Insights into the Efficacy and Binding Mode of 1,4-Disubstituted 1,2,3-Triazoles─A New Class of Agricultural Nitrification Inhibitors

Abstract

Recently, 1,4-disubstituted 1,2,3-triazoles were reported by us as a new class of nitrification inhibitors, which can outperform the commercial compound 3,4-dimethylpyrazole phosphate (DMPP) in soil incubations. In this work, the mechanism of inhibition of five 1,2,3-triazoles with different substitution patterns was explored using a bacterial assay based on the measurement of nitrite (NO 2 − ) production by pure cell cultures of Nitrosomonas europaea and Nitrosospira multiformis. While polar functional groups, such as amines, esters, and alkoxy residues, were detrimental to inhibiting production of NO 2 − , triazoles carrying only aliphatic substituents showed the highest inhibition of up to 98%. The observed correlation between lipophilicity and inhibitory activity suggests that more lipophilic compounds could more easily access the membrane-bound ammonia monooxygenase (AMO), which catalyzes the first step of the nitrification process. Measurement of the Michaelis−Menten kinetics suggests that the disubstituted 1,2,3-triazoles studied in this work act as reversible, noncompetitive inhibitors. Real-time measurements of the oxygen (O 2 ) consumption showed that the O 2 uptake rate by AMO follows zero-order kinetics in the presence of the triazoles, confirming the nonmechanistic mode of inhibition.

4-Methyl-1-(prop-2-yn-1-yl)-1H-1,2,3-triazole (MPT): A Novel, Highly Efficient Nitrification Inhibitor for Agricultural Applications

“…The cells were subsequently washed thoroughly with NaPB to remove any unbound and bound inhibitor compound, followed by reincubation with NH 4 + for 30 min and measurement of the NO 2 – production. Reversible binding of the NI to AMO would be expected to result in a recovery of the NO 2 – production activity after washing and reincubation, approximately to the level of the uninhibited cells, as we have recently found for DMP, DCD, and a series of 1,4-disubstituted 1,2,3-triazoles. , Control experiments in the absence of inhibitor confirmed that the washing and reincubation protocol did not impact (within error) the NO 2 – production rate of both bacterial strains.…”

“…europaea and N. multiformis, the standard activity and activity recovery assays, the measurements of the O 2 consumption, Michaelis–Menten kinetics, and acute toxicity tests were conducted as previously reported. ,, All bacterial incubations were performed in sodium phosphate buffer (NaPB) with 1% (v/v) DMSO at pH = 7.5 and 30 °C with different inhibitors and concentrations, as indicated. This amount of DMSO, which was required to solubilize the lipophilic inhibitor compounds, is not detrimental to the bacterial cells .…”

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