Journal of Agricultural and Food Chemistry
Article
Table 3. Post-emergence Crop Selectivity of Compounds 2a−2c and 6a−6c and Saflufenacil
crop injury (%)
compound
dosage (g of ai/ha)
corn
rice
wheat
soybean
cotton
peanut
2a
2b
2c
6a
6b
6c
150
150
150
150
150
150
75
30
30
40
20
40
50
50
10
50
10
40
40
60
80
70
0
80
50
95
60
60
60
80
70
50
95
60
70
80
90
85
90
85
60
60
90
70
70
80
70
50
30
saflufenacil
dione (11a, 0.5 g), 2-mercaptoacetic acid (0.14 g), and
paraformaldehyde (89 mg) in toluene (50 mL) was heated to reflux
with stirring under a nitrogen atmosphere for 6 h. Then, the reaction
solution was cooled to room temperature, and H2O (20 mL) was
added to the reaction solution. The organic layer was separated and
washed with H2O (20 mL) and brine (20 mL × 2), dried by MgSO4,
and concentrated by rotary evaporation. The crude compound was
purified by column chromatography to give white solid 2a (0.56 g,
86%). 1H NMR (400 MHz, DMSO-d6): δ 12.59 (s, 1H), 7.52 (d, J =
9.2 Hz, 1H), 6.92 (d, J = 6.8 Hz, 1H), 6.57 (s, 1H), 6.47 (t, J = 6.8
Hz, 1H), 4.48 (dd, J = 6.4 and 3.2 Hz, 2H), 3.42 (s, 3H), 3.33 (s,
2H). 13C NMR (101 MHz, DMSO-d6): δ 172.30, 160.44, 150.99,
150.66, 148.26, 141.36, 141.03, 139.94, 139.92, 122.40, 122.26,
119.48, 119.39, 117.47, 117.23, 113.83, 103.25, 46.96, 32.83, 32.21.
19F NMR (376 MHz, CDCl3): δ −65.75 (s), from −114.94 to
−115.60 (m). HRMS (ESI): calculated for C15H12ClF4N3NaO4S [M
+ Na]+, 464.0071; found, 464.0021.
with three replications for each compound and concentration. After
21 days post-treatment, the herbicidal activity of each compound was
evaluated, and the results are illustrated in Tables 1 and 2.
Crop Safety. Six compounds 2a−2c and 6a−6c with a wide
spectrum of weed control were selected for crop selectivity studies. Six
representative crops, corn, rice, wheat, peanut, soybean, and cotton,
were used in the crop safety experiments. The crop safety assay
methods were similar to our previous reports.11,24,25 Briefly, the seeds
of the six kinds of crops were sown in plastic pots and grown in the
greenhouse. When the tested crops reached the four-leaf stage, they
were treated by compounds 2a−2c and 6a−6c at the rate of 150 g of
ai/ha. Saflufenacil was used as a positive control, with three replicates
for each compound. After 25 days, the crop damage (percent injury,
%) of each compound was evaluated, and the results are shown in
Table 3.
Stability Assay of Compounds 2c and 6c in the NtPPO
Reaction Buffer. The assay method of compounds 2c and 6c in the
NtPPO assay buffer was the same as our previous report.11 Briefly, a
reaction solution (400 μL) containing 200 μM compound 2c or 6c,
assay buffer, NtPPO (20 μg), and protoporphyrinogen IX (1.8 μM)
was incubated at room temperature for 3, 5, and 7 min, separately.
Then, CH3OH (135 μL) was added to the reaction solution. The
remaining percentage of compounds 2c and 6c in the reaction buffer
was then detected using high-performance liquid chromatography
(HPLC, Agilent 1200 Infinity Series) with a C18 column (4.6 × 150
mm, 5.0 μm). For each compound, at least three repeats were
performed.
Metabolism Studies of Compounds 2c and 6c in ABUJU.
One of the tested weeds ABUJU was selected as the representative in
the metabolism study.11,24 Briefly, the five-leaf-stage ABUJU was
treated by a solution of compound 2c or 6c (100 μM, containing 1‰
DMF and Tween 80) in the greenhouse. When the leaves of ABUJU
started to drop (about 2−3 h), 5 g of the leaves was cut for further
analysis. The leaves were first washed with a solution containing 1‰
DMF and Tween-80 3 times and then triturated to powder in the
presence of liquid nitrogen. The powder was then transferred to a
flask, which contained ethyl acetate (50 mL) and H2O (100 mL), and
stirred vigorously for 30 min at room temperature. The organic layer
was separated and concentrated under reduced pressure. The residue
was analyzed by UPLC−HRMS (Waters Acquity UPLC H-class,
Waters Xevo G2-XS Q-TOF).
Molecular Docking. The structure of compounds 2c and R- and
S-6c were constructed and optimized by Sybyl 6.9 (Tripos, Inc.)
before use. The crystal structure of NtPPO (PDB code 1SEZ) was
download from the Protein Data Bank (PDB) database, and chain A
of the structure was used for the subsequent molecular dynamic
(MD) simulation studies. PYMOL was used to extract the receptor
and visualized the simulation results.33 AutoDockTools version 1.5.6
was used to prepare the ligands and receptors, and AutoDock 4.2 was
used to predict the binding modes of compounds 2c and R- and S-6c
to NtPPO.34 The docking parameters used in this research were the
same as our previous reports.11,24,25
The synthetic details, nuclear magnetic resonance (NMR), and
high-resolution mass spectra (HRMS) characterization data of
compounds 2b−2q and 3−6 are shown in the Supporting
NtPPO Inhibitory Activity. In the PPO inhibition assay studies,
we selected Nicotiana tabacum mitochondrial PPO2 (NtPPO) as the
representative plant enzyme because it is widely used as a model
enzyme for PPO research.15,21−23 In this study, the NtPPO
expression, purification, and enzyme kinetics assay methods were
performed as described previously.11,24−27 Briefly, in a 96 well plate
(Thermo), each well was added 200 μL of the reaction solution. The
solution consisted of 200 mM imidazole, 0.03% Tween 80 (v/v), 100
mM potassium phosphate buffer (PBS, pH 7.4), 5 mM dithiothreitol
(DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 0−35 μg of
recombinant NtPPO protein, and 0.00001−200 μM inhibitors. The
enzymatic reaction was initiated by adding 0.5−3 μM protoporphyri-
nogen IX. After 0.5−3 μM protoporphyrinogen IX was added to the
reaction solution to initiate the enzymatic catalytic reaction, the
fluorescent intensity of each well was recorded at the emission
wavelength of 630 nm under excitation at 410 nm. The inhibition
constant of the enzymatic reaction (Ki) of each compound was
calculated by the method reported by Shepherd and Dailey28 For each
compound, three repeats were performed independently, and the
NtPPO inhibition results are shown in Tables 1 and 2.
Herbicidal Activity. Three representative broadleaf weeds
Abutilon juncea (ABUJU), Amaranthus retroflexus (AMARE), and
Eclipta prostrata (ECLPR) and three representative grass weeds
Digitaria sanguinalis (DIGSA), Echinochloa crus-galli (ECHCG), and
Setaria faberi (SETFA) were used to evaluate the post-emergent
herbicidal activity the synthesized compounds 1a1 and 2−6. The
assay methods were the same as our previous reports.29−32 Briefly,
plastic pots (diameter of 7.5 cm) were filled with the clay soil to 3/4 of
their depth, then the seeds of the six kinds of weeds were sown in the
pots and grown in the greenhouse. The test compounds were
dissolved in dimethylformamide (DMF) and diluted with 0.1%
Tween 80 solution before use. When the weeds grew to the three-leaf
stage, they were treated by the tested compounds with the application
rates of 37.5−150 g of ai/ha. The test solution (Tween 80 + DMF)
and saflufenacil were used as blank and positive controls, respectively,
MD Simulations. AMBER 14 software package with ff14SB force
fields was used to perform MD simulations of the 2c− and (R and S)
6c−NtPPO systems.35 The parameters of ligands for MD simulations
were prepared by the Antechamber program with the AMBER force
fields (GAFF). The ligand−protein system was solvated with TIP3P
4085
J. Agric. Food Chem. 2021, 69, 4081−4092
Wang, Da-Wei
