Nitrification inhibitor

Abstract

A compound of formula (I) when used to inhibit nitrification: wherein: R1 is CH2C = CH; r2 is methyl; and R3 is H. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to compounds that inhibit nitration and methods for inhibiting nitration. Background Art

[0002] This application claims priority to Australian Provisional Application No. 2022902533, filed on September 2, 2022, and entitled “Nitrification Inhibitors”. The entire contents of this application are incorporated herein by reference.

[0003] Nitrification is the main cause of nitrogen loss in agriculture. While nitrogen is a nutrient for plants, it is also a nutrient for soil microorganisms. When nitrogen fertilizer is applied to the soil, only a small portion ends up in the plants. The vast majority of the nitrogen fertilizer is taken up by soil microorganisms, which convert the nitrogen into more oxidized species that are less readily absorbed by plants, such as nitrite (NO2 - ), nitric oxide and nitrate (NO3 - ). In addition, the nitrification-denitrification process produces nitrous oxide (N2O). N2O is a greenhouse gas with a global warming potential about 300 times greater than carbon dioxide (CO2). Currently, agriculture generates about 90% of the global N2O.

[0004] Because soil microorganisms chemically transform nitrogen fertilizers, which compete with plant uptake, farmers are forced to introduce more and more of these fertilizers to increase crop yields. This practice perpetuates a vicious cycle of nitrogen fertilizer application and nitrogen loss to the environment.

[0005] Several nitrification inhibitors (NIs) are already commercially available. It is believed that these NIs bind to the key (initial) enzyme ammonia monooxygenase (AMO), which is conserved in bacteria and archaea and catalyzes the first step of nitrification. The structure of this membrane-bound copper-containing metallooxidase is unknown, as attempts to crystallize this enzyme have so far been unsuccessful. Existing NIs, such as the commercial compounds 3,4-dimethylpyrazole phosphate (DMPP), dicyandiamide (DCD) and the recently discovered 1,4-substituted 1,2,3-triazoles, bind reversibly to the AMO. Therefore, the activity of these NIs is only transient.

[0006] Therefore, a more permanent method than current solutions is needed to prevent nitrification activity of soil microorganisms. Summary of the invention

[0007] In a first aspect of the present invention, there is provided a compound of formula (I), which, when used to inhibit nitration:

[0008]

[0009] in:

[0010] R 1 is CH2C≡CA, wherein A is H, methyl or ethyl;

[0011] R 2 C n H 2n+1 ;

[0012] R 3 C m H2 m+1 ;

[0013] And wherein n and m+n are each independently 1, 2, 3, 4, 5 or 6 and m is 0, 1, 2, 3, 4 or 5.

[0014] The following options may be used in conjunction with the first aspect, either individually or in any suitable combination.

[0015] A can be C x H 2x+1 , wherein x is 0, 1 or 2. It can be H, methyl or ethyl. It can be H. n can be 1. m can be 0.

[0016] The compound can bind irreversibly to the nitrifying enzyme. It can irreversibly inactivate nitrifying microorganisms.

[0017] The compound may increase the nitrification activity of N. europaea or N. multiformis by less than 10% from step (ii) to step (iv) in the following test:

[0018] i. adding ammonium sulfate and the compound to the nitrifying microbial cells in sodium phosphate buffer (NaPB),

[0019] For example, N. europaea or N. multiformis cells:

[0020] ii. Measurement of NO2 via Griess assay - produce;

[0021] iii. The cells are then washed with buffer and ammonium sulfate is added;

[0022] iv. Measure NO2 again using the Griess assay - produce.

[0023] In one embodiment, a compound of formula (I) is provided, which, when used to inhibit nitration:

[0024]

[0025] in:

[0026] R 1 CH2C≡CH;

[0027] R 2 is CH3;

[0028] R 3 is H;

[0029] A second aspect of the present invention provides a composition which, when used to inhibit nitrification in soil, comprises a compound of formula (I) as defined in the first aspect and at least one agriculturally acceptable adjuvant or diluent. The composition may also include a urease inhibitor.

[0030] The third aspect of the present invention provides a fertilizer composition, which comprises a fertilizer and a compound of formula (I) as defined in the first aspect. The fertilizer may be a urea-based fertilizer or may be an ammonium-based fertilizer. It may also include a urease inhibitor.

[0031] A fourth aspect of the present invention provides a method for reducing nitrification in soil, comprising treating the soil with a compound of formula (I) as defined in the first aspect or a composition as defined in the second aspect.

[0032] The following options may be used in conjunction with the fourth aspect, either alone or in any suitable combination.

[0033] The soil can be acidic soil. It can be slightly acidic soil. It can be non-acidic soil. It can be slightly alkaline. It can be alkaline. It can be neutral. Treatments can be repeated no more than once in any 2-week period.

[0034] In a fifth aspect of the present invention, there is provided a method for inhibiting the nitrifying activity of a nitrifying microorganism (e.g. N. europaea or N. multiformis), comprising exposing the microorganism to a compound of formula (I) as defined in the first aspect or a composition as defined in the second aspect. The nitrifying microorganism may be a nitrifying bacterium.

[0035] In a sixth aspect of the invention, there is provided a method for inactivating a nitrating enzyme, comprising exposing the enzyme to a compound of formula (I) as defined in the first aspect or a composition as defined in the second aspect. The nitrating enzyme may be an ammonia monooxygenase (AMO) or may be a hydroxylamine oxidoreductase (HAO). In one embodiment, the enzyme is an AMO.

[0036] In the seventh aspect of the present invention, there is provided the use of a compound of formula (I) as defined in the first aspect for reducing nitrification in soil, or for inhibiting the nitrification activity of nitrifying microorganisms (e.g. nitrifying bacteria, such as N. europaea or N. multiformis) or for inactivating nitrifying enzymes.

[0037] In an eighth aspect of the present invention, there is provided a composition for reducing nitrification in soil, or for inhibiting the nitrification activity of nitrifying microorganisms, or for inactivating nitrifying enzymes using the compound of formula (I) as defined in the first aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Certain embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0039] Figure 1 The effect of various functional groups on nitrification inhibition is shown, determined using N. europaea (NE) and N. multiformis (NM). NO2 in ammonia oxidizing bacteria (AOB) was determined following the "Standard Nitrification Inhibition Assay Protocol" described herein. - Production. Incubation was carried out in NaPB with 1 v / v% DMSO at pH = 7.5 and 30°C, where [NH4 + ]=3mM, [inhibitor]=0.3mM (10mol% [NH4 + ]), incubated at 100 rpm in the dark for 60 min. Standard error was determined by three biological and three technical replicates. No inhibition was indicated as NI.

[0040] Figure 2 The results of enzyme activity recovery assays using N. europaea are shown. I) 30 min + Inh means treatment with inhibitor (1.5 mM, 50 mol % NH4 + ) for 30 min compared to AOB treated with uninhibited cells for 30 min. II) "30 min-Inh" shows the percentage activity after a wash step and with (NH4 + )2SO4 re-incubation of AOB. Incubation was performed in NaPB at pH = 7.5 and 30°C with 1 v / v% DMSO, where [NH4 + ]=3mM, [inhibitor]=1.5mM (50mol% [NH4 + ]), incubated at 100 rpm for 60 min in the dark. Standard errors were determined by three biological and three technical replicates.

[0041] Figure 3Results of oxygen (O2) consumption experiments using N. europaea are shown. The trajectory describing the change in O2 concentration over time after adding (NH4)2SO4 (initial 300-600 seconds) was used as the baseline for uninhibited cells, while the consumption trajectory describing the presence of inhibitors (605-1800 seconds time span) was used to determine the rate of the inhibitors. The dotted line represents the predicted [O2] in the absence of inhibitors. All experiments were performed three times at 20°C and under constant stirring.

[0042] Figure 4 a) "Inhibition pattern" and b) "dose response curve" using N. europaea are shown. Incubation was performed in NaPB with 1 v / v% DMSO at pH = 7.5 and 30°C, with different concentrations of NH4 + and inhibitors and incubated at 100 rpm for 60 min in the dark. Standard errors were determined from three biological and three technical replicates.

[0043] Figure 5 The results show that (1) fertilizer ((NH4)2SO4,) 100 mg kg -1 Soil incubation studies were performed with (1) fertilizer + 0.5 mol% 4-MPT (4-methyl-1-(prop-2-yn-yl)-1H-1,2,3-triazole) or (3) fertilizer + 2.5 mol% 4-MPT, (4) fertilizer + 5 mol% 4-MPT, (5) fertilizer + 5 mol% Compound 1 of applied fertilizer-N. Standard errors were determined from three biological replicates.

[0044] Figure 6 It shows that NO2 - Produced with NH4 + The concentrations of 50 μM and 10 μM were plotted to determine the inhibition pattern using Michaelis-Menten kinetics.

[0045] Figure 7 The N2O production rates in soils B to E over 21 days are shown. (NH4)2SO4 at 50 mg kg -1 Soil "fertilizer only" application. [DMP] and [4-MPT] accounted for 0.5 mol%, 2.5 mol%, and 5 mol% of the applied fertilizer-N, respectively. Soil E: Note the different axis scale to include higher N2O production rates; for 5 mol% DMP (3,4-dimethylpyrazole) and all 4-MPT treatments, no N2O production was detected after day 5. Data are calculated from three biological replicates.

[0046] definition

[0047] As used herein, the term "comprising" means "including". Variations of the word "comprising" (e.g., "comprise" and "comprises") have correspondingly different meanings. As used herein, the terms "comprising" and "including" are non-exclusive and allow for the presence of other integers, optionally unspecified. As used herein, the terms "comprising" and "including" do not mean that the specified integer represents a major portion of the whole. The term "consisting essentially of means that the specified integer is the only integer that is intentionally present, although other integers (usually minor) may be present incidentally. The term "consisting of means that the specified integer is the only integer that is present.

[0048] The limitation of the meaning of general terms is not affected by specific examples introduced by "include", "includes", "including", "for example", "in particular", "such as" or similar expressions.

[0049] When defining any word or phrase, any other part of speech or other grammatical form of that word or phrase has a cognate meaning.

[0050] When an option is provided, its antonym is also explicitly contemplated. Thus, for example, if the specification states "stereochemistry may be R", this would indicate that "stereochemistry is S" is also contemplated.

[0051] As used herein, the term "irreversible" for the binding of an inhibitor to an enzyme means that the binding cannot be reversed without denaturing and / or inactivating the enzyme. The term may refer to mechanical inactivation. DETAILED DESCRIPTION

[0052] The present invention relates to the use of N-propargyl-1H-1,2,3-triazole as an inhibitor of nitrification enzymes in soil microorganisms. The inventors unexpectedly discovered that the presence of the N-propargyl group renders the binding of the inhibitor to the enzyme effectively irreversible, thereby significantly prolonging the inhibitory effect. This in turn may allow farmers to reduce the frequency of nitrogen fertilizer applications, resulting in significant cost savings and reduced environmental pollution.

[0053] The inventors hypothesize that the presence of an N-propargyl group on the triazole ring may allow a different binding mechanism to operate than other triazoles, resulting in irreversible binding to nitrating enzymes (eg, AMO or HAO).

[0054] They also hypothesized that if the inhibitor molecule is too lipophilic, the compound will not penetrate the soil effectively and will therefore be less effective. Therefore, the compounds used in the present invention are limited to compounds in which the substituents on the two ring carbon atoms have a total of 6 or fewer carbon atoms and the substituents on the distal end of the alkynyl group are limited to no more than 2 carbon atoms. R in Structure I2 It may be methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl or its structural isomer or n-hexyl or its structural isomer. 3 It can be H, methyl, ethyl, propyl, isopropyl, n-butyl or its structural isomers or n-pentyl or its structural isomers, taking into account R 2 and R 3 There are 1, 2, 3, 4, 5 or 6 carbon atoms in total. Thus, m can be 0, 1, 2, 3, 4 or 5 and n can be 1, 2, 3, 4, 5 or 6, taking into account that the sum of m+n does not exceed 6. When optical isomers are possible, both the R and S forms are included. When stereoisomers are possible, all possible stereoisomers (e.g., enantiomers, diastereomers) are included.

[0055] The compound used in the present invention can be a solid, such as a crystalline solid, or can be an oil or a liquid. They can exist as solvates (such as hydrates), and these are all encompassed in the present invention. They can also exist as salts, such as salts of halides or phosphates or organic acids (such as acetic acid or glycolic acid). Again, the present invention also encompasses such salts.

[0056] In some cases, a mixture of inhibitors may be used. In such cases, at least one (optionally each) inhibitor may be an N-propargyl-1,2,3-triazole as described herein.

[0057] Triazole nitrification inhibitors and methods of use thereof have been described in WO2021 / 042169, and the entire contents of the specification are incorporated herein by reference. However, the compound used in the present invention is characterized in that there is an alkynyl group bonded to the ring nitrogen of the triazole group through a methylene spacer. The compound used in the present invention can be prepared by the general procedure described by Clark et al. (PR Clark, GD Williams et al. A Scalable Metal-, Azide-, and Halogen-Free Method for the Preparation of Triazoles, Angew. Chem. Int. Ed. 2020, 59, 6740–6744.), the entire contents of which are incorporated herein by reference.

[0058]

[0059] Compared with the previous methods for synthesizing N-substituted 1H-1,2,3-triazoles, this method has the advantage of being tolerant to various functional groups. 1 When an alkynyl moiety is present, high yields can be obtained to produce the compounds used in the present invention.

[0060] Nitrification inhibition can be effective at temperatures between about 0 and about 50°C, or between about 0 and 25, 0 and 10, 10 and 50, 20 and 50, or 20 and 40°C, for example about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50°C, or above or below these ranges.

[0061] Preparations and compositions according to the present invention can be powders, gels, granules, tablets, emulsions, emulsified concentrates, microemulsion dispersions or other suitable forms. They can include one or more suitable adjuvants, such as mineral oil, vegetable oil or animal oil, solvent (including organic solvent), surfactant etc.

[0062] Suitable surfactants include emulsifiers, wetting agents, suspending agents, spreading agents, etc. They can be anionic or cationic, nonionic or zwitterionic. Mixtures of surfactants can be used.

[0063] Other adjuvants that may be present include crystallization inhibitors, viscosity modifiers, dyes, antioxidants, light absorbers, defoamers, complexing agents, pH adjusters, pH buffers, micronutrients, lubricants, dispersants, microbicides, and the like.

[0064] The composition may include an oil, such as a mineral oil or a vegetable oil. Suitable oils include soybean oil, olive oil, rapeseed oil, liquid hydrocarbons, C8-C 18 Fatty acids, esters thereof, and the like. Mixtures of oils may also be used. The oil may comprise from about 0.01 to about 10%, or from about 0.1 to 10, 1 to 10, 5 to 10, 0.01 to 5, 0.01 to 1, 0.01 to 0.1, 0.1 to 1, or 1 to 5%, such as about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% by weight of the formulation or composition.

[0065] The compositions used in the present invention may include from about 1 to about 99% by weight of a triazole compound of formula (I) described herein, or about 1 to 50, 1 to 20, 1 to 10, 10 to 99, 50 to 99, 10 to 50, 10 to 20 or 10 to 50%, for example about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95 or 99% thereof. They may include from about 1 to about 99% by weight of one or more adjuvants, or about 1 to 50, 1 to 20, 1 to 10, 10 to 99, 50 to 99, 10 to 50, 10 to 20 or 10 to 50 thereof, for example about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95 or 99%. They may include from 0 to about 25% by weight of one or more surfactants, or about 0 to 10, 0 to 5, 0 to 1, 1 to 25, 5 to 25, 10 to 25, 5 to 20 or 10 to 20%, for example about 0, 1, 2, 3, 4, 5, 10, 15, 20 or 25% thereof.

[0066] Inhibitors of the present invention can be applied to controlled release or sustained release formulations / compositions. They can be microencapsulated or can be embedded in a matrix. The diameter of the microcapsule can be about 0.1 to about 500 microns, or about 1 to 500, 10 to 500, 100 to 500, 200 to 500, 0.1 to 100, 0.1 to 10, 0.1 to 1, 1 to 200, 1 to 100 or 10 to 100 microns, for example, a diameter of about 0.1, 0.5, 1, 2, 5, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 microns. Sealants can be inorganic or can be organic. It can be a polymer. Suitable matrix or carrier includes organic matrix (such as wood powder lignin, etc.) and inorganic matrix (such as silicon dioxide, clay, titanium dioxide and calcium carbonate). Matrix can be a matrix that dissolves or degrades over time to realize sustained release. Those skilled in the art will readily appreciate the range of carriers and / or matrices that can be used.

[0067] The inhibitor of the present invention can be applied directly, usually in solution or suspension, or as part of a fertilizer composition. Therefore, they can be dissolved, suspended or emulsified in a suitable carrier and then applied to the soil. Alternatively or additionally, they can be combined with fertilizers and optional other components and then applied to the soil. Suitable carriers can be aqueous or can be non-aqueous, such as organic. In some cases, they may not contain organic solvents. Suitable fertilizers include nitrogen-based fertilizers, such as urea-based or amino-based fertilizers. They can be organic or can be inorganic. Examples of inorganic fertilizers that can be used include NPK fertilizers, ammonium phosphate, ammonium sulfate, ammonium nitrate, ammonium sulfate nitrate and calcium ammonium nitrate, and mixtures of two or more of these. Fertilizers can include animal manure. Fertilizers can be applied as solid fertilizers, such as powder or granular forms, or as liquid fertilizers. When applied in a fertilizer composition, the inhibitor can be present in the fertilizer composition at a concentration of at least about 0.5 mol%, or at least about 1, 1.5 or 2 mol%, or about 0.5 to 5 mol%, 1 to 5, 2 to 5, 0.5 to 2, 0.5 to 1 or 1 to 3 mol%, for example about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mol%, or more than 5 mol%.

[0068] The inhibitor may be used in combination with a urease inhibitor such as N-(butyl)thiophosphoryltriamide (NBPT).

[0069] The triazole compounds described herein can be applied no more than once in any 2-week period or in any 3, 4, 5, 6, 7, 8, 9 or 10-week period. They can be applied once every 2 weeks, or once every 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 weeks. They can be applied to soil where crops grow or are planned to grow. They can be applied to the soil once in each growth period of the crop, or applied twice, three times or four times in each growth period. The soil to which the triazole compounds are applied can be acidic. They can be weakly acidic or can be strongly acidic. Their pH value can be about 6.6 to about 4.5 or about 6.5 to 5.5, 5.5 to 4.5, 6.5 to 6, 6 to 5.5, 5.5 to 5, 5 to 4.5 or 5 to 6, for example, about 4.5, 5, 5.5, 6 or 6.5. Alternatively, they can be non-acidic, for example, neutral, alkaline, weakly alkaline or strongly alkaline.

[0070] Those skilled in the art will appreciate that the specific compounds, ranges, ratios, etc. provided herein are provided as guidelines only and may vary from those described herein in specific circumstances.

[0071] Example

[0072] Materials and methods

[0073] All chemicals used for the synthesis of triazoles were provided by Sigma Aldrich Australia. Na2S2O4 was purchased from Chem-Supply. N. europaea (ATCC19718) was purchased from American Type Culture Collection. N. europaea was obtained from an aquarium kit (Aquasonic TM , BIO-NATURE STANDARD) and its purity was confirmed by 16S rRNA gene sequencing.

[0074] Cultivation of Nitrosomonas eutropha

[0075] Ammonia oxidizing bacteria (AOB) were grown in Duran glass bottles containing 600 mL of mineral salt medium (MSM, pH 7) at 100 rpm at 30°C in the dark for 3-5 days. The lid was loosely sealed with an O2 permeable membrane to ensure ventilation ( Sealing film, Sigma Aldrich). MSM culture medium contains the main bulk culture medium, composed of K2HPO4 (2.27 g L -1 )、KH2PO4(0.95g L -1 ) and (NH4)2SO4 (0.67 g L -1 ) was used. The pH was adjusted to 7.0, which is suitable for the growth of AOB. To 1 L of this large culture medium, 2 mL of a sterile filtered (0.2 μm Millipore filter) metal solution (Na2EDTA (6.37 g L -1 )、ZnSO4.7H2O(1.0g L -1 )、CaCl2.2H2O(0.5g L -1 )、FeSO4.7H2O(2.5g L -1 )、NaMoO4.2H2O(0.1g L -1 ), CuSO4.5H2O (0.1 g L-1), CoCl2.6H2O (0.2 g L-1), MnSO4.H2O (0.52 g L-1) and MgSO4.7H2O (60.0 g L-1). The culture medium solution was supplemented with 1 v / v% aqueous Na2CO3 (50 g L-1) -1 ).

[0076] Standard cell harvesting protocol

[0077] Cells of N. multiformis and N. europaea were cultured to the mid-exponential growth phase (OD 600 =0.1,[NO2 -] approximately 800 μm) and harvested by filtration onto a 0.2 μm membrane filter (mixed cellulose esters (MCE), Sigma Aldrich). The cells were washed with 2x 100 mL 0.1 M sodium phosphate buffer (NaPB) containing 0.2 mM MgSO4, pH 7.5. The cells were washed off the filter paper by transferring the cells to a sterile 50 mL tube and resuspending in NaPB (15 mL) followed by 5 s vortexing (Ratek, Australia) and 3 s sonication (Vevor, Australia). The inoculum had an OD of 1.2–0.9 600 , which was diluted to OD 600 = 0.03 and stored at 4°C until used for the assay (cells can be stored for up to 24 hours without loss of activity). The experiment was performed in triplicate.

[0078] Standard Nitrification Inhibition Assay Protocol

[0079] In a deep 96-well plate (2 mL capacity), 980 μL of bacterial inoculum (OD 600 =0.03) and inhibitor (10 μL, 10 μL, 30 mmol L -1 in Milli-Q water), the solution was mixed thoroughly and pre-incubated at 100 rpm and 30°C in the dark for 5 minutes. Then, (NH4)2SO4 (10 μL, 150 mM, from a 19.8 g L -1 A sterile solution of (NH4)2SO4 in Milli-Q water was prepared). The plate was covered with an O2 permeable membrane to ensure aeration (Breathe- The reaction solution (50 μL) was transferred to a 96-well spectrophotometer plate, to which 50 μL of Griess reagent was added. The color was allowed to develop for 15 minutes at room temperature and the absorbance was measured at 540 nm. Each assay was accompanied by a control treatment to determine the 0% and 100% NO2 - The inhibition percentage was calculated according to Equation 1, from the cellular NO2 in NaPB - The results were (i) without additives ("untreated cells": 0% signal), (ii) with [NH4 + ] = 3 mM ("uninhibited" cells: 100% signal) and (iii) containing [NH4 + ]=3mM and [inhibitor]=0.3mM and 0.03mM (10mol% and 1mol% of [NH4 + ], “suppressed cells”). The experiments were performed in triplicate.

[0080]

[0081] O2 consumption measurement

[0082] The O2 consumption rate of N. europaea cell suspensions was measured using a Clark-type oxygen electrode (Rank Brothers, Cambridge, UK) installed in a water-jacketed electrode chamber (3 mL capacity) connected to a recirculating chiller (Lauda, ​​Austria). TM (World Precision Instruments, UK) sensor data collection system recorded data. All measurements were performed at 20 ° C and 1 mL final reaction solution volume. The polarization voltage was set to 0.6 V. To calibrate the oxygen signal, an excess (about 50 mg) of Na2S2O4 was added to 1 mL of Milli-Q water to chemically remove dissolved O2.

[0083] Additional O2 flux was prevented by applying a stopper, and the residual voltage was referred to as "0% O2". The voltage at saturated O2 concentration ("100% O2") was determined by measuring the voltage of a balanced aerated reaction system consisting of 1 mL of Milli-Q water. Samples were measured as follows: 1 mL of the reaction mixture (consisting of 980 μL of N. europaea cell solution in NaPB (OD 600 = 0.8; equivalent to about 468 μg / L protein) was equilibrated in the chamber for 5 minutes until the voltage reading was stable. The reaction was then initiated by adding (NH4)2SO4 (10 μL of a 150 mM aqueous stock solution, a final concentration of 3 mM in the reaction solution) and the chamber was immediately sealed with a stopper. After 4 minutes of oxygen consumption (the linear rate coefficient determined was approximately k obs = k = 274.7 ± 20.0 nmol O2 L -1 s -1 ), 10 μL of DMSO inhibitor MPT stock solution (1.2 mM and 0.6 mM) was added through the capillary opening via a 10 μL E Eppendorf pipette, ensuring that the tip of the pipette was exposed to the solution. The voltage was recorded for 25 minutes at 5 second intervals. The trajectory of O2 concentration versus time (initial 15-140 seconds) after the addition of (NH4)2SO4 was used as the baseline O2 consumption of uninhibited cells, while the consumption trajectory describing the presence of inhibitors (255-1740 second time window) was used to determine the O2 consumption rate in the presence of inhibitors. All experiments were performed three times at 20°C under constant stirring. The voltage was converted to [O2] according to formula 2:

[0084]

[0085] Kinetic parameters were determined by GraphPad Prism software using simple linear regression for uninhibited cells and nonlinear fitting (curve fitting) for inhibited cells. First order decay, R 2 >0.9.

[0086] Activity recovery assay

[0087] Harvest cells using the "Standard Cell Harvesting Protocol". Adjust the bacterial solution to OD 600 = 0.8 (100 mg / L protein), and transfer 980 μL aliquots to 1.5 mL centrifuge tubes ( Polypropylene). Add 10uL inhibitor solution (150mM in DMSO) and pre-equilibrate for 5 minutes; add 10μL of 150mM (NH4)2SO4 aqueous stock solution (final concentration in tube = 3mM) by multichannel pipette to ensure simultaneous addition to each tube. Place the tubes in a temperature-regulated rotating incubator (Ratek, Australia) and incubate at 30°C for 30 minutes at 100rpm in the dark. After 30 minutes, transfer 50μL aliquots to a 96-well plate (Greiner Polystyrene), 50 μL of Griess reagent was added, and the mixture was incubated for 15 minutes. The concentration was measured at 540 nm using a photometer ( The absorbance was measured with 1% paraformaldehyde (BMG Labtech, Australia). The cells were washed repeatedly (3x) by regular centrifugation (Boeco, Germany; 10.000 rpm, 10 min) and the pellet was resuspended in 1 mL NaPB. After the final centrifugation step, the pellet was resuspended in 990 μL NaPB and re-incubated with (NH4)2SO4 (10 μL of a 150 mM stock solution in water) and NO2 was measured under the conditions described previously. - Each measurement was accompanied by a control treatment to determine the concentration of 0% and 100% NO2 - Signal. According to the NO2 in the cells of NaPB - The percentage activity was normalized to: (i) without additives ("untreated cells": 0% signal), [NH4 + ] = 3 mM (“uninhibited” cells: 100% signal) and (ii) [NH4 + ]=3mM and [inhibitor]=1.5mM. The experiment was repeated three times. Figure 2 shown.

[0088] Acute toxicity test

[0089] Harvest cells using the "Standard Cell Harvesting Protocol". Adjust the bacterial solution to OD 600= 0.8 (100 mg / L protein). The solution was divided into 1 mL aliquots and transferred to 24-well tissue culture plates (Greiner Polystyrene tissue culture treatment). Each well contained 980 μL of bacterial solution, 10 μL of 150 mM aqueous stock solution (NH4)2SO4, and 10 μL of 150 mM inhibitor solution. The plate was sealed with an O2 permeable membrane to ensure ventilation (Breathe- Seal film, Sigma Aldrich) and incubate at 30°C and 100 rpm for 4 hours in the dark. After incubation, the cells were transferred to a centrifuge tube and pelleted at 10,000 rpm for 10 minutes. The supernatant was discarded and the cells were resuspended in 1 mL NaPB. A 5 μL aliquot was transferred to a 96-well plate and a bacterial stain (LIVE / DEAD for microscopy) was added following the manufacturer's instructions. TM BacLight TM BacterialViability Kit, ThermoFisher Scientific). 10 μL of the solution was transferred to a microscope slide (FisherScientific, Australia, microscope slide 7.6 cm x 2.5 cm (length x width), thickness 1-1.2 mm). Ten images were taken for each treatment using a fluorescence microscope (Leica DM6000, Germany), and dead cells and live cells were detected using a red filter setting (excitation: 575 / 30 nm (dichroic) DC: 600: emission: 635 / 40 nm) and a green channel filter setting (excitation: 500 / 20 nm DC: 515: emission: 535 / 30 nm), respectively. The percentage of live and dead cells was calculated via Formula 3 and Formula 4:

[0090]

[0091] Soil cultivation studies to determine soil mineral N transformations

[0092] Soils used in these studies were air-dried, ground, and sieved (2 mm) before use. Soil microbiomes were incubated in 250 mL polypropylene specimen containers (Sarstedt, Germany) containing 20 g of oven-dry weight of soil. Microbiomes were treated with half the volume of water required to fill 60% of the pore space (WFPS%) and pre-incubated at 25 °C for seven days to restore soil microbial activity. After pre-incubation, the remaining volume to reach WFPS% was used as one of the following treatment solutions: (1) Fertilizer ((NH4)2SO4) 100 mg kg -1, (2) fertilizer + 0.5 mol% 4-MPT, (3) fertilizer + 2.5 mol% 4-MPT, (4) fertilizer + 5 mol% 4-MPT or (5) fertilizer + 5 mol% compound 1, each treatment was repeated three times (n = 3) in each time interval. During the entire incubation period, the lid was removed for 10 minutes every 2-3 days to keep the soil microecology aerated to allow gas exchange, and the humidity level was supplemented by adding Milli-Q water as needed. At the end of the desired incubation period (i.e., after 0, 3, 7, 14 and 21 days, respectively), the soil microecology was removed and treated with 2M potassium chloride aqueous solution (KCl, 100 mL) and shaken for 1 hour for destructive sampling. The soil-KCl solution (Whatman No. 42) was filtered and the filtrate was stored at -20 °C until the end of the experiment. All KCl extracts were appropriately diluted and analyzed for ammonium (NH4 + -N) and from nitrates (NO3 - -N; NO2 in soil - To NO3 - The results are reported as the mean of three replicates, and the reported errors are the standard error of the mean. The errors associated with the raw data were calculated using the standard error propagation scheme.

[0093] Soil cultivation studies to determine N2O production

[0094] Soil cultivation was performed in triplicate. Each sample consisted of 6 g of sieved soil (2 mm). The soil was air-dried after collection and pre-cultivated at 50% water holding capacity (WHC) for seven days before the experiment. The soil was transferred to a gas chromatography vial (22 mL capacity, transparent glass bottle, Macherey-Nagel, Germany) and compacted to allow 5.5-6.2 cm of head space. The soil in the vial was incubated at a constant temperature of 21° C. for a total of 21 days with the lid open to ensure gas circulation. The control cultivation contained "untreated" soil (deionized H2O only) and "fertilizer only" soil ((NH4)2SO4; 50 mg kg -1 soil), and each measurement was accompanied by (NH4)2SO4 in aqueous solution (1g NH4 + mL -1H2O) and applied to the soil surface to simulate field conditions. Three inhibitor solutions were prepared with DMP and 4-MPT at concentrations of 0.5 mol%, 2.5 mol% and 5 mol% of the applied fertilizer-N, respectively, and applied to the soil surface. After the application treatment, the soil reached 60% WHC and was maintained at this humidity level by regular addition of deionized water to compensate for evaporation losses. On the day of measurement (1, 3, 5, 7, 14 and 21 days after fertilization), the small glass bottle was airtightly closed with a rubber septum and an aluminum cap and opened again after each measurement. N2O emissions were analyzed using a gas chromatograph equipped with an electron capture detector and a flame ionization detector (GC-ECD / FID; Clarus 580, Perkin Elmer). The N2O-N production rate F was calculated using the linear regression slope according to Formula 5:

[0095]

[0096] Where ΔC / Δt represents the change in N2O concentration in time interval t (in ppbv), V represents the head volume (in L), M represents the molar mass of N in N2O, m represents the amount of soil (in g dry weight), and V m represents the molar volume of an ideal gas at 0°C and 101.325 kPa (22.414 L mol -1 ), using T0 (273.15K) and T a (air temperature in K) to calibrate the gas sampler.

[0097] statistics

[0098] Statistical analysis was performed using Minitab 18, using P < 0.05 as the statistical significance level. All results are reported as mean ± standard error of the mean.

[0099] One-way ANOVA was used to compare the significance among the three treatments at the least significant difference level of P < 0.05.

[0100] Synthesis of Substituted 1,2,3-Triazoles

[0101] The progress of the reaction was monitored by mass spectrometry (HRMS, Thermo, Bremen, Germany) operated in positive mode. Flash column chromatography purification was performed using Divisil Chromatographic Silica Media LC60A 40-63 with ethyl acetate (Chem Supply, Australia) and petroleum ether (Thermo Fisher, Australia). All 1 H NMR and 13C NMR and spectra were recorded on a 500 MHz Bruker spectrometer (500 or 126 MHz, respectively) using the reported solvent resonance as an internal standard. Chemical shifts are expressed in parts per million (ppm) and the splitting patterns are as follows: s, singlet; d, doublet; t, triplet; q, quartet; p, quintet; m, multiplet; dd, doublet within a doublet. Coupling constants, J, are expressed in Hertz (Hz). Inhibitors N001, N003, N013, N014, N018, N023 and compounds 2, 3, and 4 were kindly provided by Bethany Taggert from the University of Melbourne.

[0102] General procedure:

[0103]

[0104] For larger quantities (1 g and above), the reaction is carried out in a round bottom flask with or without heating as required, while for smaller quantities, the reaction is carried out in a microwave tube ( Sweden). The desired α-ketoacetal (1.05 equiv) was dissolved in methanol in a reaction flask (to give a 0.4 M solution). 4-Methylbenzenesulfonylhydrazine (toluenesulfonylhydrazine, TsNHNH2) (1.0 equiv) was added in portions and the reaction was stirred at room temperature for a few minutes until ESI-MS confirmed complete conversion of the starting material to the hydrazone. Primary amine (1.1 equiv) and triethylamine (1.1 equiv) were then added to the reaction mixture. In a sealed The reaction was carried out in a microwave tube above the boiling point of methanol and primary amine and exposed to microwave radiation ( Initiator+, Sweden) or refluxed in a fume hood under an inert atmosphere. After the desired reaction time, the reaction was cooled to room temperature and concentrated in vacuo (Buechi, Germany). The crude oil was dissolved in dichloromethane (DCM): water (1: 1), and the aqueous layer was extracted three times with DCM. The combined organic layer was washed with saline, dried with Na2SO4, concentrated in vacuo, and purified as needed via rapid column chromatography.

[0105] 4-Methyl-1-(prop-2-ynyl)-1H-1,2,3-triazole (4-MPT):

[0106]

[0107] Following the general procedure, 1,1-dimethoxypropane-2-one (250 mg, 2.20 mmol) in methanol (5 mL) was mixed with tosylhydrazide (388 mg, 2.08 mmol) in a 10 ml microwave tube ( Sweden). After complete consumption of the starting material, propargylamine (129 mg, 2.29 mmol) and triethylamine (232 mg, 2.30 mmol) were added and the tube was sealed. After heating at 120°C for 20 minutes and cooling to room temperature, the reaction mixture was concentrated in vacuo to give an orange oil. The crude oil was dissolved in DCM: water (1:1) and the aqueous layer was extracted three more times with DCM. The combined organic portions were washed with brine, dried over Na2SO4, concentrated in vacuo, and purified via flash column chromatography (ethyl acetate: petroleum solvent (1:1)) to give an orange oil 4-MPT (85%). The oil was cooled at 4°C overnight to give 4-MPT as a colorless crystalline solid.

[0108] 4-MPT (crude orange oil): 1 H NMR (500 MHz, CDCl3) δ 7.50 (d, J = 1.0 Hz, 1H, triazole-H), 5.11 (d, J = 2.6 Hz, 2H, aliphatic-H), 2.54 (t, J = 2.6 Hz, 1H, alkyne-H), 2.34 (d, J = 0.9 Hz, 3H, aliphatic-H).

[0109] 4-MPT (crude orange oil): 13 C NMR (126 MHz, CDCl3) δ 143.8 (triazole-C), 120.9 (triazole-C), 76.1 (broad, 2C, alkyne-C), 39.6 (aliphatic-C), 10.8 (aliphatic-C).

[0110] ES1-HRMS calculation for C6H8N3 + ([M+H] + ): m / z 122.0713; found m / z 122.0713.

[0111] 4-MPT (crystallization): 1 H NMR (500 MHz, CDCl3) δ 7.49 (s, 1H, triazole-H), 5.10 (d, J = 2.6 Hz, 2H, aliphatic-H), 2.53 (t, J = 2.6 Hz, 1H, alkyne-H), 2.33 (s, 3H, aliphatic-H).

[0112] 4-MPT (crystallization): 13 C NMR (126 MHz, CDCl3) δ 143.8 (triazole-C), 120.9 (triazole-C), 76.1 (broad, 2C, alkyne-C), 39.6 (aliphatic-C), 10.8 (aliphatic-C).

[0113] Crystal structure of 4-MPT.

[0114]

[0115] Table 1: Crystallographic data of 4-MPT. [a]

[0116]

[0117] [a] Data were collected using a Rigaku XtaLAB Synergy-SDual Microfocus X-ray diffractometer at 100 K using Cu-Kα radiation (λ = 1.54184 Å). Crystals were transferred directly from the flask into antifreeze oil and then immediately mounted on the diffractometer at the data collection temperature to prevent solvent loss. Data were processed using CrysAlisPro. Structures were refined using the F2-based full-matrix least-squares procedure SHELXL in OLEX2. For all structures, the positions of all non-hydrogen atoms were refined using anisotropic displacement parameters. Hydrogen atoms were placed geometrically and their positions were constrained to geometric estimates using a riding model.

[0118] 4-Methyl-1-(n-propyl)-1H-1,2,3-triazole (Compound 1):

[0119]

[0120] Following the general procedure, 1,1-dimethoxypropan-2-one (250 mg, 2.20 mmol) in methanol (5 mL) and toluenesulfonyl hydrazide (388 mg, 2.08 mmol) were heated in a 10 mL microwave tube ( Sweden). After complete consumption of the starting material, n-propylamine (129 mg, 2.29 mmol) and triethylamine (232 mg, 2.30 mmol) were added and the tube was sealed. After 20 minutes at 120 ° C and cooled to room temperature, the reaction mixture was concentrated in vacuo to an orange oil. The crude oil was dissolved in dichloromethane: water (1: 1) and the aqueous layer was extracted three more times with dichloromethane. The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated in vacuo to give 1 as a yellow oil (72%).

[0121] 1 H NMR (500 MHz, CDCl3) δ 7.23 (s, 1H, triazole-H), 4.22 (t, J = 7.1 Hz, 2H, aliphatic-H), 2.29 (d, 3H, aliphatic-H), 1.86 (m, 2H, aliphatic-H), 0.89 (t, J = 7.4 Hz, 3H, aliphatic-H).

[0122] 1 13 C{ 1H} NMR (126 MHz, CDCl3) δ 142.8 (triazole-C), 118.7 (triazole-C), 59.9 (aliphatic-C), 33.6 (aliphatic-C), 25.2 (aliphatic-C), 10.9 (aliphatic-C).

[0123] ESI-HRMS is ([M+H] + ): calculated for m / z 126.1026; found m / z 126.1026.

[0124] Results and Discussion

[0125] Compound 4-methyl-1-(propane-2-vinyl)-1H-1,2,3-triazole (4-MPT)

[0126] 4-MPT is a 1-alkynyl-4-alkyl substituted 1,2,3-triazole. It showed significant inhibition of 75% and 77% against ammonia oxidizing bacteria N. multiformis and N. europaea, respectively.

[0127] Sensitivity of Ammonia Oxidizing Bacteria (AOB) to 1,4-Disubstituted 1,2,3-Triazoles

[0128] a) 1,4-disubstituted 1,2,3-triazole containing no acetylenic group

[0129] Previously, compounds containing various substituents (with or without other functional groups) were tested in 28-day soil incubation studies. Their activity has not been shown in pure bacterial cultures of AOB. The inventors here tested their percent inhibitory effect on pure cultures of two AOB strains, N. multiformis and N. europaea. The reported compounds showed an inhibitory effect on R 2 The position contains an alkyl group (refer to the structure in Table 2) and in R 1 There are different functional groups in the olefinic hydrocarbons, including (I) aliphatic chains; (II) ester functional groups; (III) amine functional groups; (IV) alkoxy functional groups; and (V) olefin functional groups.

[0130] The percent inhibition assay was prepared and analyzed as described previously.

[0131] Table 2: Substitution patterns of 1,4-diyl substituted 1,2,3-triazoles containing different functional groups tested following the "Standard Nitrification Inhibition Assay Protocol".

[0132]

[0133]

[0134] Figure 1The structure-activity relationship (SAR) of 1,4-disubstituted 1,2,3-triazoles is shown using N. europaea (NE) and N. multiformis (NM) by following a standard nitrification inhibition assay protocol to monitor NO2 - The production was determined by incubation in NaPB at pH = 7.5 and 30°C with 1 v / v% DMSO, where [NH4 + ]=3mM, [inhibitor]=0.3M(10mol%[NH4 + ]), incubated at 100 rpm in the dark for 60 min. Standard error was determined by three biological and three technical replicates. No inhibition was indicated as NI.

[0135] b) Alkynyl containing 1,4-disubstituted 1,2,3-triazole

[0136] In this work, the importance of the alkyne group was investigated by SAR studies of reported 1,4-diyl-substituted 1,2,3-triazoles containing an alkyne group at the 1-position.

[0137] Table 3: Percent inhibition of N. europaea (NE) and N. multiformis (NM) by 1-alkynyl substituted 1,2,3-triazoles. - Production was determined according to the "Standard Nitrification Inhibition Assay Protocol". Incubation was performed in NaPB at pH = 7.5 and 30°C with 1 v / v% DMSO, where [NH4 + ]=3mM, [inhibitor]=0.3mM (10mol% [NH4 + ]), incubated at 100 rpm in the dark for 60 min. Standard error was determined by three biological and three technical replicates. No inhibition was indicated as NI.

[0138]

[0139] The inhibitory effects of the three compounds fluctuated. Although compound 3 had the highest activity, the alkynylalkyl-substituted compound 2 showed significantly reduced efficacy against both bacterial strains. This suggests that the presence of an alkenyl substituent anywhere at the 1-position does not necessarily result in a potent inhibitor.

[0140] Effects of 4-MPT on ammonia monooxygenase and hydroxylamine oxidoreductase

[0141] AOB oxidizes NH3 to NO2 - The oxidation is performed by two enzymes, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). N. europaea and N. multiformis naturally contain both enzymes. To identify the affected enzyme, NH3 is supplemented by NH2OH, the substrate of the second enzyme.

[0142] Table 4: Effect of 4-MPT on hydroxylamine oxidoreductase (HOA). Following the “Standard Nitrification Inhibition Assay Protocol”, NO2 - Production test. Incubation was performed in NaPB at pH = 7.5 and 30°C with 1 v / v% DMSO, where [NH4 + ]=3mM or [NH2OH]=3mM or [inhibitor]=0.3mM (10mol% [NH4 + ] and [NH2OH]]) and incubated at 100 rpm for 60 min in the dark. Standard errors were determined from three biological and three technical replicates.

[0143]

[0144] The test clearly revealed that NO2 - The main reason for the reduction in production is the inhibition of AMO. Although HAO also showed NO2 - The production is significantly lower, but the loss of activity may be due to inactivation by AMO. The two enzymes are biochemically connected to each other through an electron shuttle mechanism, in which HAO generates two excess electrons, which are transferred to AMO to fill the gap of two electrons lost due to NH3 oxidation and accompanying oxygen reduction, thereby producing water as a byproduct. (DJArp, LASayavedra-Soto, NGHommes. Molecular biology and biochemistry ofammonia oxidation by Nitrosomonas europaea. Arch. Microbiol. 2002, 178, 250-255). Therefore, the interruption of the electron transfer chain caused by AMO inhibition (in this case 4-MPT) is likely to also lead to (partial) inhibition of HAO. Considering that when NH3 is the substrate, NO2 - The inhibition of formation was more pronounced, thus suggesting that AMO is indeed the enzyme targeted by 4-MPT.

[0145] Combination research

[0146] Binding studies were performed following an "activity recovery assay" with the nitrifying bacteria N. europaea and N. multiformis. The initial measurements represent the activity of the cells in the presence of the inhibitor. Figure 2Results of the recovery assay are shown. Overall, the reactivity of cells exposed to 1,4-diyl substituted 1,2,3-triazoles with various substitution patterns was able to recover after three wash steps. However, the activity of cells exposed to 4-MPT could not be recovered, suggesting a mechanism-based inactivator.

[0147] Mechanical inhibition

[0148] The binding studies suggest that the inhibitory effect is of a mechanistic nature. To validate the results, compound 1 was synthesized, which contains an n-propyl substituent instead of the propargyl substituent in 4-MPT. To determine its efficiency as a nitrification inhibitor, its percent activity was tested with pure cultures of N. europaea and N. multiformis. The results with both AOB strains indicate that replacement of the alkyne moiety with an alkyl group having the same number of carbon atoms results in a complete loss of inhibitory activity.

[0149] Table 5: Inhibitory effect of alkyl and alkylalkynyl substituted 1,2,3-triazoles. No inhibition is indicated as NI.

[0150]

[0151] Removal of the triple bond in N013 increased activity when N013 was compared to compound 2 compared to the prior art, supporting the suggestion that these compounds act as inhibitors of different modes.

[0152] Oxygen potential measurement

[0153] Ammonia monooxygenase belongs to the metallooxidase family and its oxygen turnover is related to NO2 - According to the "O2 consumption measurement", the oxygen concentration was measured within 20 minutes. After the bacterial solution was pre-equilibrated in the electrochemical chamber (0-300 seconds, not shown in the figure), [NH4 + ] (3 mM aqueous solution), initial oxidation. In the absence of inhibitors, O2 consumption followed zero-order kinetics with an average rate coefficient k = 275 ± 20 nmol O2 L in the time interval of 300-600 s. -1 s -1 In two independent experiments, 4-MPT was added at two different concentrations, 0.6 mM and 1.2 mM, at the 600 s time point (indicated by ※), which corresponds to NH4 + The oxygen consumption rate was significantly reduced at both inhibitor concentrations (P < 0.005) after the addition of 4-MPT. The decay curves after the addition of 4-MPT followed first-order kinetics. The observed rate coefficient k for O2 consumption was obsIn the presence of MPT, the O2 consumption rates at [MPT] = 0.6 mM and 1.2 mM were 2.7 x 10 -3 s -1 and 4.5x10 -3 s -1 Since the rate of O2 consumption in the presence of 4-MPT is a measure of enzyme inhibition, the observation of increasing inhibition with increasing [4-MPT] (in our experiments, doubling [4-MPT] increased inhibition approximately two-fold) suggests the presence of mechanical inhibition, where the amount of active enzyme decreases over time.

[0154] Combination research

[0155] These studies were performed using a modified procedure described previously. Figure 4 In a, NO2 - The production rate is [NH4 + ] was measured as a function of ] to determine whether the inhibitor follows Michaelis-Menten kinetics. [NH4 + ] were 0.003mM, 0.03mM, 0.5mM, 1.5mM, 3mM, 10mM, 15mM and 30mM respectively. [4-MPT] was kept constant at 0.3mM. The Michaelis-Menten plot shows the saturation kinetics, where NO2 - Produced at 0.5 mmol L -1 When the above + ] is irrelevant. In addition, even if [NH4 + ] increased by up to four orders of magnitude, and cells treated with 4-MPT did not result in NO2 - The Michaelis parameters determined by hyperbolic analysis revealed K m(app) The value of remains unchanged within the experimental error range, while V max(app) and decreases as [4-MPT] increases (the suffix “app” indicates that these constants were determined from bacterial cells rather than purified enzymes).

[0156] Table 6: N. europae dependence on [NH4 + ] Produces NO2 - Michaelis-Menten kinetic parameters. [a,b]

[0157]

[0158] [a] Select [4-MPT] to achieve NO2 - Partial inhibition of the effect. [b]V max(app) and K m(app)The suffix “app” in the data indicates that these constants were determined using bacterial cells rather than purified enzymes. Standard errors are calculated based on three biological replicates, each with three technical replicates.

[0159] This behavior suggests a noncompetitive mode of inhibition, i.e., 4-MPT does not compete with NH3 for the same binding site in AMO. Figure 4 b) shows the dose response curve of 4-MPT, where [4-MPT] is 1500μM, 750μM, 375μM, 188μM, 94μM, 47μM, 23μM, 12μM and 6μM. In general, the percentage inhibition increases with the increase of inhibitor concentration, indicating that the inhibitor concentration determines the NO2 - produce.

[0160] As a proof of concept, the inhibition patterns of the remaining inhibitors N001, N003, N014, and N018 were studied. Figure 6 Compared with the Figure 4 a is similar, it can be seen that NO2 - The initial sharp increase in production indicated that these inhibitors were reversible inhibitors, supporting the hypothesis that the alkyne group in 4-MPT facilitates irreversible binding to the enzyme.

[0161] Acute toxicity test

[0162] The binding studies were followed by toxicity assays, the details of which have been described previously in this article.

[0163] Table 7: Acute toxicity of 4-MPT to N. europaea cells. The control treatment contained cells without inhibitor, and both treatments contained NH4 + To avoid cell death due to starvation, the cells were incubated in NaPB at pH 7.5 and 30°C with 1 v / v% DMSO, where [NH4 + ]=3mM、[4-MPT]=1.5mM(50mol%[NH4 + ]), incubated at 100 rpm for 12 h in the dark. Standard error was determined from ten microscopic images.

[0164]

[0165] Soil cultivation research

[0166] Table 8: Specifications of the soils studied in this study. [a]

[0167]

[0168]

[0169] [a] Average from three replicates; TOC = total organic carbon, TC = total carbon, TN = total nitrogen, min-N = mineral nitrogen, WHC - water holding capacity (WHC). Soil A was collected from an agricultural site in Victoria, Australia; soil BE was collected from an agricultural site in Germany.

[0170] NH4 in soil A + and NO3 - Measurement

[0171] To determine the nitrification inhibition effect of 4-MPT in vivo, the NH4 + and NO3 - Concentration-time curves of 4-MPT were obtained over 21 days of soil incubation. 4-MPT was tested at three different concentrations (0.5 mol%, 2.5 mol%, and 5 mol% of fertilizer-N) and compared with the alkyl-substituted compound 1 (5 mol% of fertilizer-N) to confirm the requirement of the N-propargyl substituent for inhibitory activity ( Figure 5 ).

[0172] In the absence of NI and in the presence of compound 1, NH4 + The loss of NH4 + The residue level is negligible ( Figure 5 a) and quantitatively converted into NO3 - ( Figure 5 b) In addition, although 4-MPT at the lowest application rate significantly delayed the NH4 + loss, but not enough to convert NH4 + In contrast, at higher application rates of 2.5 and 5 mol%, 4-MPT was able to quantitatively inhibit NH4 in the soil throughout the incubation period. + The results showed that the inhibitor treatments were statistically significant at P < 0.001 when compared with the control treatments using (NH4)2SO4 alone and with the 5 mol% treatment using compound 1. These findings also reflect the lack of NO3 during the incubation period. - In fact, the inhibitory performance of 4-MPT at the 2.5 mol% application rate was only slightly lower than that at the higher application rate of 5 mol% (P<0.01 for 2.5 mol% MPT on day 7, and P<0.001 for 2.5 and 5 mol% 4-MPT on other days). These data clearly illustrate the superior inhibitory performance of MPT, which can be attributed to the unique inhibitory mode of MPT.

[0173] Measurement of N2O production in soils B to E

[0174] Soil incubations were performed on four German soils (soils B to E) with different pH values ​​and the N2O production in the presence of 4-MPT was compared with that measured using DMP at the same application rates (0.5 mol%, 2.5 mol% and 5 mol% of applied fertilizer-N) and in the absence of NI. Figure 7 and as shown in Table 9. In the first week after fertilization in soil B (pH = 6.3), the N2O production rate of the uninhibited soil was the highest, which was 0.68 ng g on the first day. -1 Soil -1 , 0.19ngg on the 7th day -1 Soil -1 , and then gradually decreased to 0.05 ng g on the 21st day -1 Soil -1 ( Figure 7 a).

[0175] This behavior reflects an initial high N availability that is gradually depleted over time. Treatment with DMP slowed N2O formation compared to the uninhibited soil, depending on the application rate. Thus, with 0.5 mol% DMP, the N2O production rate on day 1 was approximately 50% of that in the uninhibited soil, i.e., 0.32 ng g -1 Soil -1 When DMP was used at higher application rates (2.5 mol% and 5 mol%), N2O production was approximately 67% of the N2O production without the inhibitor. In contrast, at the lowest application rate, soil treated with 4-MPT produced only 0.06 ng g -1 Soil -1 N2O was produced at a rate of 1.3 % and 1.5 % respectively, otherwise the amount of N2O remained below the detection limit during the entire experiment, which clearly revealed that 4-MPT was more effective than DMP in reducing N2O formation.

[0176] In the more acidic soil C, the amount of NH3 available for oxidation by AMO was reduced due to protonation, resulting in a lower N2O production rate in the uninhibited soil compared to soil B ( Figure 7 b). Soils treated with 0.5 mol% and 2.5 mol% DMP did not show a significant decrease in N2O formation rate during the first two weeks compared to uninhibited soil (P>0.05). Only at the highest DMP application rate did N2O production rate gradually decrease to 0.095 ng g on day 21. -1 Soil -1 In contrast, soil treated with 0.5 mol% MPT produced approximately 0.12 ng h -1At higher 4-MPT application rates, N2O production was almost completely suppressed.

[0177] The most acidic soil, soil D, produced the lowest amounts of N2O, ranging from 0.08 to 0.14 ng g throughout the experiment. -1 Soil -1 ( Figure 7 c). There was no significant difference in N2O production rate between uninhibited soil and soil treated with DMP (P>0.05). In contrast, soil treated with 0.5 mol% 4-MPT slowed N2O production by 60%, while the two higher application rates of 4-MPT reduced N2O production to almost zero from the beginning of the experiment, indicating that the inhibitory performance of 4-MPT at these concentrations was essentially independent of pH.

[0178] Soil E (pH = 7.5) was collected from agricultural reclaimed land in a former open-pit lignite mine. Reclaimed soils are known to retain low total N content over decades. This lack of N retention capacity resulted in abnormal N2O distribution in unfertilized soils, where N2O production rates increased rapidly within the first five days after fertilization, reaching a maximum of 2.33 ng g on day 5. -1 Soil -1 , and then dropped again to almost zero on day 14, indicating that N availability was exhausted ( Figure 7 d). Since N2O formation is correlated with nitrification rates, this behavior suggests that the soil has very high N nitrification / denitrification activity and can quickly adapt to N fertilization. Treatment with DMP and 4-MPT inhibited N2O production, with 4-MPT being significantly more effective than DMP during the first five days. After this time point, N2O production rates remained very low in the presence of either inhibitor.

[0179] Table 9: Results of soil incubation studies in soils B to E to determine N2O production from 4-MPT and DMP at different inhibitor concentrations.

[0180]

[0181]

[0182]

[0183] [a] The measurements were carried out as described above and the productivity was calculated according to the formula provided in the experimental section above. The application rate for all fertilizer treatments was (NH4)2SO4 = 50 mg kg -1Soil. Mean values ​​(n = 3); errors are standard errors of the mean for biological replicates. Statistical significance: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) when comparing inhibitor treatments with the control treatment with (NH4)2SO4 alone; P < 0.05 (#), P < 0.01 (##), P < 0.001 (###) when comparing inhibitor treatments with the highest concentration of DMP, respectively. The raw N2O production rates (μg g) were analyzed using the Tuckey HSD test with multiple comparisons using GraphPad Prism 9.5.0 (2-way ANOVA). -1 Soil -1 ) for statistical analysis.

Claims

1. A compound of formula (I) which, when used to inhibit nitration: in: R 1 is CH2C≡CA, where A is H, methyl or ethyl; R 2 C n H2 +1 ; R 3 C m H2 m+1 ; And wherein n and m+n are each independently 1, 2, 3, 4, 5 or 6 and m is 0, 1, 2, 3, 4 or 5.

2. The compound according to claim 1, wherein A is H.

3. A compound according to any one of the preceding claims, when used, wherein n is 1.

4. A compound according to any one of the preceding claims, when used, wherein m is 0.

5. A compound according to any one of the preceding claims, when used, wherein The compounds bind irreversibly to the nitrase enzyme.

6. A compound according to any one of the preceding claims, when used, wherein The compounds irreversibly inactivate nitrifying microorganisms.

7. A compound according to any one of the preceding claims, wherein the nitrification activity of N. europaea or N. multiformis increases by less than 10% from step (ii) to step (iv) in the following test when used: i. Add ammonium sulfate and the compound to N. europaea or N. multiformis cells; ii. Measurement of NO2 via Griess assay - produce; iii. The cells are then washed with buffer and ammonium sulfate is added; iv. Measure NO2 again using the Griess assay - produce.

8. A compound of formula (I) which, when used to inhibit nitration: in: R 1 CH2C≡CH; R 2 is methyl; and R 3 For H.

9. A composition which, when used for inhibiting nitrification in soil, comprises a compound of formula (I) as defined in any one of the preceding claims and at least one agriculturally acceptable adjuvant or diluent.

10. The composition according to claim 9, when used, further comprising a urease inhibitor.

11. A fertilizer composition comprising a fertilizer and a compound of formula (I) as defined in any one of claims 1 to 8.

12. The fertilizer composition according to claim 11, wherein The fertilizer is a urea-based or ammonium-based fertilizer.

13. A fertilizer composition according to claim 11 or claim 12, further comprising a urease inhibitor.

14. A method for reducing nitrification in soil, the method comprising treating the soil with a compound of formula (I) as defined in any one of claims 1 to 8 or a composition as defined in claim 9 or claim 10.

15. The method according to claim 14, wherein: The soil is acidic.

16. The method according to claim 14 or claim 15, wherein: The treatment was repeated no more than once in any 2-week period.

17. A method for inhibiting the nitrifying activity of a nitrifying microorganism, the method comprising exposing the microorganism to a compound as defined in any one of claims 1 to 8 or a composition as defined in claim 9 or claim 10.

18. A method for inactivating a nitrating enzyme, the method comprising exposing the enzyme to a compound as defined in any one of claims 1 to 8 or a composition as defined in claim 9 or claim 10.

19. Use of a compound as defined in any one of claims 1 to 8 for reducing nitrification in soil, or for inhibiting the nitrifying activity of nitrifying microorganisms or for inactivating nitrifying enzymes.

20. Use of a compound as defined in any one of claims 1 to 8 for the manufacture of a composition for reducing nitrification in soil, for inhibiting the nitrifying activity of nitrifying microorganisms or for inactivating nitrifying enzymes.

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