Applying the Crystalline Sponge Method to Agrochemicals: Obtaining X-ray Structures of the Fungicide Metalaxyl-M and Herbicide S-Metolachlor.

The crystalline sponge method is a technique that provides the ability to elucidate the absolute structure of noncrystalline or hard to crystallize compounds through single-crystal X-ray diffraction by removing the need to obtain crystals of the target compound. In this study the crystalline sponges {[(ZnX2)3(2,4,6-tris(4-pyridyl)-1,3,5-trazine)2].x(solvent)} n (X = I, Br) were used to obtain X-ray structures of the agrochemical active ingredients metalaxyl-M and S-metolachlor. The effect of the temperature used during guest uptake and the influence of changing the host framework ZnX2 nodes on guest encapsulation were investigated. Additionally, three compounds containing chemical fragments similar to those of metalaxyl-M and S-metolachlor (phenylacetaldehyde, N-ethyl-o-toluidine, and methyl phenylacetate) were also encapsulated. This allowed for the effect of guest size on the position that guests occupy within the host frameworks to be examined. The disorder experienced by the guest compounds was documented, and an analysis of the intermolecular host-guest interactions (CH···π and π ···π) used for guest ordering within the host frameworks was also undertaken in this study.

NEMO oligomerization and its ubiquitin-binding properties.

The IKK [IkappaB (inhibitory kappaB) kinase] complex is a key regulatory component of NF-kappaB (nuclear factor kappaB) activation and is responsible for mediating the degradation of IkappaB, thereby allowing nuclear translocation of NF-kappaB and transcription of target genes. NEMO (NF-kappaB essential modulator), the regulatory subunit of the IKK complex, plays a pivotal role in this process by integrating upstream signals, in particular the recognition of polyubiquitin chains, and relaying these to the activation of IKKalpha and IKKbeta, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a tetrameric assembly. Interaction with peptides derived from IKKbeta disrupts formation of the tetrameric NEMO complex, indicating that interaction with IKKalpha and IKKbeta and tetramerization are mutually exclusive. Furthermore, we show that NEMO binds to linear di-ubiquitin with a stoichiometry of one molecule of di-ubiquitin per NEMO dimer. This stoichiometry is preserved in a construct comprising the second coiled-coil region and the leucine zipper and in one that essentially spans the full-length protein. However, our data show that at high di-ubiquitin concentrations a second weaker binding site becomes apparent, implying that two different NEMO-di-ubiquitin complexes are formed during the IKK activation process. We propose that the role of these two complexes is to provide a threshold for activation, thereby ensuring sufficient specificity during NF-kappaB signalling.

Scalable total synthesis and comprehensive structure-activity relationship studies of the phytotoxin coronatine.

Natural phytotoxins are valuable starting points for agrochemical design. Acting as a jasmonate agonist, coronatine represents an attractive herbicidal lead with novel mode of action, and has been an important synthetic target for agrochemical development. However, both restricted access to quantities of coronatine and a lack of a suitably scalable and flexible synthetic approach to its constituent natural product components, coronafacic and coronamic acids, has frustrated development of this target. Here, we report gram-scale production of coronafacic acid that allows a comprehensive structure-activity relationship study of this target. Biological assessment of a >120 member library combined with computational studies have revealed the key determinants of potency, rationalising hypotheses held for decades, and allowing future rational design of new herbicidal leads based on this template.

Single-crystal X-ray diffraction and NMR crystallography of a 1:1 cocrystal of dithianon and pyrimethanil.

A single-crystal X-ray diffraction structure of a 1:1 cocrystal of two fungicides, namely dithianon (DI) and pyrimethanil (PM), is reported [systematic name: 5,10-dioxo-5H,10H-naphtho[2,3-b][1,4]dithiine-2,3-dicarbonitrile-4,6-dimethyl-N-phenylpyrimidin-2-amine (1/1), C14H4N2O2S2·C12H13N2]. Following an NMR crystallography approach, experimental solid-state magic angle spinning (MAS) NMR spectra are presented together with GIPAW (gauge-including projector augmented wave) calculations of NMR chemical shieldings. Specifically, experimental 1H and 13C chemical shifts are determined from two-dimensional 1H-13C MAS NMR correlation spectra recorded with short and longer contact times so as to probe one-bond C-H connectivities and longer-range C...H proximities, whereas H...H proximities are identified in a 1H double-quantum (DQ) MAS NMR spectrum. The performing of separate GIPAW calculations for the full periodic crystal structure and for isolated molecules allows the determination of the change in chemical shift upon going from an isolated molecule to the full crystal structure. For the 1H NMR chemical shifts, changes of 3.6 and 2.0 ppm correspond to intermolecular N-H...O and C-H...O hydrogen bonding, while changes of -2.7 and -1.5 ppm are due to ring current effects associated with C-H...π interactions. Even though there is a close intermolecular S...O distance of 3.10 Å, it is of note that the molecule-to-crystal chemical shifts for the involved sulfur or oxygen nuclei are small.

Catalytic mechanism of C-C hydrolase MhpC from Escherichia coli: kinetic analysis of His263 and Ser110 site-directed mutants.

C-C hydrolase MhpC (2-hydroxy-6-keto-nona-1,9-dioic acid 5,6-hydrolase) from Escherichia coli catalyses the hydrolytic C-C cleavage of the meta-ring fission product on the phenylpropionic acid catabolic pathway. The crystal structure of E. coli MhpC has revealed a number of active-site amino acid residues that may participate in catalysis. Site-directed mutants of His263, Ser110, His114, and Ser40 have been analysed using steady-state and stopped-flow kinetics. Mutants H263A, S110A and S110G show 10(4)-fold reduced catalytic efficiency, but still retain catalytic activity for C-C cleavage. Two distinct steps are observed by stopped-flow UV/Vis spectrophotometry, corresponding to ketonisation and C-C cleavage: H263A exhibits very slow ketonisation and C-C cleavage, whereas S110A and S110G exhibit fast ketonisation, an intermediate phase, and slow C-C cleavage. H114A shows only twofold-reduced catalytic efficiency, ruling out a catalytic role, but shows a fivefold-reduced K(M) for the natural substrate, and an ability to process an aryl-containing substrate, implying a role for His114 in positioning of the substrate. S40A shows only twofold-reduced catalytic efficiency, but shows a very fast (500 s(-1)) interconversion of dienol (317 nm) to dienolate (394 nm) forms of the substrate, indicating that the enzyme accepts the dienol form of the substrate. These data imply that His263 is responsible for both ketonisation of the substrate and for deprotonation of water for C-C cleavage, a novel catalytic role in a serine hydrolase. Ser110 has an important but non-essential role in catalysis, which appears not to be to act as a nucleophile. A catalytic mechanism is proposed involving stabilisation of reactive intermediates and activation of a nucleophilic water molecule by Ser110.

Catalytic role for arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD.

The alpha/beta-hydrolase superfamily, comprised mainly of esterase and lipase enzymes, contains a family of bacterial C-C hydrolases, including MhpC and BphD which catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid pathway and Burkholderia xenovorans LB400 biphenyl degradation pathway, respectively. Five active site amino acid residues (Arg-188, Asn-109, Phe-173, Cys-261, and Trp-264) were identified from sequence alignments that are conserved in C-C hydrolases, but not in enzymes of different function. Replacement of Arg-188 in MhpC with Gln and Lys led to 200- and 40-fold decreases, respectively, in k(cat); the same replacements for Arg-190 of BphD led to 400- and 700-fold decreases, respectively, in k(cat). Pre-steady-state kinetic analysis of the R188Q MhpC mutant revealed that the first step of the reaction, keto-enol tautomerization, had become rate-limiting, indicating that Arg-188 has a catalytic role in ketonization of the dienol substrate, which we propose is via substrate destabilization. Mutation of nearby residues Phe-173 and Trp-264 to Gly gave 4-10-fold reductions in k(cat) but 10-20-fold increases in K(m), indicating that these residues are primarily involved in substrate binding. The X-ray structure of a succinate-H263A MhpC complex shows concerted movements in the positions of both Phe-173 and Trp-264 that line the approach to Arg-188. Mutation of Asn-109 to Ala and His yielded 200- and 350-fold reductions, respectively, in k(cat) and pre-steady-state kinetic behavior similar to that of a previous S110A mutant, indicating a role for Asn-109 is positioning the active site loop containing Ser-110. The catalytic role of Arg-188 is rationalized by a hydrogen bond network close to the C-1 carboxylate of the substrate, which positions the substrate and promotes substrate ketonization, probably via destabilization of the bound substrate.