with protoporphyrin IX formation by regression equations. Applying constant concentrations (0.3 and 1 μ M ) of each compound a correlation could be established with the proto IX level attained after a short-term (1-hr) incubation under moderate light intensity. Long-term treatment of the cells with cyclic imides (4 hr and more) produced excess protoporphyrin, apparently not needed for the phytotoxic effects. Including our previous data, proto IX formation could also be quantitatively linked to the inhibition of root tip growth of Echinochloa (sawa millet) by cyclic imides. All these cell parameters, obtained with green algae or higher plants, are correlated and can be taken as reliable means to quantitate the phytotoxic activity of peroxidizing herbicides.

In order to explain the close phytotoxic activities of N-aryl-3,4,5,6-tetrahydrophthalimides and their hydrolyzed products, N-aryl-3,4,5,6-tetrahydrophthalamic acids, five sets of both types of compounds possessing the same aryl residues were prepared. Their phytotoxic activity against sawa millett (E. utitts) and green microalga (S. acutus), and the relationship of interconversion between the imides and amide acids during the bioassay were investigated. In almost all cases, the imides showed stronger activity than the corresponding amide acids. The hydrolysis of the imides and the cyclization of amide acids were observed in respect of the aryl residues. The phytotoxicity caused by the imides and amide acids tested was influenced by this interconversion.

The effects of Photosystem II inhibiting herbicides, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), atrazine and two novel 2-benzylamino-1,3,5-triazine compounds, on photosynthetic oxygen evolution and chlorophyll a fluorescence induction were measured in thylakoids isolated from Chenopodium album (wild type and atrazine-resistant plants) and cyanobacterial intact cells. The resistant plants have a mutation of serine for glycine at position 264 of the D1 protein. Diuron and two members of a novel class of 2-benzylamino-1,3,5-triazine compounds were almost as active in wild-type as in atrazine-resistant thylakoids, indicating that the benzylamino substitution in the novel triazines may be important for the lack of resistance in these atrazine-resistant plants. The inhibition by the herbicides of oxygen evolution in the cyanobacteria was somewhat lower than in the thylakoids of Chenopodium album wild type, probably caused by a slower uptake in the intact cells. The so-called OJIP fluorescence induction curve was measured during a one second light pulse in the absence and in the presence of high concentrations of the four herbicides. In the presence of a herbicide we observed an increase of the initial fluorescence at the origin (Fo'), a higher J level, and a decreased steady state at its P level (Fp). The increase to Fo' and the decreased leveling Fp are discussed. After dark adaptation about 25% of the reaction centers are in the S(0) state of the oxygen evolving complex with an electron on the secondary electron accepting quinone, Q(B). The addition of a herbicide causes a transfer of the electron on Q(B) to the primary quinone acceptor, Q(A), and displacement of Q(B) by the herbicide; the reduced Q(A) leads to a higher Fo'. The decrease of Fp in the presence of the herbicides is suggested to be caused by inhibition of the photo-electrochemical stimulation of the fluorescence yield.