Synthesis and evaluation of heterocyclic analogues of bromoxynil

Article abstract of DOI:10.1021/jf404209d

One attractive strategy to discover more active and/or crop-selective herbicides is to make structural changes to currently registered compounds. This strategy is especially appealing for those compounds with limited herbicide resistance and whose chemistry is accompanied with transgenic tools to enable herbicide tolerance in crop plants. Bromoxynil is a photosystem II (PSII) inhibitor registered for control of broadleaf weeds in several agronomic and specialty crops. Recently at the University of Tennessee - Knoxville several analogues of bromoxynil were synthesized including a previously synthesized pyridine (2,6-dibromo-5-hydroxypyridine-2-carbonitrile sodium salt), a novel pyrimidine (4,6-dibromo-5-hydroxypyrimidine-2-carbonitrile sodium salt), and a novel pyridine N-oxide (2,6-dibromo-1-oxidopyridin-1-ium-4-carbonitrile). These new analogues of bromoxynil were also evaluated for their herbicidal activity on soybean (Glycine max), cotton (Gossypium hirsutum), redroot pigweed (Amaranthus retroflexus), velvetleaf (Abutilon theophrasti), large crabgrass (Digitaria sanguinalis), and pitted morningglory (Ipomoea lacunose) when applied at 0.28 kg ha^-1. A second study was conducted on a glyphosate-resistant weed (Amaranthus palmeri) with the compounds being applied at 0.56 kg ha ^-1. Although all compounds were believed to inhibit PSII by binding in the quinone binding pocket of D1, the pyridine and pyridine-N-oxide analogues were clearly more potent than bromoxynil on Amaranthus retroflexus. However, application of the pyrimidine herbicide resulted in the least injury to all species tested. These variations in efficacy were investigated using molecular docking simulations, which indicate that the pyridine analogue may form a stronger hydrogen bond in the pocket of the D1 protein than the original bromoxynil. A pyridine analogue was able to control the glyphosate-resistant Amaranthus palmeri with >80% efficacy. The pyridine analogues of bromoxynil showed potential to have a different weed control spectrum compared to bromoxynil. A pyridine analogue of bromoxynil synthesized in this research controlled several weed species greater than bromoxynil itself, potentially due to enhanced binding within the PSII binding pocket. Future research should compare this analogue to bromoxynil using optimized formulations at higher application rates.

Full text of DOI:10.1021/jf404209d

Article
pubs.acs.org/JAFC
Synthesis and Evaluation of Heterocyclic Analogues of Bromoxynil
,
†,¶
†
†
‡
†
Matthew A. Cutulle,* Gregory R. Armel, James T. Brosnan, Michael D. Best, Dean A. Kopsell,
#
§
⊥
Barry D. Bruce, Heidi E. Bostic, and Donovan S. Layton
†
Department of Plant Sciences, University of Tennessee, Knoxville, Tennessee 37996, United States
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996, United States
‡
#
Department of Biochemistry Cell Biology and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996, United
States
§
Department of Chemistry, Wofford College, Spartanburg, South Carolina 29303, United States
⊥
Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, Tennessee 37996, United States
ABSTRACT: One attractive strategy to discover more active and/or crop-selective herbicides is to make structural changes to
currently registered compounds. This strategy is especially appealing for those compounds with limited herbicide resistance and
whose chemistry is accompanied with transgenic tools to enable herbicide tolerance in crop plants. Bromoxynil is a photosystem
II (PSII) inhibitor registered for control of broadleaf weeds in several agronomic and specialty crops. Recently at the University
of TennesseeKnoxville several analogues of bromoxynil were synthesized including a previously synthesized pyridine (2,6-
dibromo-5-hydroxypyridine-2-carbonitrile sodium salt), a novel pyrimidine (4,6-dibromo-5-hydroxypyrimidine-2-carbonitrile
sodium salt), and a novel pyridine N-oxide (2,6-dibromo-1-oxidopyridin-1-ium-4-carbonitrile). These new analogues of
bromoxynil were also evaluated for their herbicidal activity on soybean (Glycine max), cotton (Gossypium hirsutum), redroot
pigweed (Amaranthus retroflexus), velvetleaf (Abutilon theophrasti), large crabgrass (Digitaria sanguinalis), and pitted
−1
morningglory (Ipomoea lacunose) when applied at 0.28 kg ha . A second study was conducted on a glyphosate-resistant
−1
weed (Amaranthus palmeri) with the compounds being applied at 0.56 kg ha . Although all compounds were believed to inhibit
PSII by binding in the quinone binding pocket of D1, the pyridine and pyridine-N-oxide analogues were clearly more potent than
bromoxynil on Amaranthus retroflexus. However, application of the pyrimidine herbicide resulted in the least injury to all species
tested. These variations in efficacy were investigated using molecular docking simulations, which indicate that the pyridine
analogue may form a stronger hydrogen bond in the pocket of the D1 protein than the original bromoxynil. A pyridine analogue
was able to control the glyphosate-resistant Amaranthus palmeri with >80% efficacy. The pyridine analogues of bromoxynil
showed potential to have a different weed control spectrum compared to bromoxynil. A pyridine analogue of bromoxynil
synthesized in this research controlled several weed species greater than bromoxynil itself, potentially due to enhanced binding
within the PSII binding pocket. Future research should compare this analogue to bromoxynil using optimized formulations at
higher application rates.
KEYWORDS: bromoxynil, herbicide resistance, glyphosate resistance, Palmer amaranth
INTRODUCTION
No new herbicidal mode of action has been incorporated into
weed management strategies in over 20 years. Prior to 1991 a
electron to a proximal pheophytin (Ph) molecule, which leads
to the formation of a P680 Ph radical pair. The occurrence
of the radical pair is brief, as an electron is transferred from the
■
•
+
•−
1
new mode of action was introduced to the market
approximately every three years. The dearth of novel herbicidal
modes of action has led to selection pressure for herbicide-
resistant weed biotypes in many different cropping systems.
Currently, over 400 weedy biotypes exist that are resistant to at
Ph anion to the nonmobile plastoquinone electron acceptor Q
A
•+
•−
6,7
to form the P680 Q
radical. Subsequently, an electron is
A
transferred to QB via several intermediates. P680+ is then
rereduced by extracting an electron from Tyr161 of the D1
2
•
+
•
protein, which leads to the formation of a TyrZ (H )Q
least one herbicide active ingredient. To that end, optimization
A
8
,9
of molecules with the potential to either provide new modes of
action or target established sites where resistance is not
radical pair. This cation is then stabilized by the Mn Ca
4
cluster in the oxygen-evolving complex, which upon further
3
,4
problematic is important for the future of weed management.
illumination extracts four electrons from two H O molecules to
2
A known target site with the potential to be exploited further
evolve O . On the other side of the membrane the electron is
2
is the plastoquinone binding site (Q ) of the photosystem II
B
transferred from Q to Q via the nonheme Fe. After accepting
A
B
(
PSII) reaction center. PSII consists of multiple protein
subunits and acts as a water:quinol oxidoreductase in all
oxygenic organisms including cyanobacteria, algae, and plants.
Upon illumination, the reaction center primary donor P680
becomes excited to a singlet state, where it then rapidly
undergoes charge separation. P680 chlorophyll donates an
Received: August 1, 2013
Revised: December 16, 2013
Accepted: December 19, 2013
Published: December 19, 2013
©
2013 American Chemical Society
329
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Journal of Agricultural and Food Chemistry
Article
two electrons Q is converted to plastoquinol and released
from the D1 binding pocket.
mays), flax (Linum usitatissimum), mint (Mentha spicata), garlic
B
5
22
(
Allium sativum), and onions (Allium cepa). Commercially,
Generally, PSII-inhibiting herbicides bind at the exchange-
bromoxynil is formulated as an ester to help penetrate the
hydrophobic cuticle of weeds. Subsequently, the herbicide is
10−14
able Q site of the D1 protein in the PSII reaction center.
B
Binding of these herbicides displaces plastoquinone at the QB
27
then hydrolyzed by plant enzymes to a free acid molecule.
site; thus, the less mobile Q is unable to be oxidized. This
A
leads to a charge recombination pathway resulting in the
formation of triple chlorophyll and singlet oxygen species,
which causes lipid peroxidation and general oxidative stress.
Typically, bromoxynil absorption is greater in shoots than in
roots because bromoxynil is conjugated in the soil and does not
translocate as easily to leaves.
5
,15
28,29
However, PSII inhibitors can interact with different portions of
this binding pocket of the D1 protein. On the basis of
simulation modeling and D1 mutant studies, phenolic
herbicides such as bromoxynil and ioxynil form hydrogen
bonds with His215 in the D1 protein, whereas it is believed that
triazines and ureas do not form a hydrogen bond at this
residue; rather they form a hydrogen bond at Ser of the D1
An understanding of bromoxynil’s impact on plant
physiology, availability of the target crystal structure of the
D1 protein, and bromoxynil’s importance to weed management
make bromoxynil an interesting compound for optimization.
Research was conducted to evaluate novel heterocyclic
analogues of bromoxynil synthesized on site for crop tolerance
and weed control. Further analysis was performed by simulating
the interaction of bromoxynil analogues in the binding pocket.
2
64
1
6,17
protein.
Currently, there are approximately 70 weedy biotypes that
are resistant to triazine herbicides, and many of the biotypes
2,18
contain a Ser to Gly264 mutation.
Comparatively, only one
weedy biotype is resistant to phenolic herbicides, and the
2
mechanism of resistance is metabolism based. Also, weedy
biotypes containing the Gly mutation are hypersensitive to
2
64
19,20
phenolic herbicides.
Resistance to phenolic herbicides is
less likely to occur for two reasons: (1) bromoxynil and ioxynil
typically have limited residual soil activity when compared to
triazine herbicides; thus, there is less selection pressure on the
2
1,22
seed bank for resistant biotypes
; and (2) Hist215 and three
other histidine residues form hydrogen bonds with the
nonheme iron. This amino acid residue is more conserved
than Ser264; thus, resistance is less likely to occur due to target
site mutations.
Physiologically, the interaction of His215 with phenolic
herbicides could be exploited further with analogues of different
electronegative properties because of the proximity of phenolic
16
herbicides to QA. Essentially, a molecular bridge forms
between Q , His of the D2 protein, the nonheme iron, His215
A
214
of the D1 protein, and Q . The CO group on bromoxynil,
B
which is deprotonated at physiological pH, forms a stronger
hydrogen bond with the NH imidazole group on His than
2
15
1
6
plastoquinone. The tighter bond coupled with slightly
different electron density characteristics of the herbicide likely
affects the conformation of the Q −His −iron−His −Q
A
214
215
B
system. Specifically, the hydrogen bond strength of the Q CO
A
23
group and the imidazole NH group off His214 is likely altered.
Subsequently, the redox potential of Q is reduced, which leads
A
+
−
to a charge recombination that favors P680 pPheo , ultimately
leading to a triplet chlorophyll state. Evaluation of analogues of
Figure 1. Chemical structures of bromoxynil and the heterocyclic
analogues synthesized and tested at the University of Tennessee
(Knoxville, TN, USA) during 2012 and 2013.
phenolic herbicides is warranted given how important the Q −
A
His −iron−His −Q molecular bridge is in PSII. In
2
14
215
B
2
4
particular, heterocyclic analogues (cyclic structures containing
atoms other than carbon) of the phenolic herbicides would be
interesting candidates for evaluation because of the potential
impact that a ring structure with different electronegative
properties would have on the π electron system of the
molecular bridge, Q binding site, and redox potential of Q .
MATERIALS AND METHODS
Compound Synthesis (Figure 1). A 3,5-dibromo-4-hydroxyben-
zonitrile bromoxynil salt (bromoxynil, Figure 2) compound was
synthesized following procedures from Gulbenk and Ruetman.
total of 300 mg of 4-hydroxybenzonitrile, 1500 mg of sodium bromate,
and 2050 mg of sodium bromide was dissolved in 15 mL of deionized
water in a 250 mL round-bottom flask with a magnetic stir bar.
Subsequently, the flask was capped with a rubber stopper and vented
with a nitrogen balloon while 6 mL of concentrated hydrochloric acid
■
30
A
B
A
Bromoxynil is an ideal phenolic PSII inhibitor for structural
modification because of its current significance to the
marketplace, limited environmental impact due to rapid
degradation in the soil, and effectiveness when applied
postemergence (POST) for control of many problematic
(
HCl) was slowly added to the reaction with a syringe over a period of
10 min. The flask was kept over a magnetic stir plate for 24 h at 20 °C.
Finally, the solution was subjected to vacuum rotary evaporation
2
5,26
broadleaf weeds including pitted morningglory.
Bromoxynil
is labeled for use in alfalfa (Medicago sativa), cereals, corn (Zea
3
30
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Journal of Agricultural and Food Chemistry
Article
conformation was determined by DART HR mass spectrometry MS
(
m/z): 277.9 (found). However, because 5-hydroxypyridine-2-carbon-
itrile is more electrophilic than 4-hydroxybenzonitrile, fewer
equivalencies of sodium bromate (753 mg) and sodium bromide
(1028 mg) were required to make pyridine.
The first time step of the synthesis of 4,6-dibromo-2-cyanopyr-
imidin-5-olate sodium salt (pyrimidine; Figure 4) required 300 mg of
-benzyloxypyrimidine-2-carbonitrile (Waterstone Techology, St.
5
Carmel, IN, USA), which was dissolved in 20 mL of acetic acid and
mixed with 30 mg of a 10% carbon palladium catalyst (Strem
Chemicals, Newburyport, MA, USA) in a 250 mL round-bottom flask
with a magnetic stir bar. Subsequently, the flask was capped and
vented with hydrogen gas and reacted over a magnetic stir plate for 36
h at 20 °C. After 36 h, the solution was poured onto a Celite filter aid
(
̈
Fisher Scientific, Fair Lawn, NJ, USA) covered Buchner funnel
Figure 2. Synthesis of 3,5-dibromo-4-hydroxybenzonitrile (bromox-
ynil) evaluated in greenhouse trials at the University of Tennessee
connected to an aspirator. The liquid phase was subjected to vacuum
rotary evaporation and then dried under a high vacuum for 24 h. The
structure of 5-hydroxypyrimidine-2-carbonitrile was confirmed by
DART HR mass spectrometry (m/z): 121.1 (found). Next, 600 mg of
sodium bromate and 900 mg of sodium bromide were dissolved in 15
mL of deionized water with 250 mg of 5-hydroxypyrimidine-2-
carbonitrile in a 250 mL round-bottom flask with magnetic stir bar.
Subsequently, the flask was capped with a rubber stopper and vented
with a nitrogen balloon while 6 mL of HCl was slowly added to the
reaction with a syringe over 10 min. The flask was kept over a
magnetic stir plate for 24 h at 20 °C. The solution was then subjected
to vacuum rotary evaporation. The structure of 4,6-dibromo-2-cyano-
pyrimidin-5-olate sodium was confirmed by DART HR mass
spectrometry MS (m/z): 278.89 (found).
(
Knoxville, TN, USA) during 2012 and 2013.
(
Buchi R-114. Nine Buchi Labortechnik AG, Flawil, Switzerland).
Structural conformation was determined by direct analysis in real time
DART) HR mass spectrometry MS (m/z): 276.91 (found). A 2,4-
(
dibromo-6-cyano-5-pyridin-3-olate sodium salt (pyridine) was also
brominated using the same technique using 5-hydroxypyridine-2-
carbonitrile starting material (pyridine; Figure 3). Structural
Synthesis of 2,6-dibromo-1-oxidopyridin-1-ium-4-carbonitrile (pyr-
idine-N-oxide; Figure 5) required a different bromination technique.
Bromination of pyridine at the 2- and 6-positions within the ring
structure of pyridine is difficult and requires drastic conditions that
typically involve high pressure and vapor phase reactions at
2
4
temperatures >500 °C. However, displacement of chlorine with
bromine at the 2,6-positions on pyridine had previously been
3
1
performed under less drastic conditions. To avoid vapor phase
reactions, 213 mg of 2,6-dichloro-4-pyridinecarbonitrile was dissolved
in 10 mL of acetic acid in a capped 250 mL three-neck round-bottom
flask with a magnetic stir bar vented with a nitrogen balloon. The flask
was attached to a condenser and an addition funnel in a hot oil bath at
Figure 3. Synthesis of 2,4-dibromo-6-cyano-5-pyridin-3-olate (pyr-
idine) evaluated in greenhouse trials at the University of Tennessee
1
03 °C over a magnetic stir plate. A total volume of 2 mL of
(Knoxville, TN, USA) during 2012 and 2013.
hydrobromic acid was added to the solution through the addition
funnel over a period of 5 min. After 9 h, 2.9 mL of phosphorus
Figure 4. Synthesis of 4,6-dibromo-5-hydroxypyrimidine-2-carbonitrile (pyrimidine) evaluated in greenhouse trials at the University of Tennessee
Knoxville, TN, USA) during 2012 and 2013.
(
3
31
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Journal of Agricultural and Food Chemistry
Article
Figure 5. Synthesis of 2,6-dibromo-1-oxidopyridin-1-ium-4-carbonitrile (pyridine-N-oxide) evaluated in greenhouse trials at the University of
Tennessee (Knoxville, TN, USA) during 2012 and 2013.
tribromide was added to the reaction with a syringe over 45 min and
the reaction continued for an additional 16 h at 130 °C. The product
of this reaction was a red solution that was cooled in an ice bath and
diluted with 20 mL of dichloromethane and 40 mL of deionized water.
The phases were mixed in a separation funnel, and the organic layer
was extracted and subjected to vacuum rotary evaporation. The
structure of the product, 2,6-dibromopyridine-4-carbonitrile, was
confirmed through DART HR mass spectrometry MS (m/z): 272.5
(Fisher Scientific International Inc., Hampton, NH, USA) prior to
application. Subsequently, the solutions were mixed with 0.1%
nonionic surfactant. The solutions were then applied to the plants
using an enclosed spray chamber (Generation III track sprayer;
DeVries 10 Manufacturing, Hollandale, MN, USA) in deionized water
−1
carrier at 215 L ha with an 8004 EVS nozzle (Teejet, Wheaton, IL,
USA).
Visual percent plant injury ratings were recorded 7 days after
treatment (DAT), whereas dry aboveground biomass was recorded 14
days after treatment. Aboveground biomass was harvested from every
plant and oven-dried (Thelco 130D laboratory oven, Precision
Scientific) at 65 °C for 3 days and weighed. Chlorophyll fluorescence
(F /F ) was recorded 10 DAT from soybean using a pulse-modulated
(
found). A total of 200 mg of 2,6-dibromopyridine-4-carbonitrile was
then dissolved in 3 mL of glacial acetic acid and 1.5 mL of 30%
hydrogen peroxide. The reaction mixture was exposed to 70 W of
microwave irradiation for 20 min at normal absorption using a Biotage
Initiator robot (Biotage, Kungsgatan, Sweden) following a protocol by
V
M
32
Khrustalev et al. Next, 15 mL of deionized water was added to the
final solution and later removed using vacuum rotary evaporation. The
product, 2,6-dibromo-1-oxidopyridin-1-ium-4-carbonitrile (pyridine-
N-oxide), was confirmed using DART HR mass spectrometry MS
fluorometer (OS1-FL, Opti-Sciences, Inc., Hudson, NH, USA).
Glyphosate Resistant Palmer Amaranth (Amaranthus pal-
meri) Greenhouse Study. A second study was conducted to evaluate
bromoxynil, pyridine, pyrimidine, and pyridine-N-oxide for control of
glyphosate-resistant Palmer amaranth (gift from Dr. Chad Brommer,
BASF Research Triangle Park, NC, USA) from February 20 to March
13, 2013. Potting medium and irrigation conditions in the greenhouse
were the same as previously described for the weed and crop response
greenhouse study. The application rate in this study was increased to
(
m/z): 277.6 (found).
All mass spectrometry analyses were conducted at the Mass
Spectrometry Center located in the Department of Chemistry at the
University of Tennessee. The DART analyses were performed using a
JEOL AccuTOF-D time-of-flight (TOF) mass spectrometer with a
DART ionization source from JEOL USA, Inc. (Peabody, MA, USA).
Mass spectrometry solutions were prepared with methanol.
−1
0.56 kg ha considering that these compounds imparted minimal
−1
injury to Palmer amaranth when applied at 0.28 kg ha in prior
Weed and Crop Response Greenhouse Study. To evaluate the
crop tolerance and weed control efficacy of synthesized bromoxynil
analogues (bromoxynil, pyridine, pyrimidine, and pyridine-9N-oxide),
an array of crops and weeds were propagated at the University of
Tennessee Plant Science Greenhouse (Knoxville, TN, USA;
experiments.
Statistical Analysis. All experiments were conducted in a
completely randomized design (CRD) with three replications. All
data were subjected to analysis of variance (ANOVA), and means were
separated using Fisher’s protected LSD at α = 0.05 in SAS (SAS
Institute v. 9.2, Cary, NC, USA). Percent visual injury ratings were
arscine transformed prior to analysis. Interpretations were not different
from nontransformed data. Thus, nontransformed percent visual
ratings are presented for clarity.
Docking Calculations. A crystal structure of the D1 and D2
proteins was extracted from the PSII complex of Thermosynechococcus
elongatus at 2.9 Å resolution (Protein Data Bank entry 3BZ1) and
incorporated into the Molecular Operating Environment (MOE,
2012.10; Chemical Computing Group Inc., Montreal, QC, Canada)
program. Structural optimization was performed on the Q binding
site to include the nonheme iron, the four surrounding histidine
ligands (D1-Hist215, D1-His272, D2-His214, and D2-His268), and the D1
residues of the QB binding site that consisted of Met214, Leu218, Val219
Tyr246, Ile248, Ala 251, His252, Phe255, Ser264, Phe265, and Leu 271). The
bromoxynil compounds were constructed in MOE; subsequently,
35.946471 latitude, 83.938558 longitude) including soybean, cotton,
redroot pigweed, velvetleaf, large crabgrass, and pitted morningglory.
All plants were grown in plastic 10.2 × 10.2 cm circular pots (Dillen
Products/Myers Industries, Inc., Middlefield, OH, USA) containing
peat-based growing media (ProMix BX Mycorrhizae, Premier Tech
Horticulture, Quakertown, PA, USA) and treated 10 days after
seeding. The experiment was conducted from Octboer 6 until 30,
33
2012, and repeated from November 14 until December 8, 2012.
During both experimental runs the average air temperature in the
climate-controlled greenhouse was 23 °C, whereas the lowest/highest
air temperatures recorded were 20/30 °C. Mean daytime light levels
B
−2
−1
during this period were 280 μmol m s . Irrigation was supplied
through an overhead mist system and was withheld for 24 h after
experimental herbicide application to ensure adequate time for foliar
absorption of the experimental herbicides.
,
−1
34
The compounds were applied POST at a rate of 0.28 kg ha . This
rate was chosen because it is the lowest labeled rate of bromoxynil
Amber forcefield was used to optimize molecular mechanics.
Geometry optimization and energy minimization were applied to the
system containing bromoxynil and the 3BZI protein. Bromoxynil was
then inserted proximal to the binding pocket, and docking simulations
were performed. Heterocyclic analogues were substituted for
bromoxynil, and docking simulations were repeated with the
analogues. To simulate physiological pH conditions, the CO group
22
recommended for weed control. Additionally, the quantities of the
analogues for use in studies were limited given that the yields of the
aforementioned synthesis processes ranged from 20 to 35%. All
compounds were dissolved in a mixture of acetone (3 mL) and
deionized water (32 mL) and agitated using a (CL-18) sonicator
3
32
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Journal of Agricultural and Food Chemistry
Article
Table 1. Crop and Weed Species Injury 7 Days following Applications of Bromoxynil and the Heterocyclic Analogues of
−1
a
Bromoxynil at 0.28 kg ai ha
injury , (%)
bc
treatment
soybean
cotton
pitted morningglory
redroot pigweed
velvetleaf
large crabgrass
bromoxynil
pyridine
35 a
41 a
16 b
45 a
36 b
16 c
25 c
38 b
45 b
45 b
22 c
55 a
12 b
30 a
18 b
33 a
21 a
11 a
18 a
25 a
6 a
9 a
pyrimidine
pyridine-N-oxide
5 a
13 a
a
b
Means were combined from two runs of a greenhouse experiment conducted in Knoxville, TN, USA, in 2012. Injury was evaluated visually on a 0
c
(
no injury) to 100% (complete kill) scale relative to a nontreated check. Means within the same column followed by the same letter are not
significantly different according to Fisher’s protected least significant difference test at the 0.05 level.
Table 2. Crop and Weed Species Aboveground Biomass 14 Days following Applications of Bromoxynil and the Heterocyclic
−1
a
Analogues of Bromoxynil at 0.28 kg ai ha
aboveground biomass , (g per pot)
bc
treatment
soybean
cotton
pitted morningglory
redroot pigweed
velvetleaf
large crabgrass
bromoxynil
pyridine
1.20 a
0.92 b
1.18 a
0.84 b
1.33 a
0.30 bc
0.21 c
0.4 b
0.95 b
0.79 bc
0.90 b
0.35 e
1.30 a
0.24 ab
0.18 b
0.23 ab
0.10 c
0.25 a
0.60 a
0.36 b
0.26 b
0.30 b
0.25 b
0.50 a
0.45 bc
0.46 bc
0.48 bc
0.62 a
pyrimidine
pyridine-N-oxide
nontreated
0.19 c
0.68 a
a
b
Means were combined from two runs of a greenhouse experiment conducted in Knoxville, TN, USA, in 2012. Means within a column followed by
c
the same letter are not significantly different according to Fisher’s protected least significant difference test at the 0.05 level. Above ground biomass
was harvested from every plant and oven-dried at 65 °C for 3 days and weighed.
from all compounds was deprotonated. The simulation distance
between herbicide and target amino acids in the binding pocket was
transcribed in Symx Draw (version 4.0, Accelrys Inc., San Diego, CA,
USA) to enhance visual clarity.
emulsifiers, detergents, wetting agents, and other surfactants
35
that improve herbicide efficacy.
Pyridine reduced photosynthetic efficiency more than any
other treatment (Table 3.). Analysis of F /F data did not
V
M
RESULTS AND DISCUSSION
Table 3. Impact of Compounds Applied to Soybeans at 0.28
■
kg ai ha− on F /F Values Taken 10 Days after Treatment
1
V
M
Weed and Crop Response. No treatment-by-experimental
run interactions were detected in percent injury or above-
ground biomass data; therefore, data from both runs were
combined.
Application of bromoxynil resulted in >35% injury to
soybean, cotton, and pitted morningglory (Table 1). However,
when bromoxynil was applied to velvetleaf, redroot pigweed, or
large crabgrass, injury measured <25% (Table 1).
a
treatment
FV/FM
bromoxynil
pyridine
0.456 b
0.222 c
0.671 a
0.456 b
0.804 a
pyrimidine
pyridine-N-oxide
nontreated
a
Means within a column followed by the same letter are not
Different trends were observed with applications of
heterocyclic bromoxynil analogues. Applications of the
pyridines (pyridine and pyridine-N-oxide) generally injured
all plant species more than pyrimidine. Injury ratings were
similar when pyridine and pyridine-N-oxide were compared;
however, applications of pyridine-N-oxide resulted in more
injury to pitted morningglory and cotton than pyridine (Table
significantly different according to Fisher’s protected least significant
difference test at the 0.05 level.
always correlate with visual ratings. For example, pyridine
reduced PSII efficiency significantly more than pyridine-N-
oxide; however, pyridine-N-oxide generally induced more
phytotoxicity than pyridine (Tables 2 and 3). Pyrimidine did
not reduce F /F significantly more than the nontreated check.
1
). This response was especially pronounced following
V
M
treatment to cotton, where application of pyridine-N-oxide
resulted in 38% injury whereas application of pyridine resulted
in only 16% injury. Interestingly, pyridine and pyridine-N-oxide
were also more injurious to redroot pigweed when compared to
bromoxynil. None of the herbicides injured large crabgrass
The most interesting trend observed in the weed and crop
response study was the ability of the pyridine analogues
(
pyridine and pyridine-N-oxide) to inflict greater phytotoxicity
on redroot pigweed than bromoxynil. Although the redroot
pigweed injury observed in this study would not be considered
acceptable, the potential impact on weed management that the
pyridine herbicides would have when applied as a formulated
product at higher rates is intriguing.
>
13%.
Generally, aboveground biomass data supported visual
assessments of plant injury (Table 2). However, pyridine
reduced velvetleaf aboveground biomass more than bromox-
ynil, which caused greater visual injury than pyridine. Technical
grade material was applied in our research, and volatility in the
spray chamber may have limited the effectiveness of the
compound. Commercially formulated herbicides often have
Glyphosate-Resistant Palmer Amaranth Study. Appli-
cations of bromoxynil and pyridine all injured glyphosate-
resistant Palmer amaranth >80% (Table 4). Although it cannot
be determined statistically, it appeared that doubling the
application rate to 0.56 kg ha− in this study greatly improved
1
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Journal of Agricultural and Food Chemistry
Article
Table 4. Glyphosate-Resistant Palmer Amaranth
(
Amaranthus palmeri) Injury and Aboveground Biomass
following Applications of Bromoxynil and the Heterocyclic
Analogues of Bromoxynil at 0.56 kg ai ha in a Greenhouse
Experiment Conducted in Knoxville, TN, USA, in 2013
−1
a
b
,
c
aboveground biomass ,
cd
treatment
injury (%)
bromoxynil
pyridine
87 a
83 a
25 b
25 b
0.18 cd
0.08 d
0.41 b
0.30 bc
0.80 a
pyrimidine
pyridine-N-oxide
nontreated
a
Injury and aboveground biomass data were collected 7 and 14 days
b
after treatment, respectively. Injury was evaluated visually on a 0 (no
injury) to 100% (complete kill) scale relative to the nontreated check.
c
Means within a column followed by the same letter are not
significantly different according to Fisher’s protected least significant
d
difference test at the 0.05 level. Aboveground biomass was harvested
from every plant and oven-dried at 65 C for 3 days and weighed.
the efficacy of bromoxynil and pyridine against pigweed species.
Application of pyrimidine and pyridine-N-oxide injured
glyphosate-resistant Palmer amaranth ∼25%. This response
was surprising considering the level of redroot pigweed injury
observed with pyridine-N-oxide in the weed and crop tolerance
study (Table 1). Whereas pyridine-N-oxide reduced glyphosate-
resistant Palmer amaranth aboveground biomass more than
pyrimidine, reductions in aboveground biomass were less than
with bromoxynil and pyridine (Table 4). Glyphosate-resistant
Palmer amaranth biotypes typically exhibit increased tran-
36
scription of EPSP-synthase. Therefore, increased expression
of cytochrome-P450s, glutathione-S-transferases, and altered
ABC-transporters in Palmer amaranth are likely not responsible
for the decreased effectiveness of pyridine-N-oxide in this study.
Docking Simulations. Bromoxynil docking in our research
16
was similar to work done by Takahashi et al. Interestingly, the
distance of the hydrogen bond between the CO group from the
pyridine and the D1 His215 was closer than was observed with
bromoxynil (Figure 6). This may explain why pyridine
performed better than bromoxynil in the weed and crop
response study. However, pyridine is supposedly less injurious
Figure 6. Docking simulations of bromoxynil and heterocyclic
analogues (A) bromoxynil, (B) pyridine, (C) pyrimidine, and (D)
pyridine-N-oxide. The hydrogen bond distance between the ligand and
30
to broadleaf crops as described by Gullbenk and Ruetman.
o
The decreased susceptibility of broadleaf crops to pyridine may
be due to metabolism. The pyrimidine analogue was inverted in
the binding pocket relative to the other compounds tested, and
the CN group formed a hydrogen bond with His215 during the
simulation. Hydrogen bonding with a CN group is typically
weaker when compared to a CO group; additionally, the bond
distance was greater in the pyrimidine simulation than in the
pyridine simulation. This could explain why pyridine resulted in
more injury than pyrimidine during our greenhouse trials.
histidine215 is in angstroms (A ).
Structure−Activity Relationship. The primary metabolite
of bromoxynil in nonsusceptible plants and microbial soil
systems results from the hydrolysis of the nitrile group to a
3
7,38
carboxylic acid or amide.
Additionally, replacement of the
bromines with hydroxyl groups results in a less herbicidal
8
,37
metabolite.
This hydroxylation reaction involves the
formation of a hydroxide anion, which then initiates an SN2
reaction by attacking the meta-positioned carbons on
bromoxynil. An electron pair is then donated to bromine,
which leaves the ring and forms a Br − ion. Pyridine and
pyrimidine analogues are less electron dense compared to
bromoxynil and would be more susceptible to hydroxylation
and other nucleophilic attacks. If hydroxylation of bromine is
the metabolic mechanism that results in decreased efficacy of
bromoxynil, then pyrimidine would be the most readily
metabolized analogue tested because it is the most electrophilic.
This theory is supported by the results of the greenhouse trials
as well as what was observed in the docking calculations.
Pyrimidine did not bind as strongly to His215 in the binding
Pyrimidine did not form a strong hydrogen bond with His215
.
Different parameters likely need to be applied to the
environment to account for the unique properties of a
pyridine-N-oxide bromoxynil analogue. An alternative simu-
lation performed with pyridine-N-oxide showed that the
oxyanion interacted with the hydrogen backbone of His215 as
opposed to the imidazole ring. Additionally, a hydogen bond
formed between the ring of pyridine-N-oxide and the sulfur
atom on Met214. The presence of a cation at the QB binding
site may influence the π electron system of the Q −His
−
A
214
Iron−His −Q bridge, which is interesting information that
215
B
could be used to design future compounds.
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Journal of Agricultural and Food Chemistry
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pocket as bromoxynil or pyridine and is likely easier to
metabolize, thus reducing weed and crop injury. Pyridine
formed a stronger hydrogen bond with His215 in the binding
pocket compared to bromoxynil; however, it is likely
metabolized more easily than bromoxynil, and thus did not
significantly injure all plants more than bromoxynil. As
mentioned earlier, the pyridine-N-oxide analogue is similar to
bromoxynil with regard to electrophilicity and therefore may
not be as easily hydroxylated as the pyridine and pyrimidine
analogues. Interestingly, application of pyridine-N-oxide
resulted in greater redroot pigweed control than bromoxynil
with less damage to cotton and soybean. Alternatively, the
structure of pyridine-N-oxide may be altered by metabolism in
both crop and weed species, resulting in greater herbicidal
activity. This scenario could be possible because the docking
simulations indicated that pyridine-N-oxide hydrogen bond
distance with His215 was not optimal.
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AUTHOR INFORMATION
■
(
herbicides. In Herbicide Resistance in Plants: Biology and Biochemistry;
Powles, S. B., Holtum, J. A. M., Eds.; CRC Press: Boca Raton, FL,
USA, 1994; pp 27−60.
Present Address
M.A.C.) Syngenta Crop Protection, Vero Beach, Florida
¶
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32967.
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Notes
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The authors declare no competing financial interest.
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