Spiro N-methoxy piperidine ring containing aryldiones for the control of sucking insects and mites: discovery of spiropidion.

BACKGROUND: Crop protection solutions for the control of key economic sucking pests derive essentially from neuronal and muscular acting chemistries, wherein neonicotinoid uses largely dominated for the last two decades. Anticipating likely resistance development of some of those arthropod species to this particular class, we intensified research activities on a non-neuronal site of action targeting insect growth and development some 10 years ago. RESULTS: Our innovation path featured reactivation of a scarcely used and simple building block from the 1960s, namely N-methoxy-4-piperidone 3. Its judicious incorporation into the 2-aryl-1,3-dione scaffold of IRAC group 23 inhibitors of fatty acid biosynthesis resulted in novel tetramic acid derivatives acting on acetyl-coenzyme A carboxylase (ACCase). The optimization campaign focused on modulation of the aryl substitution pattern and understanding substituent options at the lactam nitrogen position of those spiroheterocyclic pyrrolidine-dione derivatives towards an effective control of sucking insects and mites. This work gratifyingly culminated in the discovery of spiro N-methoxy piperidine containing proinsecticide spiropidion 1. Following in planta release, its insecticidally active dione metabolite 2 is translaminar and two-way systemic (both xylem and phloem mobile) for a full plant protection against arthropod pests. CONCLUSION: Owing to such unique plant systemic properties, growing shoots and roots actually not directly exposed to spiropidion-based chemistry after foliar application nevertheless benefit from its long-lasting efficacy. Spiropidion is for use in field crops, speciality crops and vegetables controlling a broad range of sucking pests. In light of other performance and safety profiles of spiropidion, an IPM fit may be expected. © 2020 Society of Chemical Industry.

Synthesis and evaluation of hydroxyazolopyrimidines as herbicides; the generation of amitrole in planta.

BACKGROUND: Exploiting novel herbicidal modes of action is an important method to overcome the challenges faced by increasing resistance and regulatory pressure on existing commercial herbicides. Recent reports of inhibitors of enzymes in the non-mevalonate pathway of isoprenoid biosynthesis led to the design of a novel class of azolopyrimidines which were assessed for their herbicidal activity. Studies were also undertaken to determine the mode of action responsible for the observed herbicidal activity. RESULTS: In total, 30 novel azolopyrimidines were synthesised and their structures were unambiguously determined by 1 H NMR, mass spectroscopy and X-ray crystallographic analysis. The herbicidal activity of this new chemical class was assessed against six common weed species, with compounds from this series displaying bleaching symptomology in post-emergence tests. A structure-activity relationship for the novel compounds was determined, which showed that only those belonging to the hydroxytriazolopyrimidine subclass displayed significant herbicidal activity. Observed similarities between the bleaching symptomology displayed by these herbicides and amitrole suggested that hydroxytriazolopyrimidines could be acting as elaborate propesticides of amitrole, and this was subsequently demonstrated in plant metabolism studies using Amaranthus retroflexus. It was shown that selected hydroxytriazolopyrimidines that displayed promising herbicidal activity generated amitrole, with peak concentrations of amitrole generally being observed 1 day after application. Additionally, the herbicidal activity of selected compounds was profiled against tobacco plants engineered to overexpress 4-diphosphocytidyl-2C-methyl-d-erythritol synthase (IspD) or lycopene ß-cyclase, and the results suggested that, where significant herbicidal activity was observed, inhibition of IspD was not responsible for the activity. Tobacco plants overexpressing lycopene ß-cyclase showed tolerance to amitrole and the two most herbicidally active triazolopyrimidines. CONCLUSIONS: Inhibition of IspD leading to herbicidal activity has been ruled out as the mode of action for the hydroxytriazolopyrimidine class of herbicides. Additionally, tobacco plants overexpressing lycopene ß-cyclase showed tolerance to amitrole, which indicates that this is the main herbicidal mode of action for amitrole. Results from the metabolic fate study of selected hydroxytriazolopyrimidines suggested that the herbicidal activity displayed by these compounds is due to amitrole production, which was confirmed when tobacco plants overexpressing lycopene ß-cyclase also showed tolerance towards two triazolopyrimidines from this study. © 2016 Society of Chemical Industry.

Synthesis and stability of boratriazaroles.

We describe the synthesis and stability analysis of novel boratriazaroles that can be viewed as bioisosteres of imidazoles or pyrazoles. These heterocycles could conveniently be obtained by condensing a boronic acid and amidrazone 1 in various solvents. A detailed stability analysis of selected compounds at different pH values as a function of time led to the identification of steric hindrance around the boron atom as a key element for stabilization.

The crystal structure of the phosphatidylinositol 4-kinase IIα.

Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4-kinase type IIα (PI4K IIα), in complex with ATP solved by X-ray crystallography at 2.8 Å resolution. The structure revealed a non-typical kinase fold that could be divided into N- and C-lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C-lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.

Directing group enhanced carbonylative ring expansions of amino-substituted cyclopropanes: rhodium-catalyzed multicomponent synthesis of N-heterobicyclic enones.

Aminocyclopropanes equipped with suitable N-directing groups undergo efficient and regioselective Rh-catalyzed carbonylative C-C bond activation. Trapping of the resultant metallacycles with tethered alkynes provides an atom-economic entry to diverse N-heterobicyclic enones. These studies provide a blueprint for myriad N-heterocyclic methodologies.

Structural basis for the recognition of tyrosine-based sorting signals by the µ3A subunit of the AP-3 adaptor complex.

Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous µ1, µ2, µ3, and µ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on µ2 and µ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the µ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the µ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The µ3A C-terminal domain consists of an immunoglobulin-like ß-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on µ3A subdomain A, at a location similar to the YXXØ-binding site on µ2 but not µ4. The binding sites on µ3A and µ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the µ3A site and show that this protein binds YXXØ signals with 14-19 µm affinity. The surface electrostatic potential of µ3A is less basic than that of µ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.

Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules.

Sorting nexins (SNXs) are regulators of endosomal sorting. For the SNX-BAR subgroup, a Bin/Amphiphysin/Rvs (BAR) domain is vital for formation/stabilization of tubular subdomains that mediate cargo recycling. Here, by analysing the in vitro membrane remodelling properties of all 12 human SNX-BARs, we report that some, but not all, can elicit the formation of tubules with diameters that resemble sorting tubules observed in cells. We reveal that SNX-BARs display a restricted pattern of BAR domain-mediated dimerization, and by resolving a 2.8 Å structure of a SNX1-BAR domain homodimer, establish that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR-dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX-BARs, and the formation of high order SNX-BAR oligomers through selective 'tip-loop' interactions. Overall, the restricted and selective nature of these interactions provide a molecular explanation for how distinct SNX-BAR-decorated tubules are nucleated from the same endosomal vacuole, as observed in living cells. Our data provide insight into the molecular mechanism that generates and organizes the tubular endosomal network.

Novel diamide insecticides: sulfoximines, sulfonimidamides and other new sulfonimidoyl derivatives.

Novel insecticidal anthranilamides with elaborated sulfur-containing groups are described. The synthesis of compounds with functional groups such as sulfoximines and scarcely reported groups such as sulfonimidoyl hydrazides and hydroxylamides, their in vitro and in vivo biological activity as well as their physical properties are reported.

Aryldiones incorporating a [1,4,5]oxadiazepane ring. Part 2: chemistry and biology of the cereal herbicide pinoxaden.

BACKGROUND: Pinoxaden is a new cereal herbicide that provides outstanding levels of post-emergence activity against a broad spectrum of grass weed species for worldwide selective use in both wheat and barley. RESULTS: Factors influencing activity and tolerance to pinoxaden were in part linked to distinct structural parts of the active ingredient. Three complementary contributions that decisively impact upon the herbicidal potency against grasses were identified: a preferred 2,6-diethyl-4-methyl aromatic substitution pattern, a dione area suitable for proherbicide formation and beneficial adjuvant effects. The uptake and translocation pattern of pinoxaden when coapplied with its tailored adjuvant were analysed by autoradiography, indicating extensive and rapid penetration, followed by effective distribution throughout the plant. Crop injury reduction on incorporation of the [1,4,5]oxadiazepane ring into the aryldione template was reinforced with safener technology. Comparative studies on the behaviour of pinoxaden applied either alone or in combination with the safener cloquintocet-mexyl demonstrated that addition of the safener resulted in significant enhancement of metabolic degradation in wheat and barley, providing excellent crop tolerance and a substantial selectivity margin without adverse effects on weed control. CONCLUSION: The biological potential of pinoxaden and its active principle pinoxaden dione in terms of grass weed control and tolerance in cereals was fully exploited by inclusion of the safener cloquintocet-mexyl in the formulation in combination with a specific and tailor-made tank-mix adjuvant based on methylated rape seed oil.

Assembly of the biogenesis of lysosome-related organelles complex-3 (BLOC-3) and its interaction with Rab9.

The Hermansky-Pudlak syndrome (HPS) is a genetic hypopigmentation and bleeding disorder caused by defective biogenesis of lysosome-related organelles (LROs) such as melanosomes and platelet dense bodies. HPS arises from mutations in any of 8 genes in humans and 16 genes in mice. Two of these genes, HPS1 and HPS4, encode components of the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Herein we show that recombinant HPS1-HPS4 produced in insect cells can be efficiently isolated as a 1:1 heterodimer. Analytical ultracentrifugation reveals that this complex has a molecular mass of 146 kDa, equivalent to that of the native complex and to the sum of the predicted molecular masses of HPS1 and HPS4. This indicates that HPS1 and HPS4 interact directly in the absence of any other protein as part of BLOC-3. Limited proteolysis and deletion analyses show that both subunits interact with one another throughout most of their lengths with the sole exception of a long, unstructured loop in the central part of HPS4. An interaction screen reveals a specific and strong interaction of BLOC-3 with the GTP-bound form of the endosomal GTPase, Rab9. This interaction is mediated by HPS4 and the switch I and II regions of Rab9. These characteristics indicate that BLOC-3 might function as a Rab9 effector in the biogenesis of LROs.

Hybrid structural model of the complete human ESCRT-0 complex.

The human Hrs and STAM proteins comprise the ESCRT-0 complex, which sorts ubiquitinated cell surface receptors to lysosomes for degradation. Here we report a model for the complete ESCRT-0 complex based on the crystal structure of the Hrs-STAM core complex, previously solved domain structures, hydrodynamic measurements, and Monte Carlo simulations. ESCRT-0 expressed in insect cells has a hydrodynamic radius of RH = 7.9 nm and is a 1:1 heterodimer. The 2.3 Angstroms crystal structure of the ESCRT-0 core complex reveals two domain-swapped GAT domains and an antiparallel two-stranded coiled-coil, similar to yeast ESCRT-0. ESCRT-0 typifies a class of biomolecular assemblies that combine structured and unstructured elements, and have dynamic and open conformations to ensure versatility in target recognition. Coarse-grained Monte Carlo simulations constrained by experimental RH values for ESCRT-0 reveal a dynamic ensemble of conformations well suited for diverse functions.

Structure and reaction geometry of geranylgeranyl diphosphate synthase from Sinapis alba.

The crystal structure of the geranylgeranyl diphosphate synthase from Sinapis alba (mustard) has been solved in two crystal forms at 1.8 and 2.0 A resolutions. In one of these forms, the dimeric enzyme binds one molecule of the final product geranylgeranyl diphosphate in one subunit. The chainfold of the enzyme corresponds to that of other members of the farnesyl diphosphate synthase family. Whereas the binding modes of the two substrates dimethylallyl diphosphate and isopentenyl diphosphate at the allyl and isopentenyl sites, respectively, have been established with other members of the family, the complex structure presented reveals for the first time the binding mode of a reaction product at the isopentenyl site. The binding geometry of substrates and product in conjunction with the protein environment and the established chemistry of the reaction provide a clear picture of the reaction steps and atom displacements. Moreover, a comparison with a ligated homologous structure outlined an appreciable induced fit: helix alpha8 and its environment undergo a large conformational change when either the substrate dimethylallyl diphosphate or an analogue is bound to the allyl site; only a minor conformational change occurs when the other substrate isopentenyl diphosphate or the product is bound to the isopentenyl site.

Crystal structure of ethylbenzene dehydrogenase from Aromatoleum aromaticum.

Anaerobic degradation of hydrocarbons was discovered a decade ago, and ethylbenzene dehydrogenase was one of the first characterized enzymes involved. The structure of the soluble periplasmic 165 kDa enzyme was established at 1.88 A resolution. It is a heterotrimer. The alpha subunit contains the catalytic center with a molybdenum held by two molybdopterin-guanine dinucleotides, one with an open pyran ring, and an iron-sulfur cluster with a histidine ligand. During catalysis, electrons produced by substrate oxidation are transferred to a heme in the gamma subunit and then presumably to a separate cytochrome involved in nitrate respiration. The beta subunit contains four iron-sulfur clusters and is structurally related to ferredoxins. The gamma subunit is the first known protein with a methionine and a lysine as axial heme ligands. The catalytic product was modeled into the active center, showing the reaction geometry. A mechanism consistent with activity and inhibition data of ethylbenzene-related compounds is proposed.

Catalytic importance of acidic amino acids on subunit NuoB of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN and contains one FMN and 9 iron-sulfur clusters as redox groups. Electron transfer from NADH to ubiquinone is coupled with the translocation of protons across the membrane by a yet unknown mechanism. Redox-induced Fourier transform infrared difference spectroscopy showed that the oxidation of iron-sulfur cluster N2 located on NuoB is accompanied by the protonation of acidic amino acid(s). Here, we describe the effect of mutating the conserved acidic amino acids on NuoB. The complex was assembled in all mutants but the electron transfer activity was completely abolished in the mutants E67Q, D77N, and D94N. The complex isolated from these mutants contained N2 although in diminished amounts. The protonation of acidic amino acid(s) coupled with the oxidation of N2 was not detectable in the complex from the mutant E67Q. However, the conservative mutations E67D and D77E did not disturb the enzymatic activity, and the signals because of the protonation of acidic amino acid(s) were detectable in the E67D mutant. We discuss the possible participation of Glu(67) in a proton pathway coupled with the redox reaction of N2.

Retinal is formed from apo-carotenoids in Nostoc sp. PCC7120: in vitro characterization of an apo-carotenoid oxygenase.

The sensory rhodopsin from Anabaena (Nostoc) sp. PCC7120 is the first cyanobacterial retinylidene protein identified. Here, we report on NosACO (Nostoc apo-carotenoid oxygenase), encoded by the ORF (open reading frame) all4284, as the candidate responsible for the formation of the required chromophore, retinal. In contrast with the enzymes from animals, NosACO converts beta-apo-carotenals instead of beta-carotene into retinal in vitro. The identity of the enzymatic products was proven by HPLC and gas chromatography-MS. NosACO exhibits a wide substrate specificity with respect to chain lengths and functional end-groups, converting beta-apo-carotenals, (3R)-3-hydroxy-beta-apo-carotenals and the corresponding alcohols into retinal and (3R)-3-hydroxyretinal respectively. However, kinetic analyses revealed very divergent Km and Vmax values. On the basis of the crystal structure of SynACO (Synechocystis sp. PCC6803 apo-carotenoid oxygenase), a related enzyme showing similar enzymatic activity, we designed a homology model of the native NosACO. The deduced structure explains the absence of beta-carotene-cleavage activity and indicates that NosACO is a monotopic membrane protein. Accordingly, NosACO could be readily reconstituted into liposomes. To localize SynACO in vivo, a Synechocystis knock-out strain was generated expressing SynACO as the sole carotenoid oxygenase. Western-blot analyses showed that the main portion of SynACO occurred in a membrane-bound form.

The crystal structure of Aspergillus fumigatus cyclophilin reveals 3D domain swapping of a central element.

The crystal structure of Aspergillus fumigatus cyclophilin (Asp f 11) was solved by the multiwavelength anomalous dispersion method and was refined to a resolution of 1.85 A with R and R(free) values of 18.9% and 21.4%, respectively. Many cyclophilin structures have been solved to date, all showing the same monomeric conformation. In contrast, the structure of A. fumigatus cyclophilin reveals dimerization by 3D domain swapping and represents one of the first proteins with a swapped central domain. The domain-swapped element consists of two beta strands and a subsequent loop carrying a conserved tryptophan. The tryptophan binds into the active site, inactivating cis-trans isomerization. This might be a means of biological regulation. The two hinge loops leave the protein prone to misfolding. In this context, alternative forms of 3D domain swapping that can lead to N- or C-terminally swapped dimers, oligomers, and aggregates are discussed.

The structure of a retinal-forming carotenoid oxygenase.

Enzymes that produce retinal and related apocarotenoids constitute a sequence- and thus structure-related family, a member of which was analyzed by x-ray diffraction. This member is an oxygenase and contains an Fe2+-4-His arrangement at the axis of a seven-bladed beta-propeller chain fold covered by a dome formed by six large loops. The Fe2+ is accessible through a long nonpolar tunnel that holds a carotenoid derivative in one of the crystals. On binding, three consecutive double bonds of this carotenoid changed from a straight all-trans to a cranked cis-trans-cis conformation. The remaining trans bond is located at the dioxygen-ligated Fe2+ and cleaved by oxygen.

Structure of a type II thymidine kinase with bound dTTP.

The structure of human cytosolic thymidine kinase in complex with its feedback inhibitor 2'-deoxythymidine-5'-triphosphate was determined. This structure is the first representative of the type II thymidine kinases found in several pathogens. The structure deviates strongly from the known structures of type I thymidine kinases such as the Herpes simplex enzyme. It contains a zinc-binding domain with four cysteines complexing a structural zinc ion. Interestingly, the backbone atoms of the type II enzyme bind thymine via hydrogen-bonds, in contrast to type I, where side chains are involved. This results in a specificity difference exploited for antiviral therapy. The presented structure will foster the development of new drugs and prodrugs for numerous therapeutic applications.

Structural analysis of the group II intron splicing factor CRS2 yields insights into its protein and RNA interaction surfaces.

Chloroplast RNA splicing 2 (CRS2) is a nuclear-encoded protein required for the splicing of nine group II introns in maize chloroplasts. CRS2 functions in the context of splicing complexes that include one of two CRS2-associated factors (CAF1 and CAF2). The CRS2-CAF1 and CRS2-CAF2 complexes are required for the splicing of different subsets of CRS2-dependent introns, and they bind tightly and specifically to their genetically defined intron targets in vivo. The CRS2 amino acid sequence is closely related to those of bacterial peptidyl-tRNA hydrolases (PTHs). To identify the structural differences between CRS2 and bacterial PTHs responsible for CRS2's gains of CAF binding and intron splicing functions, we determined the structure of CRS2 by X-ray crystallography. The fold of CRS2 is the same as that of Escherichia coli PTH, but CRS2 has two surfaces that differ from the corresponding surfaces in PTH. One of these is more hydrophobic in CRS2 than in PTH. Site-directed mutagenesis of this surface blocked CRS2-CAF complex formation, indicating that it is the CAF binding site. The CRS2 surface corresponding to the putative tRNA binding face of PTH is considerably more basic than in PTH, suggesting that CRS2 interacts with group II intron substrates via this surface. Both the sequence and the structural context of the amino acid residues essential for peptidyl-tRNA hydrolase activity are conserved in CRS2, yet expression of CRS2 is incapable of rescuing a pth(ts)E.coli strain.