Study On Site Of Action (AHAS) And Microsomal Metabolism Of Pyribambenz-propyl

As a novel class of herbicide precursor, 2-pyrimidinyloxy-N-aryl-benzylamine derivatives, which showed potent herbicidal activities, were invented in the early 2000s in China with independent intellectual property rights. One of these compounds, pyribambenz-propyl was successfully developed as a potent herbicide for rape field. Few studies, however, have been conducted on the herbicidal form(s), the site of action and selectivity of the herbicide both at home and abroad. For the purpose to understand the mode of action of pyribambenz-propyl, it is critical to clarify the target site inhibition by herbicidal form(s) and to elucidate the metabolism of the herbicide in plant.The present study was carried out to address two major issues: 1) AHAS inhibition by pyribambenz-propyl and tentative metabolites artificially synthesized; and 2) microsomal metabolism of pyribambenz-propyl.Major results obtained are as follows:1. Procedures were established both for the partial purification and for assay of AHAS. The highest specific activity and recovery of the enzyme were obtained by 15-40% AS fractionation among three different concentration regimes of ammonium sulfate (AS saturation concentration, 25%-50%, 5-15-40% and 15-40%). PEG (w/w, 5%-15%) fractionation gave similar results as 15-40% AS. For consistency and convenience, 30 min and 37℃were selected for AHAS assay.2. During isolations of AHAS from various plant sources, we noticed that the feedback inhibition of AHAS activities by branched-chain amino acids displayed an interesting phenomenon: leaf AHAS was always inhibited to a much greater extent than those of root AHAS. The possibility of partial proteolysis of AHAS as the cause for changes of AHAS sensitivity against branched-chain amino acids was tested by inclusion of protease inhibitor DPDS during enzyme isolation from Zea mays. Nonetheless, preliminary results excluded enzyme proteolysis as the major cause for the differential feedback inhibition from leaves and roots; rather such difference was presumably resulted from the presence in roots of pyruvate decarboxylase activities, which interfered with the AHAS assay.3. With AHAS from leaves of Brassica napus and Zea mays, inhibition experiment by pyribambenz-propyl and its tentative metobolites artificially synthesized showed that only the compound A, possessing "pyrimidinyl-oxy -salicylate " , a core herbicidal structure of pyrimidinyl salicylate herbicides, strongly inhibited AHAS activity while all seven other compounds including pyribambenz-propyl and its rearrangement product showed little effect. Therefore, the experiments confirmed previous suggestion that pyribambenz-propyl was a pro-herbicide. The results would also suggest that pyribambenz-propyl might exert its herbicidal effects by first transforming to similar structures as those of pyrimidinyl salicylate herbicides.4. Root length experiments with ten cultivars of wheat showed that with 20μM pyribambenz-propyl, the largest inhibition of root growth occurred in By535 by 62.4% while those of Linmai 2# and Taishan 23# were inhibited by 38.7% and 40.7%, respectively.5. In vitro metabolism of [C ring-U-¹⁴C] pyribambenz-propyl was studied with microsomes isolated from etiolated wheat seedlings pretreated with 5% (v/v) ethanol and 10 mM pyribambenz-propyl. Labeled fractions of reaction products were first separated and determined by HPLC-LSC and then three major putative metabolites (Y1, Y2 and Y3) were identified by HPLC-MS. Analysis of the molecular structures of these products in respect to that of pyribambenz-propyl suggested that these products were likely all related to Smiles rearrangement. The study indicated that pyribambenz-propyl underwent hydroxylation in wheat microsomes and the in vitro system might also facilitate isomerization of the herbicide. Further studies are required to clarify whether rearrangement reactions of pyribambenz-propyl are ready to occur within plant cells and whether these Smiles rearrangement-related metabolites are involved in herbicidal activities and /or detoxification of the (pro-) herbicide.