Discovery of pyridine-based agrochemicals by using Intermediate Derivatization Methods

Pyridine-based compounds have been playing a crucial role as agrochemicals or pesticides including fungicides, insecticides/acaricides and herbicides, etc. Since most of the agrochemicals listed in the Pesticide Manual were discovered through screening programs that relied on trial-and-error testing and new agrochemical discovery is not benefiting as much from the in silico new chemical compound identification/discovery techniques used in pharmaceutical research, it has become more important to find new methods to enhance the efficiency of discovering novel lead compounds in the agrochemical field to shorten the time of research phases in order to meet changing market requirements. In this review, we selected 18 representative known agrochemicals containing a pyridine moiety and extrapolate their discovery from the perspective of Intermediate Derivatization Methods in the hope that this approach will have greater appeal to researchers engaged in the discovery of agrochemicals and/or pharmaceuticals.     

Bacillus-based bionematicides: development,modes of action and commercialisation

Agricultural crops are severely damaged by root-knot nematodes causing extensive financial losses globally. Historically, agrochemicals have been the preferred method to combat these pests; however, threats to humans and the environment posed by these agrochemicals led to the need for developing new biocontrol agents. Importantly, the latter should adhere to biosafety regulations while being highly effective. Root-knot nematodes live in soil and thus the use of rhizobacteria such as Bacillus for biocontrol development have shown potential. Although various Bacillus species have been tested in this capacity, little is known about their secondary metabolites and the mechanisms of action responsible for their nematicidal activity. If these secondary metabolites can be qualitatively and quantitatively characterised, metabolic features could be synthetically engineered and used to combat root-knot nematodes. Although there is great potential for bionematicides, the commercialisation and development of such products can be difficult. This review summarises the importance of Bacillus species as natural antagonists of root-knot nematodes through the production of secondary metabolites. It provides an overview of the significance of root-knot nematodes in agriculture and the advances of chemical nematicides in recent years. The potential of Bacillus species as biocontrol agents, the known mechanisms of action responsible for the nematicidal activity demonstrated by Bacillus species, non-target effects of biocontrol agents and the commercialisation of Bacillus-based bionematicides are discussed.     

Fungicide-loaded and biodegradable xylan-based nanocarriers

The delivery of agrochemicals is typically achieved by the spraying of fossil-based polymer dispersions, which might accumulate in the soil and increase microplastic pollution. A potentially sustainable alternative is the use of biodegradable nano- or micro-formulations based on biopolymers, which can be degraded selectively by fungal enzymes to release encapsulated agrochemicals. To date, no hemicellulose nanocarriers for drug delivery in plants have been reported. Xylan is a renewable and abundant feedstock occurring naturally in high amounts in hemicellulose - a major component of the plant cell wall. Herein, xylan from corncobs was used to produce the first fungicide-loaded xylan-based nanocarriers by interfacial polyaddition in an inverse miniemulsion using toluene diisocyanate (TDI) as a crosslinking agent. The nanocarriers were redispersed in water and the aqueous dispersions were proven to be active in vitro against several pathogenic fungi, which are responsible for fungal plant diseases in horticulture or agriculture. Besides, empty xylan-based nanocarriers stimulated the growth of fungal mycelium, which indicated the degradation of xylan in the presence of the fungi, and underlined the degradation as a trigger to release a loaded agrochemical. This first example of crosslinked xylan-based nanocarriers expands the library of biodegradable and biobased nanocarriers for agrochemical release and might play a crucial role for future formulations in plant protection.     

The effect of agrochemicals on indicator bacteria densities in outdoor mesocosms

Water bodies, which are monitored for microbial water quality by quantification of faecal indicator organisms (IOs), can contain various zoonotic pathogens contributed by livestock waste and other sources. Sediments can serve as reservoirs of IOs and other enteric microorganisms, including pathogens. Agrochemicals may influence the survival of these microorganisms in water bodies impacted by livestock waste by enhancing or reducing their survival. Complex, 1100 l, freshwater mesocosms containing leaf litter, zooplankton, periphyton, phytoplankton, and invertebrate and vertebrate animals were used to investigate the response of Escherichia coli and enterococci to agrochemicals. Replicate tanks were treated with atrazine, malathion, chlorothalonil and inorganic fertilizer, either alone at 1× or 2× their expected environmental concentrations (EECs) or in pair‐wise combinations at their EECs. IOs inoculated in sediment (～10⁴ cfu per 100 ml) were enumerated over 28 days. IOs generally declined over time, but manova revealed that addition of fertilizer and atrazine resulted in significantly greater IO densities. Malathion, chlorothalonil and agrochemical concentration (1× vs 2×) did not significantly affect IO densities and no significant interactions between agrochemicals were noted. The augmentation of IO densities in sediments by fertilizer and atrazine may impact their reliability as accurate predictors of water quality and human health risk, and indicates the need for a better understanding of the fate of IOs and enteric pathogens in sediments exposed to agrochemicals.     

Biofilm formation as an extra gear for Apilactobacillus kunkeei to counter the threat of agrochemicals in honeybee crop

The alteration of a eubiosis status in honeybees’ gut microbiota is directly linked to the occurrence of diseases, and likely to the honeybees decline. Since fructophilic lactobacilli were suggested as symbionts for honeybees, we mechanistically investigated their behaviour under the exposure to agrochemicals (Roundup, Mediator and Reldan containing glyphosate, imidacloprid and chlorpyrifos-methyl as active ingredients respectively) and plant secondary metabolites (nicotine and p-coumaric acid) ingested by honeybees as part of their diet. The effects of exposure to agrochemicals and plant secondary metabolites were assessed both on planktonic cells and sessile communities of three biofilm-forming strains of Apilactobacillus kunkeei. We identified the high sensitivity of A. kunkeei planktonic cells to Roundup and Reldan, while cells embedded in mature biofilms had increased resistance to the same agrochemicals. However, agrochemicals still exerted a substantial inhibitory/control effect if the exposure was during the preliminary steps of biofilm formation. The level of susceptibility resulted to be strain-specific. Exopolysaccharides resulted in the main component of extracellular polymeric matrix (ECM) in biofilm, but the exposure to Roundup caused a change in ECM production and composition. Nicotine and p-coumaric acid had a growth-promoting effect in sessile communities, although no effect was found on planktonic growth.     

Synthesis and Stereochemical Characterization of Hydroxy- and Epoxy-derivatives of Rotenone

One to four routes of synthesis are described for 8′-hydroxyrotenone, 5′-hydroxyrotenone, two epimers of 6′,7′-dihydro-6′,7′-dihydroxyrotenone, two epimers of 6′,7′-epoxyrotenone and the four rotenolones derived from each of these compounds. The stereochemical relationships are determined, in each case, by chemical interconversion, ORD and monochromatic rotation to assess the absolute configuration of the B/C ring juncture and by IR, MS and NMR for the cis- or trans-nature of this juncture. The new compounds described are useful standards for studies on the metabolites and photodecomposition products of rotenone insecticide chemical.     

Microbial models of soil metabolism: biotransformations of danofloxacin

Danofloxacin is a new synthetic fluoroquinolone antibacterial agent under development for exclusive use in veterinary medicine. Such use could lead to deposition of low levels of danofloxacin residues in the environment in manure from treated livestock. This study was conducted to evaluate the potential for indigenous soil microorganisms to metabolize danofloxacin. Cultures of 72 soil microorganisms representing a diverse panel of bacteria, fungi and yeast were incubated with danofloxacin mesylate substrate and samples analyzed periodically by high performance liquid chromatography for loss of danofloxacin and formation of metabolites. Some samples were further analyzed by liquid chromatography-mass spectrometry and mass spectrometry to confirm metabolite identification. Twelve organisms, representing eight different genera, biotransformed danofloxacin to metabolites detectable by the chromatographic methods employed. Two Mycobacterium species, two Pseudomonas species, and isolates of Nocardia sp, Rhizopus arrhizus and Streptomyces griseus all formed N-desmethyldanofloxacin. The formation of the 7-amino danofloxacin derivative, 1-cyclopropyl-6-fluoro-7-amino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid by cultures of Candida lipopytica, Pseudomonas fluorescens, two Mycobacterium species and three Penicillium species demonstrates the propensities of these cultures to completely degrade the piperazine ring. At least two additional and unidentified metabolite peaks were observed in chromatograms of Aspergillus nidulans and Penicillium sp cultures. Radiolabled [2-¹⁴C]danofloxacin added to cultures of the fungus Curvularia lunata was apparently mineralized, with approximately 31% of the radiolabel recovered as volatile metabolites after 24 h of incubation, indicating the susceptibility of the quinolone ring to microbial metabolic degradation. Received 09 December 1996/ Accepted in revised form 09 April 1997     

Identification of quinine metabolites in urine after oral dosing in humans

A gas chromatographic–mass spectrometric method was used to separate quinine and its metabolites present in urine after oral dosing of 300 mg quinine in humans. The technique allowed the separation of quinine and ten metabolites. Four of these metabolites were definitely identified as 3-hydroxyquinine, 2′-quinone, O-desmethylquinine and 10,11-dihydroxydihydroquinine, by comparing their methane chemical ionization mass spectra with those of authentic standards prepared by organic synthesis. Six other metabolites are described for the first time in human urine. From their electron impact and chemical ionization mass spectra, we propose these compounds to be 3-hydroxy-2′-quinone, O-desmethyl-2′-quinone, O-desmethyl-3-hydroxyquinine, O-desmethyl-3-hydroxy-2′-quinone, 10,11-dihydroxydihydro-2′-quinone and 10,11-dihydroxydihydro-O-desmethylquinine. These secondary metabolites probably arose from further biotransformation of the four primary metabolites.     

Chemistry and biological activity of azoprenylated secondary metabolites

N-Prenyl secondary metabolites (isopentenylazo-, geranylazo-, farnesylazo- and their biosynthetic derivatives) represent a family of extremely rare natural products. Only in recent years have these alkaloids been recognized as interesting and valuable biologically active secondary metabolites. To date about 35 alkaloids have been isolated from plants mainly belonging to the Rutaceae family, and from fungi, bacteria, and/or obtained by chemical synthesis. These metabolites comprise anthranilic acid derivatives, diazepinones, and indole, and xanthine alkaloids. Many of the isolated prenylazo secondary metabolites and their semisynthetic derivatives are shown to exert valuable in vitro and in vivo anti-cancer, anti-inflammatory, anti-bacterial, anti-viral, and anti-fungal effects. The aim of this comprehensive review is to examine the different types of prenylazo natural products from a chemical, phytochemical and biological perspective.     

Arousing sleeping genes: shifts in secondary metabolism of metal tolerant actinobacteria under conditions of heavy metal stress

Numerous microbial habitats are strongly influenced by elevated levels of heavy metals. This type of habitat has developed either due to ore mining and metal processing or by pedogenesis above metal-rich base rocks. Most actinobacteria are soil-borne microbes with a remarkable capability for the synthesis of a broad variety of biologically active secondary metabolites. One major obstacle in identifying secondary metabolites, however, is the known phenomenon of sleeping gene clusters which are present, but silent under standard screening conditions. Here, we proceed to show that sleeping gene clusters can be awakened by the induction in heavy metal stress. Both, a chemical and a biological screening with extracts of supernatant and biomass of 10 strains derived from metal contaminated and non-contaminated environments was carried out to assay the influence of heavy metals on secondary metabolite patterns of metal tolerant actinobacteria. Metabolite patterns of cultures grown in complex and minimal media were compared to nickel (or cadmium) spiked parallels. Extracts of some strains grown in the presence of a metal salt displayed intense antibiosis against Escherichia coli, Mycobacterium smegmatis, Staphylococcus aureus and Candida albicans. Contrarily to the widely held opinion of metals as hindrance in secondary metabolism, metals thus can induce or enhance synthesis of possibly potent and medically relevant metabolites in metal tolerant strains. Hence, re-screening of existing strain libraries as well as identification of new strains from contaminated areas are valid strategies for the detection of new antibiotics in the future.     

Growth inhibition effect of pyroligneous acid on pathogenic fungus,<Emphasis Type="Italic">Alternaria mali</Emphasis>, the agent of Alternaria blotch of apple

We investigated the growth inhibition effect of pyroligneous acid on the pathogenic fungus,Alternaria mali, which is known to be the agent of Alternaria blotch of apple plants. Chemical control ofA. mali could be achieved through the use of agrochemical fungicides, while the substitute for agrochemical control is gradually increasing. It was observed that pyroligneous acid exhibited antifungal activity against some plant pathogenic fungi. More specifically, the growth ofA. mali was completely inhibited in pyroligneous acid at a dilution of 1∶32. When its antifungal activity was compared to that of polyoxin B, which is used for the chemical control of Alternaria blotch of apple, it was observed that the antifungal activity of pyroligneous acid diluted at 1∶32 corresponded to 2.0 mg/mL of polyoxin B. Consequently, it is concluded that the diluted pyroligneous acid can substitute for polyoxin B, thereby reducing the use of the agrochemical for the control of Alternaria blotch of apple.     

Tools and ingredients for the biocatalytic synthesis of metabolites

Metabolic networks have been an interesting starting point not only for the design of synthetic routes in a similar sequence of reactions, e.g., in biomimetic syntheses, but also for assembling a number of biocatalytic steps by preparing the required enzymes and auxiliary reagents. Retrosynthetic analysis involving multiple biocatalytic reactions steps therefore needs to consider the practically realized biocatalytic single steps. The opportunities for route selection are enlarged if novel synthetic reactions connecting easily available starting materials and products are found, and/or both biocatalytic and classical reactions of organic chemistry are utilized. Tools and ingredients for biocatalytic synthesis are of special interest for reactions difficult to achieve by classical organic synthesis. Densely and differentially functionalized small molecules do not allow much space for protecting or activating groups. Biocatalytic reactions have therefore performed well for a number of useful metabolites in enantiopure form to achieve full functionality. Although many well-known metabolites from classical biochemistry have only been prepared in racemic form, it is of fundamental interest to have these available in enantiomerically pure form. Biocatalytic reactions with nature's privileged chiral catalysts appear to be a promising synthetic strategy towards these metabolites, especially when sensitive or stable-isotope-labeled metabolites are to be prepared. The main applications for these metabolites are as references materials in metabolomics, as enzyme substrates for the characterization of metabolic enzyme activities and as potential pharmaceuticals in biomedical research. The use of stable-isotope-labeled metabolites can thereby simplify in vivo applications and metabolic flux analyses.     

Production of Ethyl (R)-2-Hydroxy-4-phenylbutanoate via Reduction of Ethyl 2-Oxo-4-phenylbutanoate in an Interface Bioreactor

Ethyl (R)-2-hydroxy-4-phenylbutanoate [(R)-EHPB], a useful intermediate for the synthesis of various anti-hypertension drugs, was produced via microbial reduction of ethyl 2-oxo-4-phenylbutanoate [EOPB] in an interface bioreactor. Rhodotorula minuta IFO 0920 and Candida holmii KPY 12402 were selected as the best type culture and isolated yeasts, respectively. The highest enantiomeric excess of (R)-EHPB produced by R. minuta and C. holmii were 95 and 94%, respectively. C. holmii was used for the reduction of EOPB in a pad-packed interface bioreactor (inner volume, 3 liter). After incubation for 4 days, 4.4 g of (R)-EHPB was obtained via extraction with methanol followed by column chromatography. The overall yield, chemical purity, and enantiomeric excess of (R)-EHPB were 58%, 99.1%, and 90%, respectively.     

The Effect of the Intra- and Extracellular Metabolites of Microorganisms Isolated from Various Ecotopes on the Catalase Activity of Staphylococcus aureus ATCC 6538 P

The cell extracts (i.e., intracellular metabolites) and culture liquids (i.e., extracellular metabolites) of microorganisms isolated from various ecotopes were found to inhibit the catalase activity of Staphylococcus aureus ATCC 6538 P, which resulted in a considerable inhibition of the growth of metabolite-treated S. aureus cells by hydrogen peroxide. The inhibitory effect of microbial metabolites on S. aureus catalase can be considered as a mechanism of intercellular interactions responsible for the formation of microbiocenoses.     

Distinction between α- and γ-Glutamyl Dipeptides by Means of NMR Spectrometer and Amino Acid Analyzer

Conjugated metabolites of abscisic acid (ABA) have been characterized by their chemical ionization (CI) mass spectra using isobutane and ammonia as reagent gases. The CI mass spectra usually consist of quasimolecular ions ([QM]⁺), and constituent aglycone- and.sugar-derived ions. Hence, the spectra are simply interpreted and afford useful information for structutal characterization. CI examinations of ABA and its unconjugated metabolites and collisionally activated dissociation (CAD) measurements of selected ions were helpful in characterizing the constituent aglycones. The distinction between the two glycosidal forms, glycoside and glycosyl ester, has been also discussed in the corresponding conjugates. Confirmation of the fragmentation pathways via recognition of the diagnostic ions can be made by the extensive use of ammonia- d₃ as an additional reagent gas.     

Physical and biological compatibility of diafenthiuron with micro/macro nutrients fungicides and biocontrol agents used in cardamom

In the context of complex field problems, compatibility of an efficacious insecticide with other agrochemicals normally used in the field is essential. In this view diafenthiuron, a novel insecticide which inhibits ATP synthesis, used widely for pest management in cardamom, was tested for its compatibility with agrochemicals viz., fungicides and nutrients normally used in the crop. The results revealed that all the chemicals tested were physically and biologically compatible with diafenthiuron by means of physical stability and phytotoxicity ratings in the field. But the bioefficacy study on Conogethes punctiferalis in the laboratory and bioefficacy studies in the field against Sciothrips cardamomi reveal that diafenthiuron is incompatible and should not be sprayed along with fungicides like mancozeb and copper oxychloride. Another study on the compatibility of diafenthiuron with antagonistic microorganisms of plant pathogens viz., Trichoderma viride and Pseudomonas fluorescens revealed that diafenthiuron had some inhibitory effect on the mycelial growth of T. viride. Diafenthiuron did not affect the growth of P. fluorescens and thus can be used simultaneously for the control of insect pests and seed- and soil-borne diseases.     

Seasonal prevalence and susceptibility to agrochemicals of Tyrophagus similis (Acari: Acaridae) in spinach buds and agricultural soil under greenhouse conditions

Seasonal prevalence of Tyrophagus similis was investigated from 1997 to 1998 in two spinach greenhouses in central Japan. Susceptibility of T. similis to agrochemicals was also tested in the laboratory. Tyrophagus similis density in the soil was low during the high temperature period from May to August. The density rapidly increased in late autumn and remained at a high level during the cool season from December to February. The number further increased in April and then rapidly decreased in May. The high temperatures in the greenhouses from spring to early autumn are considered the main causes of population decrease. Mites on spinach buds increased the number after those on and in the soil increased. Mites attacked spinach buds mostly in late autumn and early spring. Dichlorvos did not reduce the number of mites in either greenhouse even though it was highly toxic under laboratory condition. This discrepancy suggests that the mites in both the soil and spinach buds had little direct contact with the chemicals. These data suggest that once crop damage by mites is detected, it is usually too late to use chemicals, and that mites that live in the buds are protected from agrochemicals. This revised version was published online in July 2006 with corrections to the Cover Date.     

核开关结构、机制及应用新进展

核开关(riboswitch)是Breaker等在2002年发现的一种全新的转录后调节机制．它可以通过小分子与mRNA结合来直接调控基因的表达,不需要任何蛋白质的参与．与常见的经由蛋白质的调控方式相比,riboswitch响应更迅速,对细胞内代谢物的变化更敏感．它的发现为RNA研究展示了新的领域． 目前在这个领域,既有基础研究,如riboswitch晶体结构解析、作用机制和动力学研究,又有前沿应用研究,如基于riboswitch的生物传感器和药物设计．Topp等通过设计riboswitch成功地改变了大肠杆菌的趋化性,这为合成生物学和人工生物网络的设计提供了新思路．目前对于riboswitch结构、机制及动力学的研究为基于riboswitch的合理药物设计奠定了基础,有望针对这一新的机制开发新一代抗菌药物．     

Distribution and metabolism of exogenous ecdysteroids in the egyptian cotton leafworm Spodoptera littoralis (lepidoptera: noctuidae)

After ingestion of various amounts of either [³H]ecdysone or [³H]20-hydroxyecdysone (0.8 ng to 10 μg) by sixth instar larvae of the Egyptian cotton leafworm Spodoptera littoralis, apolar metabolites are rapidly detected in the gut and frass. Hydrolysis of the apolar products with Helix hydrolases releases solely [³H]ecdysone or [³H]20-hydroxyecdysone, respectively. This, coupled with the formation of chemical derivatives (acetonide and acetate) which cochromatograph with authentic reference compounds on hptlc and hplc demonstrates that these apolar metabolites consist of ecdysone or 20-hydroxyecdysone esterified at C-22 with common long-chain fatty acids. The major fatty acids have been identified by RP-hplc and their contribution to the mixture determined. In contrast, [³H]ecdysone injected into the haemolymph of S. littoralis is metabolized to yield 20-hydroxyecdysone, ecdysonoic acid, and 20-hydroxyecdysonoic acid. Thus, two different pathways exist for the metabolism of ecdysteroids in this species. In addition to an essentially polar pathway operating on injected and endogenous ecdysteroids, exogenous ecdysteroids entering the gut of S. littoralis are detoxified, yielding apolar ecdysteroid 22-fatty acyl esters which are rapidly excreted. The significance of these results in relation to the effects of ingested ecdysteroids on S. littoralis is discussed. Arch. Insect Biochem. Physiol. 34:329–346, 1997. © 1997 Wiley-Liss, Inc.