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• Chlorsulfuron (Fig. 1) was firstly reported by G. Levitt as it effectively controls a large number of weed species among cereal crops at ultra-low application rate in both pre- and postemergence treatment [1]. The invention of chlorsulfuron has led further efforts to develop a new class of important sulfonylurea herbicides whose target enzyme is AHAS (acetohydroxyacid synthase) or ALS (acetolactate synthase) that it exists only in plants and microbes, not in mammals [2-4].

  Figure 1

  

  Figure 1.  Structures of Chlorsulfuron and 5- substituted compounds Ia and Ib.

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  In consideration of the particular crop rotation systems (2-3 successive crops/same plot/same year) in many regions of China and the long persistence behavior which is detrimental to sensitive following rotation crops including wheat, corn, paddy, cotton and bean [5-8], the Ministry of Agriculture of China suspended the field application of 3 classical SUs chlorsulfuron, metsulfuron-methyl and ethametsulfuron since 2014 [9]. In view of the environment protection and sustainable development, the ban has attracted our attention to undertake a green chemical approach to solve this problem. Soil pH is one of the most important factors influencing the degradation rate of sulfonylurea herbicides in soils due to their acidic nature [10-13]. Thirunarayanan et al. reported that DT₅₀ of chlorsufuronwas 88.5 days in pH 6.2 soil and 144 days in pH 8.1 soil [14]. Wiese et al. measured persistence of chlorsulfuron in field study in Pullman clay loam under a 3-year winter wheat-sorghumfallow crop rotation pattern with an application of 34 g/ha on growing wheat. After a lengthy period of 25 months in soil pH >7.5, the residue of chlorsulfuron injured the newly planted sorghum [15]. Previous studies demonstrated that soil degradation of sulfonylurea herbicides is pH-dependent and has strong negative correlation with pH [16-19].

  Our previous research firstly discovered that electron-donating groups on the 5th position of the benzene ring of SU structures, such as dimethylamino and diethylamino substituents accelerated their degradation rate than their parent structure chlorsulfuron in acidic soil (pH 5.41) while the herbicidal activities were retained [20, 21] (Table 1). In order to further examine the degradation behavior of these new structures in high pH soil, an alkaline soil (pH 8.46) was selected according to the Chinese National Standard GB/T 31270.1-2014 from Cangzhou, Hebei Province [22]. The dimethylamino and diethylamino substituted compounds Ia and Ib (Fig. 1) were taken as the tested samples and chlorsulfuron as a control.

  Table 1

  

  Table 1.  The herbicidal activity of dimethylamino and diethylamino substituted compounds Ia and Ib.

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  The synthesis procedures of dimethylamino and diethylamino substituted compounds Ia and Ib have been reported by our previous researches [20, 21]. The crystal structure of compound Ia was diffracted by the following procedures. A colorless crystal of Ia was obtained by self-evaporation in the mixture solvent of dichloromethane and n-hexane. The crystal was analyzed by Xray diffraction with dimensions of 0.20mm ×0.18mm ×0.12mm. The data was collected on a Rigaku Saturn 724 CCD diffractometer equipped with a graphite monochromated Mo Kα radiation (λ=0.71073Å) at 113(2) K with θ_max=27.59°. The molecular formula is C₁₄H₁₇ClN₆O₄S, and the formula weight is 400.85 g/mol. The crystal was a triclinic system, space group P-1, with cell parameters existed as α=7.920(4)Å, b=8.661(4)Å, c=13.968(6)Å; α=101.766(9)°, β=93.734(6)°, γ =107.614(13)°; cell ratio as α/b=0.9144, b/c=0.6201, c/α=1.7636; calculated density was 1.503g/cm³; V=885.73(70) ų; Z=2 and linear absorption coefficient was 0.368mm^-1. A total 11448 integrated reflections were collected, and 4022 were independent with R_int=0.0558 and completeness of data was 97.5%. The structure was elucidated by direct methods with the program of SHELXS-97 [23]. Full-matrix least-squares refinement which was based on F² gave final values of R=0.0412, wR=0.1187 using the weight of 1/[σ²(F_o²) + (0.0843P)²+0.0029P]. Hydrogen atoms were located by a fixed value of isotropic displacement parameter. The data was corrected for absorption with multi-scan, T^min=0.9301, T_max=0.9572.

  The crystal structure of Ia was showed in Fig. 2 (CCDC number. 1546964). Fromthe data, the bond length of N(1)-C(5), N(2)-C(9), N (3)-C(9) were 1.361(2) nm, 1.375(2) nm and 1.384(2) nm respectively, which were shorter than general C-N bond that they may be caused by the transfer of π electrons. The sum angles of O (3)-C(9)-N(2) 123.31(16)°, O(3)-C(9)-N(3) 120.92(15)° and N(2)-C (9)-N(3) 115.77(15)° was 360°, indicating the plane sp² hybridization state of C(9) atom. The bond angles of O(1)-S(1)-C(1), O(2)-S (1)-C(1), N(2)-S(1)-C(1) were 108.05(8)°, 110.69(8)° and 104.36(8)° respectively which indicated state of the S(1) atom was sp³ hybridization. The torsion angle of O(3)-C(9)-N(2)-H(2) was 169.806(1510)° while the O(3)-C(9)-N(3)-H(3) showed as 0.331 (1237)°. The benzene ring and the triazine ring were non-planar for their dihedral angle being 79.676(48)°. The conformation of dimethylamino group was nearly located on the plane of the benzene ring and their dihedral angle was 3.473(110)°. From Fig. 2, two kinds of H-bonds could also be found: N(2)-H(2)…N(6) and N (3)-H(3)…O(3), it could be explained as the reason of the torsion angles of O(3)-C(9)-N(2)-H(2) and O(3)-C(9)-N(3)-H(3).

  Figure 2

  

  Figure 2.  Crystal structure of Ia.

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  The surflex-dock method [24] was applied to study the binding mode of our newly-designed Ia with the target AHAS (ALS) for its high herbicidal activity. Ia was manually docked into the active site of yeast AHAS using the SYBYL 6.9 software package referring to the crystal complex of chlorimuron-ethyl and yeast AHAS [25] which was retrieved from the RCSB Protein Data Bank (PDB code: 1N0H).

  The molecular docking study of Ia was illustrated in Fig. 3. The conformation of aromatic ring and triazine ring connected to the sulfonyl urea bridge were approximately mutual vertical (Fig. 3a) which was similar to the binding structure of AHAS and chlorimuron-ethyl that the heterocyclic part blocks the substrate access channel through intramolecular interactions (H-bonds and π-π stacking) [4]. H-bonds interaction between the urea-triazine moiety of Ia and the guanidine group of B/Arg 380 was showed in yellow dashed lines. There was a π-π stacking interaction between the triazine moiety of Ia and the indole part of B/Trp 586 (Fig. 3b).Ia was ascertained to be a typical AHAS interaction by the docking result. This conformation was favorable to retaining its biological activity for the suitable angle between the dimethylamino group and the benzene ring which was consistent with its crystal structure. Comparing the combining conformation with the crystal structure of Ia, the C(9)-N(3) bond was twisted that N(2)-H(2) and N(3)-H(3) was homo-direction. It was consistent with the active conformation of chlorimuron-ethyl and monosulfuron [26-28].

  Figure 3

  

  Figure 3.  Binding mode of Ia to yeast AHAS.

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  The research of soil degradation of samples (Ia and Ib) was performed by the following procedures. The alkaline soil was derived from the upper-layer (0–25 cm) in fresh farm land, and airdried in the shade, shifted through 2 mm sieve according to the Chinese National Standard GB/T 31270.1-2014 [22]. The soil texture, pH value, organic matter, cation exchange capacity (CEC) and particle size analysis of tested alkaline soil were determined by Tianjin Institute of Agricultural Resources and Environment Science. The properties of tested alkaline soil were listed in Table 2.

  Table 2

  

  Table 2.  The properties of tested alkaline soil.

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  Each sample was tested by UV spectra for suitable wave length of each tested compound. Methanol and H₃PO₄ aqueous solution (pH 3.0) were selected as the mobile phase. The samples were analyzed by HPLC according to the Chinese National Standard GB/T 16631-2008 [29]. The method employed a Shimadzu HPLC (series LC-20AT), equipped with a binary pump (Shimadzu, LC-20AT), an UV/vis detector (Shimadzu, SPD-20A), an auto sampler (Shimadzu, SIL-20A), a column oven (Shimadzu, CTO-20AC) and a Shimadzu shim-pack VP-ODS column (5 mm, 250 mm × 4.6 mm) connected to a Shimadzu shim-pack GVP-ODS (10 mm × 4.6 mm) pre-column, and a computer (model Dell) for carrying out the data analysis. The analytical data for alkaline soil degradation were listed in Table 3. After that, each sample was detected twice to establish standard curves for quantitative analysis with 10 mL injection volume.

  Table 3

  

  Table 3.  Analytical conditions for alkaline soil degradation.

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  According to the Chinese Agricultural Industry Standard NY/T 788-2004 [30], each sample was extracted through mixed extraction solvent to meet the standard requirement for recovery rate, standard deviation and coefficients of variation (Table 3). The initial additive concentration 5 mg/kg, 2 mg/kg and 0.5 mg/kg of Ia, Ib or cholrsulfuron was added into 20 g soil in 100 mL conical flask, followed by the regulation of 60% water holding capacity. Suitable extraction solvent was poured into the flask, and then was shook in the thermostatic oscillator for 3 h. The sample was centrifuged at 6500 rpm in a Thermo Scientific centrifuge for 2 min, the supernatant was concentrated in vacuum at room temperature. Dichloromethane (30 mL × 2) and distilled water (30 mL) were used for the extraction of the residues, the organic phase was dried by anhydrous sodium sulfate, filtered and concentrated. The concentrated sample was dissolved into 10 mL acetonitrile, and was then shook in oscillator for 1 h, filtered through millipore filter for HPLC detection. Each concentration of one compound was replicated 5 times for the repeatability and consistency.

  Soil sample (20 g) was weighed into 100 mL conical flasks for 6 groups, each group was triplicated. Compound sample with its concentration at 5 mg/kg was added, 60% water holding capacity was then regulated. The sealed soil sample was cultivated in a biochemical incubator at 25 ±1 ℃ and 80% humidity in the dark. The water holding capacity was adjusted regularly during the cultivation. The samples were extracted periodically by 6 interval times to establish the degradation curves, and the statistical analysis was also guaranteed by the triplicated data.

  The establishment of degradation curves followed the standard first-order kinetic curves C_t = C₀ e^-kt (C_t: sample concentration (mg/kg) at t days; C₀: original sample concentration; k: the degradation rate constant; t: time (days)) [1, 14, 31-32] with the correlation coefficient (R²) keeping above 0.99. The standard derivation (SD) of each sample was calculated according to the triplicated data. After that, half-lives of degradation (DT₅₀) were calculated according to the formula: DT₅₀=ln2/k (Table 4). As the data in Table 4 showed, the degradation rate of Ia (DT₅₀ 3.36 days) accelerated nearly 30 folds comparing with chlorsulfuron (DT₅₀ 84.53 days) while Ib (DT₅₀ 6.25 days) accelerated nearly 15 folds. In our previous study in acidic soil (pH 5.41), DT₅₀ of Ia, Ib and chlorsulfuron were 0.43, 1.60 and 12.91 days, respectively [21]. Comparing the degradation results in acidic soil and alkaline soil, an equal or rather larger acceleration rate was appeared in alkaline soil. This may be an efficient approach to solve the degradation problems of SUs in alkaline soil.

  Table 4

  

  Table 4.  Kinetic parameters for alkaline soil (pH 8.46) degradation.

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  On the basis of our previous research in acidic soil (pH 5.41) of the 5-substituted chlorsulfuron, further degradation study in alkaline soil (pH 8.46) were undertaken with compounds Ia and Ib while chlorsulfuron was taken as a control. Meanwhile conformation of Ia was elucidated by its crystal data, and the surflex docking result of Ia onto yeast AHAS confirmed the typical SU ligand/AHAS receptor interaction. There was almost no interference of the 5th substituents on the benzene ring at the site of action which well explained the retention of their potent herbicidal activity. As the alkaline soil (pH 8.46) degradation results illustrated, Ia (DT₅₀ 3.36 days) degraded nearly 30 folds faster comparing with chlorsulfuron (DT₅₀ 84.53 days). Under the same condition, Ib (DT₅₀ 6.25 days) degraded 15 folds faster. As a whole, the research indicated that Ia and Ib both accelerated their degradation rate much faster in alkaline soil as wellas in acidic soil than their parent structure. It revealed a favorable resolution to the residue problems of SUs in alkaline soil. This research re-affirmed our previous discovery that the 5th position of the benzene ring in SU is a key point to greatly influence its degradation rate. This approach foresees a considerable strategy to reduce its impact of traditional herbicides on the ecological environment.

  Acknowledgments

  This work was supported by the National Natural Science Foundation of China (No. 21272129), the State Key Laboratory of Elemento-Organic Chemistry (Nankai University), Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), and Syngenta PhD Scholarship.

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• 

  Figure 1  Structures of Chlorsulfuron and 5- substituted compounds Ia and Ib.

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  Figure 2  Crystal structure of Ia.

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  Figure 3  Binding mode of Ia to yeast AHAS.

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  Table 1.  The herbicidal activity of dimethylamino and diethylamino substituted compounds Ia and Ib.

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  Table 2.  The properties of tested alkaline soil.

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  Table 3.  Analytical conditions for alkaline soil degradation.

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  Table 4.  Kinetic parameters for alkaline soil (pH 8.46) degradation.

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•