Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
  • A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
  • A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
  • A01N43/56—1,2-Diazoles; Hydrogenated 1,2-diazoles
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
  • C12Y103/05—Oxidoreductases acting on the CH-CH group of donors (1.3) with a quinone or related compound as acceptor (1.3.5)
  • C12Y103/05002—Dihydroorotate dehydrogenase (1.3.5.2)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/902—Oxidoreductases (1.)
  • G01N2333/90206—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)

Definitions

• This invention relates to the production of herbicidal compounds and compositions comprising the same which inhibit dihydroorotate dehydrogenase.
• sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named BA9596WOPCT_ST25.txt created on October 26, 2016 and having a size 93 kilobytes and is filed concurrently with the specification.
• sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
• One aspect of the present invention is a method for the production of herbicidal compounds comprising the following steps:
• step (c) preparing a herbicidal composition comprising the compound identified in step (a) and tested in step (b).
• the screening step (a) of the method may include an assay step selected from in-vitro activity assays, computer modeling assays and binding assays.
• the method may further include the step of verifying that the provided candidate compound is not a general enzyme inhibitor.
• the screening step makes use of a DHOD polypepetide having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
• the invention also includes the manufacturing or use of a herbicidal composition comprising at least one additional component selected from the group consisting of surfactants, solid diluents and liquid diluents.
• Another aspect of the invention is a method for the production of herbicidal compounds, comprising testing a candidate compound in a DHOD activity inhibition assay wherein the assay utilizes DHOD from a weed to be controlled.
• Another aspect of the invention is a method of controlling weeds comprising applying a herbicidally effective amount of a DHOD inhibitor produced by the method described herein to a locus in need of such treatment.
• Yet another aspect of the invention is a herbicidal composition
• a herbicidal composition comprising a DHOD inhibitor produced by the method described herein in combination with another herbicide.
• Yet another aspect of the invention is a herbicidal composition
• a herbicidal composition comprising a DHOD inhibitor produced by the method described herein wherein the inhibitor is at least in part an indirect DHOD inhibitor
• Yet another aspect of the invention is a DHOD inhibition assay utilizing a biosensor or computer modeling.
• compositions comprising, “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any limitation explicitly indicated.
• a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, or method.
• transitional phrase consisting essentially of is used to define a composition, method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention.
• a DHOD inhibition assay refers to assays which measure inhibition of enzymatic activity as well as binding assays and in-silico methods of computer aided molecular design.
• a "general enzyme inhibitor” as referred to herein is a compound that inhibits enzymes generally but not by specific interaction with DHOD. For example some compounds might denature certain enzymes and thus render them inactive generally. By way of example but not limitation one might use assays for enzymes unrelated in structure and function from DHOD such as beta-galactosidase or alkaline phosphatase to identify general enzyme inhibitors.
• Indirect inhibitors are compounds that inhibit DHOD following metabolism of those inactive compounds to an active inhibitor in plants.
• a herbicidally effective amount of DHOD inhibitor refers to an amount of DHOD inhibitor sufficient to kill or inhibit the growth of the weed it is desired to control.
• weed(s) relates to any unwanted vegetation and includes, for example, undesired carry-over or “rogue” or “volunteer” crop plants in a field of desired crop plants.
• DHOD mitochondrial membrane bound dihydroorotate dehydrogenase
• DHOD inhibition assays to identify antifungal compounds useful against agronomically important fungi are described in U.S. Pat. No. 5,976,848.
• U.S. Pat. No. 7,320,877 describes the use of an assay to identify plant dihydroorotase inhibitors as herbicidal active ingredients and describes a test system coupling plant dihydroorotase and plant dihydroorotate dehydrogenase.
• Dihydroorotate dehydrogenase was included in the test system not as an intended target of interest for inhibition but rather as an enzymatic means to generate NADH which was then detected as a means of measuring dihydroorotase activity indirectly.
• U.S. Patent Publication No. 2002/0058244 describes a method of detecting uracil biosynthesis inhibitors using a plant whole tissue assay measuring the conversion of
• Herbicidal compounds which are unequivocal inhibitors of plant DHOD are desireable. We have discovered that compounds described in WO 2015/084796 (DuPont) are potent inhibitors of plant DHOD. Therefore this is a previously undescribed mode of action for herbicides of practical application.
• Embodiment IP is a method for the production of herbicidal compounds comprising the following steps:
• step (c) preparing a pesticidal composition comprising the compound identified in step (a) and verified in step (b).
• Embodiment 2P The method of Embodiment IP wherein screening step (a) is selected from the group of in-vitro activity assays, computer modeling assays and binding assays.
• Embodiment 3P The method for production of herbicidal compounds which comprises testing a candidate compound in a DHOD activity inhibition assay wherein the assay utilizes DHOD from a weed to be controlled
• Embodiment 4P The screening step makes use of a DHOD having 50%, 51%, 53%, 54%, 55%, 56% 57% 58%, 59% 60%, 61%, 62%, 63%, 64%, 65% 66% , 67%, 68% 69% 70% 71%, 72% 73%, 74% 75%, 76%, 77% 78%, 79%, 80%, 81% 82%, 83%, 84%, 85% 86%, 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the amino acid sequence of SEQ ID NO: 2 or 3.
• Arabidopsis Landsberg erecta (Ler-0) seeds were sterilized in chlorine gas and plated on 0.5x Murashige and Skoog salts (MS) plant growth media, containing 0.05% 2-(N- Morpholino)-ethanesulfonic acid (MES, CA# 1266615-59-1), 0.5% sucrose, and 0.8% Phytagar) in a 24-well plate.
• MS Murashige and Skoog salts
• Ethyl methanesulfonate (EMS)-mutagenized M 2 seeds of Arabidopsis Ler-0 (M2E- 04-07) were purchased from Lehle Seeds (Round Rock, TX). In order to screen resistant mutant Arabidopsis plants to Compound 5, about 12,000 sterilized M 2 seeds were distributed on MS plates containing Compound 5. Plate conditions for EMS-mutagenized Col-0 seeds were identical to those used for the IC 50 determination, except the concentration with 10 times of the IC 50 concentration of Compound 5 (5 ⁇ ). Resistant seedlings were selected after seven to 10 days on selective medium and transferred to soil.
• One such resistant line designated 45R1 was grown in a growth chamber in a 4-inch (10 cm) pot using Metromix 360 potting soil under cool -white fluorescent light and a 16 h/8 h day/night photoperiod. Compound 5 resistance was confirmed in the next generation with the same concentration of herbicide that was used in the original selection.
• Crosses of the mutant to a diverged genome were required in order to reduce unlinked genetic background within selected resistant mutants.
• BC1 Fl seeds and out-cross 1 (OC1) Fl seeds the selected Compound 5-resistant mutant males were crossed with wild-type Ler-0 females and wild-type Columbia (Col-0) females, respectively.
• Compound 5-resistant Fl seeds were generated by pollinating emasculated flowers of wild-type plants with pollen from mutant plants.
• BC2 and OC2 were created from BC1 with wild-type Ler-0 and OC1 with wild- type Col-0 plants, respectively.
• the created Fl (BC1 and OC1) and F2 (BC2 and OC2) seeds were plated on MS media containing the same concentration of Compound 5 as was used in the original screen.
• DHOD Arabidopsis DHOD
• a metabolomics approach was conducted using a combination of the LC and GC/MS analysis.
• Two analogs e.g, Compound 80 and Compound 102
• Plant samples were collected at 3 time points (6, 24 and 72 hours after treatments) and four replicates were analyzed per each condition.
• Approximately 100 mg of frozen samples were homogenized and extracted in 1 mL of a mixture of chloroform/methanol/water (1 :2.5: 1) for 30 min at 4 °C. After spinning down the insoluble material, supernatants were transferred to fresh tubes and dried down in a Speed- Vac centrifuge.
• PCA Principle component analysis
• 4,5-dihydoroorotic acid which is the substrate of DHOD in pyrimidine de novo synthesis
• significant alteration of N-carbamyl-D,L- aspartic acid which is another intermediate of pyrimidine de novo biosynthesis was observed: increased at 6 h (2.8 fold) and peaked at 24 h (7.4 fold) decreasing at 72 h (4.1 fold) compared to the control.
• the method for the production of potential herbicides does not require use of any particular DHOD inhibition assay. Suitable assays are described hereinafter, but those skilled in the art can readily substitute functionally equivalent test methods. For example, although the in-vitro screening assay described hereinafter uses DHOD produced by A. thaliana, the DHOD produced by other plants may be substituted. For example the DHOD of a commercially significant weed (either purified by conventional biochemical techniques or preferably recombinantly) may be used.
• the assays to be employed include but are not limited to the group selected from one or more of the following assays: in-vitro activity assays, computer modeling assays and binding assays.
• active in the DHOD inhibition assay means that a measurable reduction in DHOD activity is observed.
• DHOD inhibitor encompasses any compound that: (a) produces measurable inhibition in a DHOD inhibition assay using DHOD from a plant; (b) is not a general enzyme inhibitor. It should be understood that no herbicidal compound was known to have DHOD inhibition as its mode of action at the time the present invention was made.
• Preferred DHOD inhibitors are those which produce at least a measurable reduction in DHOD activity when tested at 10 ⁇ g/mL in the A. thaliana DHOD Inhibition Assay described hereinafter. We have found it convenient in our work to restrict further testing to those compounds that cause at least a 50% reduction in DHOD activity. This is a novel herbicidal mode of action and its discovery opens up the opportunity of identifying novel chemicals that inhibit the same enzyme target either directly or following metabolism to an active inhibitor in-plantae ("indirect inhibitors").
• More preferred DHOD inhibitors are those which produce at least a 25% reduction in
• DHOD variants described herein lack the N-terminal mitochondrial signal peptide as it is not a catalytically essential structural feature and its presence hinders solubility and expression. C-terminal "tags" were attached to facilitate purification.
• host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
• Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY 1986 and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
• bacterial cells such as E. coli, Streptomyces and Bacillus subtilis cells
• fungal cells such as yeast cells and Aspergillus cells
• insect cells such as Drosophila S2 and Spodoptera Sf9 cells
• animal cells such as CHO, COS and HeLa as well as plant cells.
• Bacterial systems are generally preferred.
• Such systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
• viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
• vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmid
• the expression systems may contain control regions that regulate as well as engender expression.
• any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used.
• the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL.
• secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
• the cells may be harvested prior to use in the screening assay. If DHOD is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
• DHOD can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, high performance liquid chromatography. Most preferably, affinity chromatography is employed when the protein has been "tagged". Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.
• DHOD for use in the assay was obtained in the following exemplary fashion. While the method described below is one using a recombinant approach native enzyme prepartions can be purified from whole plants, plant parts, and plant cells in culture by conventional biochemical means well within the expertise of the skilled artisan.
• Example 1
• Plant DHOD enzyme(s) derived from A. thaliana, Setaria italica, wheat, rice, corn, soybean, sugar beet and Amaranthus hypochondriacus were produced in a heterologous, E. coli, expression system.
• DHOD coding sequences were cloned into E. coli expression vectors encoding a lOx-His-tag in fusion with the C-terminus of each DHOD enzyme.
• the DHOD sequences were truncated at the 5'-end to remove a mitochondrial leader sequence and membrane-associated domain resulting in a soluble expressed protein with a N-terminal truncation of 75 to 80 amino acids, depending on the plant species.
• BL21 DE3 cells transformed with expression vectors encoding for DHOD were grown in liquid culture at 37 °C until O.D. 0.6, cooled to 16 °C and induced with 0.2 mM isopropyl thiogalactopyranoside (TPTG) for approximately 16 h.
• E. coli cells were collected by centrifugation and lysed by sonic disruption in Buffer A (25 mM KP04 (7.4), 5% glycerol, 300 mM KCl, lx protease inhibitor tablets).
• Quinone stock solutions (2 mM) [Qio-] were prepared fresh by solubilization in 5% Brij-35 detergent liposomes using micro cavitation.
• reactions were pre-equilibrated for 5 min at 25° C with either DMSO or synthetic compounds and reactions were initiated by addition of dihydroorotate at a final concentration of 1 mM in a total reaction volume of 0.2 mL.
• the final DMSO concentration in the assay was 0.1%.
• the progress of the reaction was monitored by UV-Vis spectroscopy at either 300 or 600 nM.
• DHOD inhibition was measured by spectrophotometrically observing (at 610 nm) the reduction of DCIP by electrons liberated when dihydroorotate was oxidized to orotate (electrons were transferred to DCIP in the reaction mixture via ubiquinone- 10).
• DCIP was observed as a dark blue color that strongly absorbing at 610 nm but not observed when reduced.
• IC50 values were determined for the compounds and enzyme species listed below in Table 1 where a "-" means not tested.
• Table 1 setaria IC50 (nM) rice IC50 (nM) soy IC50 (nM) corn IC50 (nM)
• Cmpd # Refers to the compound number in Index Table A, B or C.
• SEQ ID NO: Refers to the Sequence Identification Number.
• the assay method described in Example 2 can be modified to accommodate high throughput screening by methods well known in the art.
• the assay is preferably conducted using Falcon® 96-well, flat bottom polystyrene plates having 96 wells arrayed in 12 columns and 8 rows.
• the inhibition of DHOD by Compound 204 may desirably be used to standardize the effects of other test compounds on DHOD activity. Certain well(s) are used for determining background and uninhibited activity measurements.
• Preparation of the plates is preferably automated, using robotic workstations to dilute the stock compounds and add appropriate volumes of reaction solutions and compounds to the individual wells of the 96-well microtiter plate.
• the assay is initiated by adding 5 ⁇ of concentrated DHOD solution to each well, which is conveniently done using an Eppendorf 8-channel dispenser. After this solution is added, the concentration of test compound in each well is 10 ⁇ g/mL. The contents of each well on the plate is mixed, and changes in absorbance at 300 nm are recorded every 10 s for 5 min using the THERMO maxTM (Molecular Devices) plate reader (set at 30 °C incubation temperature).
• THERMO maxTM Molecular Devices
• the rate of absorbance change per minute (mAbsgio nm/min) due to reduction of DCIP is then calculated for each sample and the background controls.
• a plot of absorbance versus time for each well yields a downward sloping line, reflecting decreased absorbance as the DCIP is reduced. Under the conditions of the assay described above, the plot is essentially linear.
• the rate of absorbance change per minute (mAbs3oo nm/min) due to reduction of DCIP is then calculated for each sample and the background controls.
• a plot of absorbance versus time for each well yields an upward sloping line, reflecting increased absorbance as the DCIP is reduced. Under the conditions of the assay described above, the plot is essentially linear. Compounds that inhibit DHOD reduce the reaction rate and result in a linear plot with a reduced slope.
• the present invention is directed to herbicidal use of compounds that inhibit DHOD, as opposed to herbicidal use of compounds that inhibit enzymes generally.
• An example of a compound that inhibits enzymes generally is diethyl pyrocarbonate, which reacts preferentially with protein thiol and amino groups.
• the active compound may be tested in a second enzyme assay.
• a suitable assay for this purpose is, for example, the E. coli alkaline phosphatase assay described by Garen and Levinthal, Biochim. Biophys. ACTA 1960, 38, 470. If the compound is not inhibitory in the second enzyme assay, it may generally be safely concluded that the compound does not inhibit enzymes generally.
• Biological efficacy of a herbicidal compound in whole organisms is influenced by many factors, including not only intrinsic activity of the compound, i.e. efficiency of its interaction with the target molecule, but also stability of the compound and ability of the compound to be translocated to the target site.
• the DHOD inhibition assay measures the intrinsic activity of the compound. It will be appreciated by those skilled in the art that once a potential herbicide is detected using the DHOD assay, conventional techniques must be used to determine the usefulness of the compound in various environments.
• a herbicidally effective amount of the compounds of this invention is determined by a number of factors. The exact concentration of compound required varies with the weed to be controlled, the type of formulation employed, the method of application, climate conditions and the like. Generally, a herbicidally effective amount of compounds of this invention is about 0.005 to 20 kg/ha with a preferred range of about 0.01 to 1 kg/ha. One skilled in the art can easily determine the herbicidally effective amount necessary for the desired level of weed control.
• compositions that inhibit DHOD include blackgrass (Alopecurus myosuroides), downy bromegrass ⁇ Bromus tectorum), green foxtail (Setaria viridis), Italian ryegrass (Lolium multiflorum), wild oat (Avena fatua), catchweed bedstraw (Galium aparine), bermudagrass (Cynodon dactylon), Surinam grass (Brachiaria decumbens), common cocklebur (Xanthium strumarium), large crabgrass (Digitaria sanguinalis), woolly cupgrass (Eriochloa villosa), giant foxtail (Setaria faberii), goosegrass (Eleusine indica), johnsongrass (Sorghum halepense), kochia (Kochia scoparia), lambsquarters (Chenopodium album), morningglory (Ipomoea coccinea), eastern black nightshade ⁇ Solarium ptycant
• compositions comprising the compound that inhibit DHOD include corn (Zea mays), soybean (Glycine max), wheat (TRZAW, Triticum aestivum), winter barley (Hordeum vulgare), rice (Oryza sativa), oilseed rape (Brassica napus) and sunflower (Helianthus annuus).
• TRITICUM AESTIVUM DHOD (Traes 2AL 27580E224.3 CDS, DNA, TRITICUM
• TRITICUM AESTIVUM DHOD (Traes 2AL 27580E224.3, PROTEIN, TRITICUM
• GLYCINE MAX DHOD (Glyma.10G286200.1 CDS, DNA, GLYCINE MAX)
• GLYCINE MAX DHOD (Glyma.10G286200.1, PROTEIN, GLYCINE MAX)
• GLYCINE MAX DHOD EXPRESSION CONSTRUCT (Glyma.10G286200.1, PROTEIN,
• GLYCINE MAX DHOD (Glyma.20G103100.1 CDS, DNA, GLYCINE MAX)
• GLYCINE MAX DHOD (Glyma.20G103100.1, PROTEIN, GLYCINE MAX)
• GLYCINE MAX DOHD EXPRESSION CONSTRUCT (Glyma.20G103100.1, PROTEIN,
• the proteins and encoding DNA sequences depicted above are all capable of being used in the present invention.
• the present invention is not limited to use of sequences such as those exemplified above but rather any plant DHOD protein or encoding nucleic acids are intended.
• the invention includes the use of any DHOD having 50% to 100% to identity to SEQ ID NO: 2 or SEQ ID NO: 3 (without taking into account mismatches at the N-terminus) where every integer value in between is intended.
• amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
• the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• the comparison of sequences and determination of percent sequence identity between two sequences may be accomplished using a mathematical algorithm.
• the percent identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (J. Mol. Biol. 1970, 48, 444-453), which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
• the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
• a particularly preferred set of parameters is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
• the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of Meyers and Miller (CABIOS 1989, 4, 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
• the above sequences can be conventionally synthesized by the modified phosphotriester method using fully protected deoxyribonucleotide building blocks. Such synthetic methods are well known in the art and can be carried out in substantial accordance with the procedure of Itakura et al., Science 1977, 198, 1056 and Crea et al., Proc. Nat. Acad. Sci. U.S.A. 1978, 75, 5765. In addition, an especially preferred method is disclosed in Hsiung et al., Nucleic Acid Research 1983, 11, 3227 and Narang et al., Methods in Enzymology 1980, 68, 90. In addition to the manual procedures referenced above, the DNA sequence can be synthesized using automated DNA synthesizers, such as the ABS (Applied Biosy stems, 850 Lincoln Centre Drive, Foster City, Calif. 94404) 380B DNA Synthesizer.
• ABS Applied Biosy stems, 850 Lincoln Centre Drive, Foster City, Calif. 94404
• Candidate DHOD inhibitors can be screened within the meaning of the claims herein using computer modeling. The utility of this approach has been demonstrated in the screening of DHOD inhibitors for medicinal use in humans. McLean, L. R. et al., Bioorg. and Med. Chem. Lett. 2010, 20(6), 1981-1984. The computational design of inhibtors of DHOD has been described in WO 2004/056747.
• a further embodiment of the present invention utilizes a database searching program which is capable of scanning a database of small molecules of known three-dimensional structure for candidates which fit into the target protein site.
• Suitable software programs include 4SCan® (U.S. Pat. No. 7,247,736 and DE 10009479, EP 1094415), FLEXX (Rarey et al., J. Mol. Biol. 1996, 261, 470-489), and UNITY (Tripos Inc., St. Louis, Mo.).
• 4SCan® was developed to scan/screen large virtual databases up to several millions of small molecules in a reasonable time-frame.
• a further embodiment of the present invention utilizes a database searching program which is capable of scanning a database of small molecules of known three-dimensional structure for candidates which align properly with the co-crystallized ligand, both in shape and interaction properties.
• Suitable software programs include 4SCan® (U.S. Pat. No. 7,247,736 and EP 1094415) and FLEXS (Lemmen et al., J. Med. Chem. 1998, 41, 4502- 4520).
• 4SCan® is capable of aligning large virtual databases up to several millions of small molecules in a reasonable time-frame.
• GRID computer program
• GRID seeks to determine regions of high affinity for different chemical groups (termed probes) on the molecular surface of the binding site.
• GRID hence provides a tool for suggesting modifications to known ligands that might enhance binding. Consequently, virtual combinatorial libraries covering numerous variations of the addressed scaffold, functional groups, linkers and/or monomers can be build up using suitable software programs including LEGION (Tripos Inc., St. Louis, Mo.) or ACCORD FOR EXCEL (Accelrys Inc., San Diego, Calif), followed by scanning or virtual screening or docking of these libraries using suitable software mentioned above.
• a range of factors including electrostatic interactions, hydrogen bonding, hydrophobic interactions, desolvation effects, conformational strain, ligand flexibility and cooperative motions of ligand and enzyme, all influence the binding effect and should be taken into account in attempts to design bioactive inhibitors.
• Yet another embodiment of a computer-assisted molecular design method for identifying inhibitors of DHOD comprises searching for fragments which fit into a binding region subsite and link to a pre-defined scaffold.
• the scaffold itself may be identified in such a manner.
• a representative program suitable for the searching of such functional groups and monomers include LUDI (Boehm, J., Comp. Aid. Mol. Des. 1992, 6, 61-78) and MCSS (Miranker et al., Proteins 1991, 11, 314-328).
• Yet another embodiment of a computer-assisted molecular design method for identifying inhibitors of DHOD comprises the de novo synthesis of potential inhibitors by algorithmic connection of small molecular fragments that will exhibit the desired structural and electrostatic complementarity with the active site of the enzyme.
• the methodology employs a large template set of small molecules which are iteratively pierced together in a model of the DHOD ubiquinone binding site. Programs suitable for this task include GROW (Moon et al., Proteins 1991, 11, 314-328) and SPROUT (Gillet et al., J. Comp. Aid. Mol. Des. 1993, 7, 127).
• the suitability of inhibitor candidates can be determined using an empirical scoring function, which can rank the binding affinities for a set of inhibitors.
• an empirical scoring function which can rank the binding affinities for a set of inhibitors.
• a compound which is identified by one of the foregoing methods as a potential inhibitor of DHOD can then be obtained, for example, by synthesis or from a compound library, and assessed for the ability to inhibit DHOD in vitro.
• Such an in vitro assay can be performed as is known in the art, for example, by contacting DHOD in solution with the test compound in the presence of the substrate and cofactor of DHOD and ubiquinone. The rate of substrate transformation can be determined in the presence of the test compound and compared with the rate in the absence of the test compound. Suitable assays for DHOD biological activity are described below, the teachings of each of which are hereby incorporated by reference herein in their entity.
• a candidate compound is identified in this method by providing a computer program with information comprising (a) structural information about a binding pocket plant DHOD, (b) structural information of the compound, and (c) a set of rules and instructions about binding interactions between the compound and the binding pocket of the DHOD. Based on the information and the instructions provided to the computer program, the computer program predicts whether the binding of the compound is sufficiently robust and selecting the compound as a putative DHOD inhibitor.
• Glide computer software by Schrodinger LLC is used to identify the compounds which would bind to and regulate metalloregulatory activity of the proteins of ArsR/SmtB family by binding to the novel pocket.
• the Glide software program is described in U.S. Pat. No. 8, 145,430, entitled "Predictive Scoring Function for Estimating Binding Affinity".
• the software programs which may be used in the current invention include but are not limited to: MCSS, Ludi, QUANTA (macromolecular X-ray crystallography software); Insight II (biological compound modeling and simulation software); Cerius 2 (modeling and simulation software); CHARMm (software for simulation of biological macromolecules); Modeler from Accelrys, Inc.
• Structural information of a compound is provided as input to a docking program in order to screen the compound's ability to bind to a plant DHOD.
• the docking software program predicts the probability of the compound binding to the protein. This could be followed by extensive molecular dynamics simulations using the AMBER program that investigate the ability of the candidate molecule to regulate the activity of DHOD.
• binding interactions between a small molecule compound and a target binding site include, but are not limited to, hydrophilic interactions, hydrophobic interactions, van der Waals interactions, and hydrogen bonds.
• the structural information about a binding pocket of a protein is provided as input to a docking program.
• a set of rules and instructions are also provided to the docking program about binding interactions between a small molecule compound and the binding pocket of a DHOD. Based on the structural information and the instructions, the docking program predicts the binding affinity of a particular small molecule compound a particular DHOD.
• the docking program provides a "binding score" which indicates the strength of the binding between a compound and the binding pocket on a DHOD protein. Binding score can be calculated so that a higher binding score indicates stronger binding affinity. Binding scores can also be calculated so that a higher binding score indicates weaker binding affinity. In an embodiment of the invention, only those compounds from the compound library are selected which have binding scores higher or lower than a pre-determined level.
• Homology models of plant and fungal DHOD was generated using the structure of human DHOD in complex with leflunomide (A771726, PDBID 1D3H; Liu, S., Neidhardt, E. A., Grossman, T. H., Ocain, T., Clardy, J., 2000 Struct. Fold. Des. 8, 25-33).
• leflunomide A771726, PDBID 1D3H
• Arabidopsis DHOD or AtDHOD was determined to have 51% identity to the human DHOD sequence used as the template structure in the homology modelling/structure prediction module of the Maestro (Schroedinger) molecular modelling suite.
• the Maestro generated model included all ligands from the template structure (A771726, FMN and orotic acid).
• the PDB text file for the A. thaliana DHOD model was modified to include water 603 from the 1D3H template structure using identical x,y,z coordinates from the template file.
• affinity-based detection method where an analyte binds to a DHOD may be employed as a screening method.
• affinity-based detection method where an analyte binds to a ligand immobilized on a sensing surface may be employed, provided that a change in a property of the sensing probe is measured and quantitatively indicative of binding of the analyte to the immobilized ligand.
• Fluorescence and luminescence detections or radioactive labeling of the analyte in biomolecular interactions are still vogue in many established bio-oriented techniques described in P. Yamamoto-Fujita, et al., Anal. Chem. 2005, 77 5460-5466; K. Murakami et al., Prion 2002, 2, 73-80; M. H. Ko et al., Small 2009, 5 1207-1212; and J. E. Vandenengel et al., Biochem. Biophys. Res. Commun. 2009, 378, 51-56.
• labeling methods are not favourable in some cases, because labeling materials may occupy the important binding sites or cause steric hindrance, resulting in false information about interactions.
• an additional step is required prior to the analysis of the interaction due to the difficulty of labeling procedure.
• biosensors are analytical devices for analyzing minute quantities of sample solution having an analyte of interest, wherein the analyte is analyzed by a detection device that may employ a variety of detection methods.
• detection methods include, but are not limited to, mass detection methods, such as piezoelectric, optical, thermo-optical and surface acoustic wave (SAW) device methods, and electrochemical methods, such as potentiometric, conductometric, amperometric and capacitance methods.
• representative methods include those that detect mass surface concentration, such as reflection-optical methods, including both internal and external reflection methods, angle, wavelength or phase resolved, for example, ellipsometry and evanescent wave spectroscopy (EWS), the latter including surface plasmon resonance (SPR) spectroscopy, Brewster angle refractometry, critical angle refractometry, frustrated total reflection (FTIR), evanescent wave ellipsometry, scattered total internal reflection (STIR), optical wave guide sensors, evanescent wave-based imaging such as critical angle resolved imaging, Brewster angle resolved imaging, SPR angle resolved imaging, and the like.
• SPR surface plasmon resonance
• FTIR frustrated total reflection
• evanescent wave ellipsometry scattered total internal reflection
• optical wave guide sensors evanescent wave-based imaging such as critical angle resolved imaging, Brewster angle resolved imaging, SPR angle resolved imaging, and the like.
• photometric methods based on, for example, evanescent fluorescence (TIRF) and phosphor
• SPR Surface plasmon resonance
• biosensors In the context of SPR spectroscopy, one exemplary class of biosensors is sold by Biacore AB (Uppsala, Sweden) under the tradename BIACORE® (hereinafter referred to as "the BIACORE instrument"). Such biosensors utilize a SPR based mass-sensing technique to provide a "real-time" binding interaction analysis between a surface bound ligand and an analyte of interest.
• the BIACORE instrument includes a light emitting diode, a sensor chip covered with a thin gold film, an integrated fluid cartridge and a photo detector. Incoming light from the diode is reflected in the gold film and detected by the photo detector. At a certain angle of incidence ("the SPR angle"), a surface plasmon wave is set up in the gold layer, which is detected as an intensity loss or "dip” in the reflected light. More particularly, and as is appreciated by those skilled in the art, the phenomenon of surface plasmon resonance (SPR) associated with the BIACORE instrument is dependent on the resonant coupling of light, incident on a thin metal film, to oscillations of the conducting electrons, called plasmons, at the metal film surface.
• SPR surface plasmon resonance
• surface plasmon resonance is an optical phenomenon arising in connection with total internal reflection of light at a metal film-liquid interface.
• an optically denser medium e.g., a glass prism
• an optically less dense medium e.g., a buffer
• the angle of incidence is larger than the critical angle.
• This is known as total internal reflection.
• a component of the incident light momentum called the evanescent wave penetrates a distance of the order of one wavelength into the buffer.
• the evanescent wave may be used to excite molecules close to the interface.
• the evanescent wave under certain conditions will interact with free oscillating electrons (plasmons) in the metal film surface.
• plasmons free oscillating electrons
• the resonance phenomenon will only occur for light incident at a sharply defined angle which, when all else is kept constant, is dependent on the refractive index in the flowing buffer close to the surface. Changes in the refractive index out to about 1 ⁇ from the metal film surface can thus be followed by continuous monitoring of the resonance angle.
• a detection volume is defined by the size of the illuminated area at the interface and the penetration depth of the evanescent field. It should be noted that no light passes through the detection volume (the optical device on one side of the metal film detects changes in the refractive index in the medium on the other side).
• the SPR angle depends on the refractive index of the medium close to the gold layer.
• dextran is typically coupled to the gold surface, with the ligand being bound to the surface of the dextran layer.
• the analyte of interest is injected in solution form onto the sensor surface through the fluid cartridge.
• the refractive index in the proximity of the gold film depends upon (1) the refractive index of the solution (which is constant) and, (2) the amount of material bound to the surface, the binding interaction between the bound ligand and analyte can be monitored as a function of the change in SPR angle.
• a typical output from the BIACORE instrument is a "sensorgram” which is a plot of response (measured in “resonance units” or “RU") as a function of time.
• An increase of 1,000 RU corresponds to an increase of mass on the sensor surface of approximately 1 ng/mm 2 .
• association As a sample containing the analyte contacts the sensor surface, the ligand bound to the sensor surface interacts with the analyte in a step referred to as "association.” This step is indicated on the sensorgram by an increase in RU as the sample is initially brought into contact with the sensor surface.
• association normally occurs when sample flow is replaced by, for example, a buffer flow.
• This step is indicted on the sensorgram by a drop in RU over time as analyte dissociates from the surface-bound ligand.
• each sensor chip may have a plurality of sensing surfaces, and that such sensing surfaces may be arranged in series or parallel with respect to the fluid sample pathway of the fluid cartridge.
• each of the plurality of sensing surfaces of a single sensor chip may have bound thereto a unique type of ligand that is capable of interacting with an analyte in its own characteristic way.
• a direct binding assay for plant DHOD was developed using a Biacore T200 instrument (GE lifesciences) and an NTA chip (nitrolotri acetic acid chip, GE lifesciences).
• the running buffer (RB) consisted of PBS (phosphate buffer solution) P+ (GE) supplemented with 1% DMSO and 50 ⁇ EDTA.
• the NTA chip surface was prepared by a 120 s injection of 0.35 M ethylenediaminetetraacetic acid (EDTA) at a flow rate of 10 ⁇ / ⁇ , followed by a 60 s injection of RB.
• the NTA surface was charged with Ni 2+ by injecting a 60 s pulse of 0.5 mM N1CI2 at a flow rate of 10 ⁇ / ⁇ followed by a 60 s injection with RB.
• 1 Ox-Hi s-tagged DHOD (30 ⁇ g/mL) was injected over the surface of the Ni-NTA chip at a flow rate of 10 ⁇ / ⁇ for 15 s. Injections of His-tagged DHOD were repeated until the amount of protein added to the surface reached approximately 2700 - 2800 Resonance units (RUs). The surface was then washed with 2 x 60 s pulses of RB at the same flow rate.
• Figure 1A and Figure IB shows the response of Arabidopsis DHOD and rice (Oryza sativum) DHOD respectively.
• Reference and blank subtracted kinetic data were fit to a single site model in the Biacore T200 evaluation software and values for k on , k 0 ff, KD and R j nax were determined.
• Figure 2 shows the values for the rice enzyme where R j nax values were typically in the range of 10 - 25 RUs.
• a compound of this invention will generally be used as a herbicidal active ingredient in a formulation, with at least one additional component selected from the group consisting of surfactants, solid diluents and liquid diluents, which serves as a carrier.
• the formulation or composition ingredients are selected to be consistent with the physical properties of the active ingredient, mode of application and environmental factors such as soil type, moisture and temperature.
• Liquid compositions include solutions (including emulsifiable concentrates), suspensions, emulsions (including microemulsions, oil-in-water emulsions, flowable concentrates and/or suspoemulsions) and the like, which optionally can be thickened into gels.
• aqueous liquid compositions are soluble concentrate, suspension concentrate, capsule suspension, concentrated emulsion, microemulsion, oil-in-water emulsion, flowable concentrate and suspo-emulsion.
• nonaqueous liquid compositions are emulsifiable concentrate, microemulsifiable concentrate, dispersible concentrate and oil dispersion.
• compositions are dusts, powders, granules, pellets, prills, pastilles, tablets, filled films (including seed coatings) and the like, which can be water-dispersible ("wettable") or water-soluble. Films and coatings formed from film- forming solutions or flowable suspensions are particularly useful for seed treatment.
• Active ingredient can be (micro)encapsulated and further formed into a suspension or solid formulation; alternatively the entire formulation of active ingredient can be encapsulated (or "overcoated”). Encapsulation can control or delay release of the active ingredient.
• An emulsifiable granule combines the advantages of both an emulsifiable concentrate formulation and a dry granular formulation. High-strength compositions are primarily used as intermediates for further formulation.
• Sprayable formulations are typically extended in a suitable medium before spraying. Such liquid and solid formulations are formulated to be readily diluted in the spray medium, usually water, but occasionally another suitable medium like an aromatic or paraffinic hydrocarbon or vegetable oil. Spray volumes can range from about from about one to several thousand liters per hectare, but more typically are in the range from about ten to several hundred liters per hectare. Sprayable formulations can be tank mixed with water or another suitable medium for foliar treatment by aerial or ground application, or for application to the growing medium of the plant. Liquid and dry formulations can be metered directly into drip irrigation systems or metered into the furrow during planting.
• the formulations will typically contain effective amounts of active ingredient, diluent and surfactant within the following approximate ranges which add up to 100 percent by weight.
• Solid diluents include, for example, clays such as bentonite, montmorillonite, attapulgite and kaolin, gypsum, cellulose, titanium dioxide, zinc oxide, starch, dextrin, sugars (e.g., lactose, sucrose), silica, talc, mica, diatomaceous earth, urea, calcium carbonate, sodium carbonate and bicarbonate, and sodium sulfate.
• Typical solid diluents are described in Watkins et al., Handbook of Insecticide Dust Diluents and Carriers, 2nd Ed., Dorland Books, Caldwell, New Jersey.
• Liquid diluents include, for example, water, N,N-dimethylalkanamides (e.g., N,N-dimethylformamide), limonene, dimethyl sulfoxide, N-alkylpyrrolidones (e.g., N-methylpyrrolidinone), alkyl phosphates (e.g., triethyl phosphate), ethylene glycol, Methylene glycol, propylene glycol, dipropylene glycol, polypropylene glycol, propylene carbonate, butylene carbonate, paraffins (e.g., white mineral oils, normal paraffins, isoparaffins), alkylbenzenes, alkylnaphthalenes, glycerine, glycerol triacetate, sorbitol, aromatic hydrocarbons, dearomatized aliphatics, alkylbenzenes, alkylnaphthalenes, ketones such as cyclohexanone
• Liquid diluents also include glycerol esters of saturated and unsaturated fatty acids (typically C6-C22), such as plant seed and fruit oils (e.g., oils of olive, castor, linseed, sesame, corn (maize), peanut, sunflower, grapeseed, safflower, cottonseed, soybean, rapeseed, coconut and palm kernel), animal-sourced fats (e.g., beef tallow, pork tallow, lard, cod liver oil, fish oil), and mixtures thereof.
• plant seed and fruit oils e.g., oils of olive, castor, linseed, sesame, corn (maize), peanut, sunflower, grapeseed, safflower, cottonseed, soybean, rapeseed, coconut and palm kernel
• animal-sourced fats e.g., beef tallow, pork tallow, lard, cod liver oil, fish oil
• Liquid diluents also include alkylated fatty acids (e.g., methylated, ethylated, butylated) wherein the fatty acids may be obtained by hydrolysis of glycerol esters from plant and animal sources, and can be purified by distillation.
• alkylated fatty acids e.g., methylated, ethylated, butylated
• Typical liquid diluents are described in Marsden, Solvents Guide, 2nd Ed., Interscience, New York, 1950.
• the solid and liquid compositions of the present invention often include one or more surfactants.
• surfactants also known as “surface-active agents”
• surface-active agents generally modify, most often reduce, the surface tension of the liquid.
• surfactants can be useful as wetting agents, dispersants, emulsifiers or defoaming agents.
• Nonionic surfactants useful for the present compositions include, but are not limited to: alcohol alkoxylates such as alcohol alkoxylates based on natural and synthetic alcohols (which may be branched or linear) and prepared from the alcohols and ethylene oxide, propylene oxide, butylene oxide or mixtures thereof; amine ethoxylates, alkanolamides and ethoxylated alkanolamides; alkoxylated triglycerides such as ethoxylated soybean, castor and rapeseed oils; alkylphenol alkoxylates such as octylphenol ethoxylates, nonylphenol ethoxylates, dinonyl phenol ethoxylates and dodecyl phenol ethoxylates (prepared from the phenols and ethylene oxide, propylene oxide, butylene oxide or mixtures thereof); block polymers prepared from ethylene oxide or propylene
• Useful anionic surfactants include, but are not limited to: alkylaryl sulfonic acids and their salts; carboxylated alcohol or alkylphenol ethoxylates; diphenyl sulfonate derivatives; lignin and lignin derivatives such as lignosulfonates; maleic or succinic acids or their anhydrides; olefin sulfonates; phosphate esters such as phosphate esters of alcohol alkoxylates, phosphate esters of alkylphenol alkoxylates and phosphate esters of styryl phenol ethoxylates; protein-based surfactants; sarcosine derivatives; styryl phenol ether sulfate; sulfates and sulfonates of oils and fatty acids; sulfates and sulfonates of ethoxylated alkylphenols; sulfates of alcohols; sulfates of e
• Useful cationic surfactants include, but are not limited to: amides and ethoxylated amides; amines such as N-alkyl propanediamines, tripropylenetriamines and dipropylenetetramines, and ethoxylated amines, ethoxylated diamines and propoxylated amines (prepared from the amines and ethylene oxide, propylene oxide, butylene oxide or mixtures thereof); amine salts such as amine acetates and diamine salts; quaternary ammonium salts such as quaternary salts, ethoxylated quaternary salts and di quaternary salts; and amine oxides such as alkyldimethylamine oxides and bis-(2-hydroxyethyl)-alkylamine oxides.
• amines such as N-alkyl propanediamines, tripropylenetriamines and dipropylenetetramines, and ethoxylated
• Nonionic, anionic and cationic surfactants and their recommended uses are disclosed in a variety of published references including McCutcheon 's Emulsifiers and Detergents, annual American and International Editions published by McCutcheon's Division, The Manufacturing Confectioner Publishing Co.; Sisely and Wood, Encyclopedia of Surface Active Agents, Chemical Publ. Co., Inc., New York, 1964; and A. S. Davidson and B. Milwidsky, Synthetic Detergents, Seventh Edition, John Wiley and Sons, New York, 1987.
• compositions of this invention may also contain formulation auxiliaries and additives, known to those skilled in the art as formulation aids (some of which may be considered to also function as solid diluents, liquid diluents or surfactants).
• formulation auxiliaries and additives may control: pH (buffers), foaming during processing (antifoams such polyorganosiloxanes), sedimentation of active ingredients (suspending agents), viscosity (thixotropic thickeners), in-container microbial growth (antimicrobials), product freezing (antifreezes), color (dyes/pigment dispersions), wash-off (film formers or stickers), evaporation (evaporation retardants), and other formulation attributes.
• Film formers include, for example, polyvinyl acetates, polyvinyl acetate copolymers, polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohols, polyvinyl alcohol copolymers and waxes.
• formulation auxiliaries and additives include those listed in McCutcheon 's Volume 2: Functional Materials, annual International and North American editions published by McCutcheon's Division, The Manufacturing Confectioner Publishing Co.; and PCT Publication WO 03/024222.
• compositions of this invention can also be mixed with RNA to enhance effectiveness or to confer safening properties. Accordingly, a compositions containing a compound from Index Table A, B or C can be mixed with polynucleotides including but not limited to DNA, RNA, and/or chemically modified nucleotides influencing the amount of a particular target through down regulation, interference, suppression or silencing of the genetically derived transcript that render a herbicidal effect.
• composition containing a compound from Index Table A, B or C can be mixed with polynucleotides including but not limited to DNA, RNA, and/or chemically modified nucleotides influencing the amount of a particular target through down regulation, interference, suppression or silencing of the genetically derived transcript that render a safening effect.
• the target is DHOD or an upstream or downstream pyrimidine biosynthesis inhibitor.
• the DHOD inhibitor or indirect inhibitor and any other active ingredients are typically incorporated into the present compositions by dissolving the active ingredient in a solvent or by grinding in a liquid or dry diluent.
• Solutions, including emulsifiable concentrates can be prepared by simply mixing the ingredients. If the solvent of a liquid composition intended for use as an emulsifiable concentrate is water-immiscible, an emulsifier is typically added to emulsify the active-containing solvent upon dilution with water. Active ingredient slurries, with particle diameters of up to 2,000 ⁇ can be wet milled using media mills to obtain particles with average diameters below 3 ⁇ .
• Aqueous slurries can be made into finished suspension concentrates (see, for example, U.S. Pat. No. 3,060,084) or further processed by spray drying to form water-dispersible granules. Dry formulations usually require dry milling processes, which produce average particle diameters in the 2 to 10 ⁇ range. Dusts and powders can be prepared by blending and usually grinding (such as with a hammer mill or fluid-energy mill). Granules and pellets can be prepared by spraying the active material upon preformed granular carriers or by agglomeration techniques.
• Pellets can be prepared as described in U.S. 4,172,714.
• Water-dispersible and water-soluble granules can be prepared as taught in U.S. 4,144,050, U.S. 3,920,442 and DE 3,246,493.
• Tablets can be prepared as taught in U.S. 5, 180,587, U.S. 5,232,701 and U.S. 5,208,030.
• Films can be prepared as taught in GB 2,095,558 and U.S. 3,299,566.
• a mixture of one or more of the following herbicides in a composition of this invention may be particularly useful for weed control: acetochlor, acifluorfen and its sodium salt, aclonifen, acrolein (2-propenal), alachlor, alloxydim, ametryn, amicarbazone, amidosulfuron, aminocyclopyrachlor and its esters (e.g., methyl, ethyl) and salts (e.g., sodium, potassium), aminopyralid, amitrole, ammonium sulfamate, anilofos, asulam, atrazine, azimsulfuron, beflubutamid, benazolin, benazolin-ethyl, bencarbazone, benfluralin, benfuresate, bensulfuron-methyl, bensulide, bentazone, benzobicyclon, benzofenap, bicyclopyrone, bifenox, bilana
• herbicides also include bioherbicides such as Alternaria destruens Simmons, Colletotrichum gloeosporiodes (Penz.) Penz. & Sacc, Drechsiera monoceras (MTB-951), Myrothecium verrucaria (Albertini & Schweinitz) Ditmar: Fries, Phytophthora palmivora (Butl.) Butl. and Puccinia thlaspeos Schub..
• bioherbicides such as Alternaria destruens Simmons, Colletotrichum gloeosporiodes (Penz.) Penz. & Sacc, Drechsiera monoceras (MTB-951), Myrothecium verrucaria (Albertini & Schweinitz) Ditmar: Fries, Phytophthora palmivora (Butl.) Butl. and Puccinia thlaspeos Schub..
• a composition of the present invention can further comprise (in a herbicidally effective amount) at least one additional herbicidal active ingredient having a similar spectrum of control but a different site of action.
• compositions of the invention might also include a herbicidally effective amount of an antidotally effective amount of safener.
• Antidotally effective amounts of safeners can be easily determined by one skilled in the art through simple experimentation.
• herbicide safeners include but are not limited to benoxacor, cloquintocet-mexyl, cumyluron, cyometrinil, cyprosulfamide, daimuron, dichlormid, dicyclonon, dietholate, dimepiperate, fenchlorazole-ethyl, fenclorim, flurazole, fluxofenim, furilazole, isoxadifen-ethyl, mefenpyr-diethyl, mephenate, methoxyphenone, naphthalic anhydride, oxabetrinil, N- (aminocarbonyl)-2-methylbenzenesulfonamide and N-(aminocarbonyl)-2- fluor
• composition comprising a compound of the invention (in a herbicidally effective amount), at least one additional active ingredient selected from the group consisting of other herbicides and herbicide safeners (in an effective amount), and at least one component selected from the group consisting of surfactants, solid diluents and liquid diluents.

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