Discovery of Novel Pyrazole Acyl Thiourea Skeleton Analogue as Potential Herbicide Candidates.

To discover novel transketolase (TKL, EC 2.2.1.1) inhibitors with potential herbicidal applications, a series of pyrazole acyl thiourea derivatives were designed based on a previously obtained pyrazolamide acyl lead compound, employing a scaffold hopping strategy. The compounds were synthesized, their structures were characterized, and they were evaluated for herbicidal activities. The results indicate that 7a exhibited exceptional herbicidal activity against Digitaria sanguinalis and Amaranthus retroflexus at a dosage of 90 g ai/ha, using the foliar spray method in a greenhouse. This performance is comparable to that of commercial products, such as nicosulfuron and mesotrione. Moreover, 7a showed moderate growth inhibitory activity against the young root and stem of A. retroflexus at 200 mg/L in the small cup method, similar to that of nicosulfuron and mesotrione. Subsequent mode-of-action verification experiments revealed that 7a and 7e inhibited Setaria viridis TKL (SvTKL) enzyme activity, with IC50 values of 0.740 and 0.474 mg/L, respectively. Furthermore, they exhibited inhibitory effects on the Brassica napus acetohydroxyacid synthase enzyme activity. Molecular docking predicted potential interactions between these (7a and 7e) and SvTKL. A greenhouse experiment demonstrated that 7a exhibited favorable crop safety at 150 g ai/ha. Therefore, 7a is a promising herbicidal candidate that is worthy of further development.

Design, Synthesis, and Herbicidal Activity of Pyrazole Amide Derivatives as Potential Transketolase Inhibitors.

The design and synthesis of new herbicidal active compounds based on a new target are of great significance for the development of new herbicides. Transketolase (TK) plays a key role in the Calvin cycle of plant photosynthesis and has been confirmed as a potential candidate target to develop and discover new herbicides. To obtain compounds with ultraefficient targeting of TK, a series of pyrazole amide derivatives were designed and synthesized through structural optimization for lead compound 4u based on TK as the new target. The bioassay results showed that compounds 6ba and 6bj displayed a highly inhibitory effect with the root inhibition of about 90% against Digitaria sanguinalis (DS) and 80% against Amaranthus retroflexus (AR) and Setaria viridis (SV) by the small cup method, which was better than the positive control mesotrione and nicosulfuron. Furthermore, compounds 6ba and 6bj exhibited an excellent inhibitory effect with the inhibition of about 80% (against DS) and over 80% (against SV) at the dosage of 150 g of active ingredient/ha by the foliar spray method. The TK enzyme activity inhibition test showed that the inhibition effect of target compounds against TK was consistent with the results of herbicidal activities. Also, molecular docking analysis showed that compounds 6ba and 6bj went deep into the active cavity of TK, bound to TK by a strong interaction, and might act on the enzyme TK. Above of all, compounds 6ba and 6bj are promising herbicide lead compounds targeting TK. Hence, they could be developed into more efficient herbicides by further structural optimization.

Establishment of an indirect competitive immunoassay for the detection of dicamba based on a highly specific nanobody.

Dicamba, a traditional highly effective and low toxicity herbicide, has gained new life with the development of dicamba-tolerant transgenic crops in recent years. However, dicamba is highly volatile and therefore easy to cause drift damage to sensitive crops. The development of efficient and sensitive detection methods is essential for monitoring of trace dicamba in the environment. Nanobody-based immunoassay plays an important role in on-site detection of pesticides. However, now rapid and sensitive immunoassay methods based on nanobody for dicamba detection were lacking. In this study, the nanobodies specifically recognizing dicamba were successfully obtained by immunising camels and phage display library construction, and then an indirect competitive immunoassay based on Nb-242 was constructed with IC50 of 0.93 µg/mL and a linear range of 0.11-8.01 µg/mL. Nb-242 had good specificity with no cross-reactivities against the dicamba analogs other than 2,3,6-trichlorobenzoic acid and the developed immnoassay had a good correlation with the standard HPLC in the spike-recovery studies. Finally, the key amino acid Ala 123, Tyr 55, Tyr 59 and Arg 72 of Nb-242 that specifically recognizing and binding with dicamba were identified by homologous modeling and molecular docking, laying an important foundation for further structural modification of Nb-242. This study has important guiding significance for constructing immunoassay method of dicamba based on nanobody and provides a sensitive, specific, and reliable detection method that is suitable for the detection of dicamba in the environment.

Development of an immunoassay based on a specific antibody for the detection of diphenyl ether herbicide fomesafen.

Fomesafen belongs to the diphenyl ether herbicide, and is widely used in the control of broadleaf weeds in crop fields due to its high efficiency and good selectivity. The residual of fomesafen in soil has a toxic effect on subsequent sensitive crops and the microbial community structure because of its long residual period. Therefore, an efficient method for detecting fomesafen is critical to guide the correct and reasonable use of this herbicide. Rapid and sensitive immunoassay methods for fomesafen is unavailable due to the lack of specific antibody. In this study, a specific antibody for fomesafen was generated based on rational design of haptens and a sensitive immunoassay method was established. The half maximal inhibitory concentration (IC50) of the immunoassay was 39 ng/mL with a linear range (IC10-90) of 1.92-779.8 ng/mL. In addition, the developed assay had a good correlation with the standard UPLC-MS/MS both in the spike-recovery studies and in the detection of real soil samples. Overall, the developed indirect competitive enzyme immunoassay reported here is important for detecting and quantifying fomesafen contamination in soil and other environmental samples with good sensitivity and high reproducibility.

Discovery of Bis-5-cyclopropylisoxazole-4-carboxamides as Novel Potential 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27; HPPD) represents a potential target for novel herbicide development. To discover the more promising HPPD inhibitor, we designed and synthesized a series of bis-5-cyclopropylisoxazole-4-carboxamides with different linkers using a multitarget pesticide design strategy. Among them, compounds b9 and b10 displayed excellent herbicidal activities versus Digitaria sanguinalis (DS) and Amaranthus retroflexus (AR) with the inhibition of about 90% at the concentration of 100 mg/L in vitro, which was better than that of isoxaflutole (IFT). Furthermore, compounds b9 and b10 displayed the best inhibitory effect versus DS and AR with the inhibition of about 90 and 85% at 90 g (ai)/ha in the greenhouse, respectively. The structure-activity relationship study showed that the flexible linker (6 carbon atoms) is responsible for increasing their herbicidal activity. The molecular docking analyses showed that compounds b9 and b10 could more closely bind to the active site of HPPD and thus exhibited a better inhibitory effect. Altogether, these results indicated that compounds b9 and b10 could be used as potential herbicide candidates targeting HPPD.

Synthesis of Indoles via Domino Reactions of 2-Methoxytoluene and Nitriles.

2-Arylindoles are privileged structures widely present in biologically active molecules. New sustainable synthetic routes toward their synthesis are, therefore, in high demand. Herein, a mixed base-promoted benzylic C-H deprotonation of commercially available ortho-anisoles, addition of the resulting anion to benzonitriles, and SNAr to displace the methoxy group provide indoles. A diverse array of 2-arylindoles is prepared with good yields (>30 examples, yields up to 99%) without added transition metal catalysts.

Development of immunoassay based on a specific antibody for sensitive detection of nicosulfuron in environment.

Nicosulfuron, one of the most widely used selective herbicides in corn field, can effectively control annual and perennial grass weeds, sedges, and some broadleaf weeds. The residual phytotoxicity of nicosulfuron in soil and water has become increasingly prominent. Therefore, an efficient method for detection of nicosulfuron was critical to ensure the sustainable and healthy development of agriculture and the ecological environment. In this paper, five nicosulfuron haptens which contained carboxyl group or aldehyde groups were designed and synthesized, and an indirect competitive immunoassay was developed for the first time. The assay showed an IC50 of 8.42 ng/mL and had negligible cross reactivities toward other sulfonylurea herbicides. In the spike and recovery studies, the recovery rate from soil samples was 95 %-104 %, and that of wheat roots was 92 %-98 %, which showed a good correlation with LC-MS analysis for nicosulfuron. The immunoassay was then used to quantify nicosulfuron concentration which could cause the obvious phytotoxic symptoms to wheat. Obvious symptoms of nicosulfuron phytotoxicity in wheat root was observed at the concentration of 0.068 ± 0.006 mg/kg (ELISA result) which was consistent with 0.072 ± 0.007 mg/kg obtained by LC-MS. The developed immunoassay method is an effective tool for environment contamination monitoring.

Citric acid modified ultrasmall copper peroxide nanozyme for in situ remediation of environmental sulfonylurea herbicide contamination.

Herbicide residues in the environment threaten high-quality agriculture and human health. Consequently, in situ remediation of herbicide contamination is vital. We synthesized a novel self-catalyzed nanozyme, ultrasmall (2-3 nm) copper peroxide nanodots modified by citric acid (CP@CA) for this purpose, which can break down into H2O2 and Cu2+ in water or soil. Ubiquitous glutathione reduces Cu2+ into Cu+, which promotes the decomposition of H2O2 into •OH through a Fenton-like reaction under mild acid conditions created by the presence of citric acid. The generated •OH efficiently degrade nicosulfuron in water and soil, and the maximum degradation efficiency could be achieved at 97.58% in water at 56 min. The possible degradation mechanisms of nicosulfuron were proposed through the 25 intermediates detected. The overall ecotoxicity of the nicosulfuron system was significantly reduced after CP@CA treatment. Furthermore, CP@CA had little impact on active components of soil bacterial community. Moreover, CP@CA nanozyme could effectively remove seven other sulfonylurea herbicides from the water. In this paper, a high-efficiency method for herbicide degradation was proposed, which provides a new reference for the in situ remediation of herbicide pollution.

Design, Synthesis, and Herbicidal Activity of Naphthalimide-Aroyl Hybrids as Potent Transketolase Inhibitors.

Transketolase (TK) was identified as a new target for the development of novel herbicides. In this study, a series of naphthalimide-aroyl hybrids were designed and prepared based on TK as a new target and tested for their herbicidal activities. In vitro bioassay showed that compounds 4c and 4w exhibited stronger inhibitory effects against Digitaria sanguinalis (DS) and Amaranthus retroflexus (AR) with the inhibition over 90% at 200 mg/L and around 80% at 100 mg/L. Also, compounds 4c and 4w exhibited excellent postemergence herbicidal activity against DS and AR with the inhibition around 90% at 90 g [active ingredient (ai)]/ha and 80% at 50 g (ai)/ha in the greenhouse, which was comparable with the activity of mesotrione. The fluorescent quenching experiments of At TK revealed the occurrence of electron transfer from compound 4w to At TK and the formation of a strong exciplex between them. Molecular docking analyses further showed that compounds 4w exhibited profound affinity with At TK through the interaction with the amino acids in the active site, which results in its strong inhibitory activities against TK. These findings demonstrated that compound 4w is potentially a lead candidate for novel herbicides targeting TK.

Design, Synthesis, and Evaluation of Novel 4-Chloropyrazole-Based Pyridines as Potent Fungicide Candidates.

A rational molecular design approach was developed in our laboratory to guide the discovery of novel sterol biosynthesis inhibitors. Based on the application of bioactivities of heterocyclic rings and molecular docking targeting the sterol biosynthesis 14α-demethylase, a series of 4-chloropyrazole-based pyridine derivatives were rationally designed, synthesized, and characterized and their fungicidal activities were also evaluated. Bioassay results showed that 7e, 7f, and 7m exhibited commendable, diverse antifungal actions that are comparable to those of the positive controls imazalil and triadimefon. The active compounds' mode of action was further studied by microscopy observations, Q-PCR, and enzyme inhibition assay and discovered that target compounds affect fungal sterol biosynthesis via disturbing RcCYP51 enzyme system. These findings support that their fungicidal mode of action still targets the cytochrome P450-dependent 14α-demethylase as the molecular design did at first. The above results strongly suggest that our rational molecular design protocol is not only practical but also efficient.

2-Arylindoles: Concise Syntheses and a Privileged Scaffold for Fungicide Discovery.

Indole is a popular and functional scaffold existing widely in the fields of medicine, pesticides, spices, food and feed additives, dyes, and many others. Among indoles, 2-arylindole represents a particular and interesting subset but has attracted less attention for drug discovery. In this study, we report a general, practical one-pot assembly of a variety of 2-arylindole derivatives. To develop novel fungicide scaffolds, their fungicide activity was also evaluated. The bioassay results showed that many of the synthesized 2-arylindoles exhibited considerable fungicidal activities especially toward Rhizoctonia cerealis, and several demonstrated an inhibition rate of more than 90%. Notably, 4-fluoro-2-phenyl-1H-indole 6e was obtained with a broad spectrum of fungicidal activities, which showed excellent growth inhibition activities against R. cerealis, Rhizoctonia solani, Botrytis cinerea, Magnaporthe oryza, and Sclerotinia sclerotiorum with EC50 values of 2.31, 4.98, 6.78, 10.57, and 17.80 µg/mL, respectively. Preliminary fungicidal mode of action of 6e showed a significant inhibition effect on mycelial growth and spore germination. These results indicated that 2-arylindoles as privileged scaffolds exhibited potential fungicidal activities that deserve further study.

Discovery of Novel Pyrazole Amides as Potent Fungicide Candidates and Evaluation of Their Mode of Action.

A rational molecule design strategy based on scaffold hopping was applied to discover novel leads, and then a series of novel pyrazole amide derivatives were designed, synthesized, characterized, and evaluated for their antifungal activities. Bioassay results indicated that some target compounds such as S3, S12, and S26 showed good in vivo antifungal activities; among them, S26 exhibited commendable in vivo protective activity with an 89% inhibition rate against Botrytis cinerea on cucumber at 100 µg/mL that is comparable to positive controls boscalid, isopyrazam, and fluxapyroxad. Microscopy observations suggested that S26 affects the normal fungal growth. Fluorescence quenching analysis and SDH (succinate dehydrogenase) enzymatic inhibition studies validated that S26 may not be an SDH inhibitor. Based on induction of plant defense responses testing, S26 enhanced the accumulation of RBOH, WRKY6, WRKY30, PR1, and PAL defense-related genes expression and the defense-associated enzyme phenylalanine ammonia lyase (PAL) expression on cucumber. These findings support that S26 not only displayed direct fungicidal activity but also exhibited plant innate immunity stimulation activity, and it could be used as a promising plant defense-related fungicide candidate.

Design, Synthesis, Herbicidal Activity, and Molecular Docking Study of 2-Thioether-5-(Thienyl/Pyridyl)-1,3,4-Oxadiazoles as Potent Transketolase Inhibitors.

Transketolase (TK) has been regarded as a new target for the development of novel herbicides. In this study, a series of 2-thioether-5-(thienyl/pyridyl)-1,3,4-oxadiazoles were designed and synthesized based on TK as the new target. The preliminary bioassay results indicated that compounds 4l and 4m displayed the best herbicidal activities against Amaranthus retroflexus (AR) and Digitaria sanguinalis (DS), with the inhibition exceeding 90% at 100-200 mg/L in vitro. Moreover, they also displayed higher postemergence herbicidal activities (90% control) against AR and DS than all of the positive controls at 45-90 g [active ingredient (ai)]/ha in a greenhouse. Notably, compounds 4l and 4m showed a broad spectrum of weed control at 90 g ai/ha. More significantly, compound 4l exhibited good crop selectivity against maize at 90 g ai/ha. Both fluorescent binding experiments and molecular docking analyses indicated that compounds 4l and 4m exhibited strong TK inhibitory activities with superior binding affinities than the others. Preliminary mechanism studies suggested that they might exert their TK inhibitory effects by occupying the active cavity of At TK and forming more strong interactions with amino acids in the active site. Taken together, these results suggested that compound 4l was a potential herbicide candidate for weed control in maize fields targeting TK.

Development of a Rapid Gold Nanoparticle Immunochromatographic Strip Based on the Nanobody for Detecting 2,4-DichloRophenoxyacetic Acid.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a systemic conductive herbicide widely used across the world. With the large-scale and continuous use of 2,4-D, its possible harm to the environment and non-target organisms has attracted increasing attention, and the construction of a stable rapid on-site detection method is particularly important. In order to achieve on-site rapid detection of 2,4-D, we developed a gold nanoparticle immunochromatographic strip method with the visual elimination value was 50 ng/mL, and a quantitative detection limit of 11 ng/mL based on a nanobody. By combing with the color snap, the immunochromatographic strip could quantitatively analyze the amounts of 2,4-D. Meanwhile, a colorimetric card based on the true color of the test strips was developed for the qualitative analysis of 2,4-D on-site. The samples (water, fruits and vegetables) with and without 2,4-D were detected by the immunochromatographic strips, and the results showed the accuracy and reliability. Thus, this assay is a rapid and simple on-site analytical tool to detect and quantify 2,4-D levels in environmental samples, and the analytical results can be obtained in about ten minutes. In addition, the nanobody technology used in this study provides an inexhaustible supply of a relatively stable antibodies that can be archived as a nanobody, plasmid or even its sequence.

Design, Synthesis, and Herbicidal Activity of Thioether Containing 1,2,4-Triazole Schiff Bases as Transketolase Inhibitors.

Transketolase (TK) represents a potential target for novel herbicide development. To discover novel TK inhibitors with potency against resistant weeds, 36 thioether compounds containing 1,2,4-triazole Schiff bases were designed and synthesized for herbicidal activity evaluation. The results demonstrated that compounds 5av and 5aw provided excellent weed control with inhibition of over 90% against the tested weeds, even at concentrations as low as 100 mg/L in vitro. In addition, compounds 5av and 5aw exhibited higher postemergence herbicidal activity than all of the positive controls against the tested weeds at 50-90 g [active ingredient (ai)]/ha in a greenhouse, while being safe for crops of maize and wheat at 90 g (ai)/ha. Fluorescent binding experiments of At TK indicated that compounds 5av and 5aw had strong TK inhibitory activity and could tightly bind with the enzyme At TK. Also, molecular docking analyses revealed that the structures of compounds 5av and 5aw were suitable for TK inhibitory activity. Taken together, these results suggested that compounds 5av and 5aw were promising herbicide candidates for weed control in wheat and maize fields targeting TK.

One-Pot Synthesis of N-H-Free Pyrroles from Aldehydes and Alkynes.

The first base-mediated intermolecular cyclization of arylaldehydes and terminal arylacetylenes for the synthesis of a wide range of pyrroles in a single step has been described. The developed methodology used commercially available starting materials and tolerated a broad range of functional groups affording 2,3,5-triaryl-substituted-1H-pyrroles with good yields (up to 92% yield) under mild conditions. The possible mechanism was also discussed.

Highly specific nanobody against herbicide 2,4-dichlorophenoxyacetic acid for monitoring of its contamination in environmental water.

2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, is a small organic chemical pollutant in the environment. To develop a nanobody-based immunoassay for monitoring trace levels of 2,4-D, a step-wise strategy for the generation of nanobodies highly specific against this small chemical was employed. Firstly, we synthesized three novel haptens mimicking 2,4-D and assessed their influence on the sensitivity and specificity of the existing antibody-based assay. Polyclonal antibodies (pAb) from rabbits showed good sensitivity and moderate specificity for 2,4-D, pAb from llama based on selected haptens showed similar performance when compared to those from rabbits. Secondly, nanobodies derived from llama were generated for 2,4-D by an effective procedure, including serum monitoring and one-step library construction. One nanobody, NB3-9, exhibited good sensitivity against 2,4-D (IC50 = 29.2 ng/mL) had better specificity than the rabbit pAb#1518, with no cross-reactivities against the 2,4-D analogs tested. Thirdly, one-step fluorescent enzyme immunoassay (FLEIA) for 2,4-D based on a nanobody-alkaline phosphatase (AP) fusion was developed with IC50 of 1.9 ng/mL and a linear range of 0.4-8.6 ng/mL. Environmental water samples were analyzed by FLEIA and LC-MS/MS for comparison, and the results were consistent between both methods. Therefore, the proposed step-wise strategy from hapten design to nanobody-AP fusion production was successfully conducted, and the resulting nanobody based FLEIA was demonstrated as a convenient tool to monitor 2,4-D residuals in the environment.

Synthesis of pyrazole-4-carboxamides as potential fungicide candidates.

A series of novel pyrazole-4-carboxamides were rationally designed, synthesized, and their structures were characterized by 1H NMR, 13C NMR and HRMS. Preliminary bioassay showed that four compounds 8g, 8j, 8o and 8s exhibited more than 90% and even completed inhibition against Alternaria solani at 100 µg/mL; and 8d displayed 100% inhibition against Fusarium oxysporum at the same concentration. Moreover, 8j exhibited good in vitro fungicidal activity against A. solani with EC50 value of 3.06 µg/mL, and it also displayed completed in vivo protective antifungal activity against A. solani on tomato at 10 mg/L, as boscalid did. The molecular docking results indicated that 8j exhibited the high affinity with SDH protein by H-bond and π-π stacking interactions, which may explain the reasons for its good activities. These data support that compound 8j could be used as a fungicide candidate for further study. A practical method for the synthesis of pyrazole-4-carboxamides were provided and evaluation of their antifungal activities.

On-spot quantitative analysis of dicamba in field waters using a lateral flow immunochromatographic strip with smartphone imaging.

Dicamba herbicide is increasingly used in the world, in particular' with the widespread cultivation of genetically modified dicamba-resistant crops. However, the drift problem in the field has caused phytotoxicity against naive, sensitive crops, raising legal concerns. Thus, it is particularly timely to develop a method that can be used for on-the-spot rapid detection of dicamba in the field. In this paper, a lateral flow immunochromatographic strip (LFIC) was developed. The quantitative detection can be conducted by an app on a smartphone, named "Color Snap." The tool reported here provides results in 10 min and can detect dicamba in water with a LOD (detection limit) value of 0.1 mg/L. The developed LFIC shows excellent stability and sensitivity appropriate for field analysis. Our sensor is portable and excellent tool for on-site detection with smartphone imaging for better accuracy and precision of the results. Graphical abstract.

An Innovative Nanobody-Based Electrochemical Immunosensor Using Decorated Nylon Nanofibers for Point-of-Care Monitoring of Human Exposure to Pyrethroid Insecticides.

A novel ultrasensitive nanobody-based electrochemical immunoassay was prepared for assessing human exposure to pyrethroid insecticides. 3-Phenoxybenzoic acid (3-PBA) is a common human urinary metabolite for numerous pyrethroids, which broadly served as a biomarker for following the human exposure to this pesticide group. The 3-PBA detection was via a direct competition for binding to alkaline phosphatase-embedded nanobodies between free 3-PBA and a 3-PBA-bovine serum albumin conjugate covalently immobilized onto citric acid-decorated nylon nanofibers, which were incorporated on a screen-printed electrode (SPE). Electrochemical impedance spectroscopy (EIS) was utilized to support the advantage of the employment of nanofibrous membranes and the success of the immunosensor assembly. The coupling between the nanofiber and nanobody technologies provided an ultrasensitive and selective immunosensor for 3-PBA detection in the range of 0.8 to 1000 pg mL-1 with a detection limit of 0.64 pg mL-1. Moreover, when the test for 3-PBA was applied to real samples, the established immunosensor proved to be a viable alternative to the conventional methods for 3-PBA detection in human urine even without sample cleanup. It showed excellent properties and stability over time.