Identification of a 1-deoxy-D-xylulose-5-phosphate synthase (DXS) mutant with improved crystallographic properties.

In this report, we describe a truncated Deinococcus radiodurans 1-deoxy-D-xylulose-5-phosphate synthase (DXS) protein that retains enzymatic activity, while slowing protein degradation and showing improved crystallization properties. With modern drug-design approaches relying heavily on the elucidation of atomic interactions of potential new drugs with their targets, the need for co-crystal structures with the compounds of interest is high. DXS itself is a promising drug target, as it catalyzes the first reaction in the 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway for the biosynthesis of the universal precursors of terpenes, which are essential secondary metabolites. In contrast to many bacteria and pathogens, which employ the MEP pathway, mammals use the distinct mevalonate-pathway for the biosynthesis of these precursors, which makes all enzymes of the MEP-pathway potential new targets for the development of anti-infectives. However, crystallization of DXS has proven to be challenging: while the first X-ray structures from Escherichia coli and D. radiodurans were solved in 2004, since then only two additions have been made in 2019 that were obtained under anoxic conditions. The presented site of truncation can potentially also be transferred to other homologues, opening up the possibility for the determination of crystal structures from pathogenic species, which until now could not be crystallized. This manuscript also provides a further example that truncation of a variable region of a protein can lead to improved structural data.

Synthesis of α-Chlorolactams by Cyanoborohydride-Mediated Radical Cyclization of Trichloroacetamides.

A cyanoborohydride-promoted radical cyclization methodology has been developed to access α-chlorolactams in a simple and efficient way using NaBH3 CN and trichloroacetamides easily available from allylic and homoallylic secondary amines. This methodology allowed the synthesis of a library of α-chlorolactams (mono- and bicyclic), which were tested for herbicidal activity, trans-3-chloro-4-methyl-1-(3-trifluoromethyl)phenyl-2-pyrrolidinone being the most active.

Synthesis of 4-fluoromethylsydnones and their participation in alkyne cycloaddition reactions.

We report the synthesis and some structural studies of 4-trifluoromethyl, 4-difluoromethyl-, and 4-monofluoromethylsydnones. All but the latter compounds are stable and represent effective precursors to a range of pyrazoles after cycloaddition reactions with alkynes. The cycloadditions are generally highly regioselective and provide 5-fluoromethylpyrazole products, although we have observed that Bn-substituted sydnones can provide an unexpected alkyne insertion mode that generates the 3-fluoromethyl isomer.

Designing New Protoporphyrinogen Oxidase-Inhibitors Carrying Potential Side Chain Isosteres to Enhance Crop Safety and Spectrum of Activity.

There are several methods to control weeds, which impose particular challenges for farmers in all parts of the world, although applying small molecular compounds still remains the most efficient technology to date. However, plants can evolve to become resistant toward active ingredients which is also the case for protoporphyrinogen oxidase (PPO) inhibitors, a class of highly effective herbicides in use for more than 50 years. Hence, it is essential to continuously discover and develop new herbicidal PPO inhibitors with enhanced intrinsic activity, an improved resistance profile, enhanced crop safety, favorable physicochemical properties, and a clean toxicological profile. By modifying structural key features from known PPO inhibitors such as tiafenacil, inspired by isostere and mix&match concepts in combination with modeling investigations based on a wild-type Amaranthus crystal structure, we have found new promising lead structures showing strong activity in vitro and in vivo against several notorious dicotyledon and monocotyledon weeds with emerging resistance (e.g., Amaranthus palmeri, Amaranthus tuberculatus, Lolium rigidum, and Alopecurus myosuroides). While several phenyl uracils carrying an isoxazoline motif in their thio-linked side chain showed promising resistance-breaking potential against different Amaranthus species, introducing a thioacrylamide side chain afforded outstanding efficacy against resistant grass weeds.

Design, synthesis and screening of herbicidal activity for new phenyl pyrazole-based protoporphyrinogen oxidase-inhibitors (PPO) overcoming resistance issues.

BACKGROUND: Whilst there are several methods to control weeds, which continuously plague farmers around the globe, the application of small molecular compounds is still the most effective technology to date. Plants can evolve to become resistant to PPO-inhibitors, a class of herbicides in commercial use since the 1960s. It is therefore essential to continuously develop new herbicides based on this mode-of-action with enhanced intrinsic activity, an improved resistance profile and favourable physicochemical properties. Based on an Amaranthus PPO crystal structure and subsequent modelling studies, halogen-substituted pyrazoles have been investigated as isosteres of uracil-based PPO-inhibitors. RESULTS: By combining structural features from the commercial PPO-inhibitors tiafenacil and pyraflufen-ethyl and by investigating receptor-binding properties, we identified new promising pyrazole-based lead structures showing strong activity in vitro and in vivo against economically important weeds of the Amaranthus genus: A. retroflexus, and resistant A. palmeri and A. tuberculatus. CONCLUSION: The present work covers a series of novel PPO-inhibiting compounds that contain a pyrazole ring and a substituted thioacetic acid sidechain attached to the core phenyl group. These compounds show good receptor fit in line with excellent herbicidal activity against weeds that plague corn and rice crops with low application rates. This, in combination with promising selectivity in corn, have the potential to mitigate and affect weeds that have become resistant to some of the current market standards. Remarkably, some of the novel PPO-inhibitors outlined herein show efficacies against economically important weeds that were superior to recently commercialized and structurally related tiafenacil. © 2023 Society of Chemical Industry.

First crystal structures of 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) from Mycobacterium tuberculosis indicate a distinct mechanism of intermediate stabilization.

The development of drug resistance by Mycobacterium tuberculosis and other pathogenic bacteria emphasizes the need for new antibiotics. Unlike animals, most bacteria synthesize isoprenoid precursors through the MEP pathway. 1-Deoxy-D-xylulose 5-phosphate synthase (DXPS) catalyzes the first reaction of the MEP pathway and is an attractive target for the development of new antibiotics. We report here the successful use of a loop truncation to crystallize and solve the first DXPS structures of a pathogen, namely M. tuberculosis (MtDXPS). The main difference found to other DXPS structures is in the active site where a highly coordinated water was found, showing a new mechanism for the enamine-intermediate stabilization. Unlike other DXPS structures, a "fork-like" motif could be identified in the enamine structure, using a different residue for the interaction with the cofactor, potentially leading to a decrease in the stability of the intermediate. In addition, electron density suggesting a phosphate group could be found close to the active site, provides new evidence for the D-GAP binding site. These results provide the opportunity to improve or develop new inhibitors specific for MtDXPS through structure-based drug design.

A general and regioselective synthesis of 5-trifluoromethyl-pyrazoles.

Two synthetic approaches to 4-trifluoromethylsydnones, a novel class of these mesoionic reagents, are reported. These compounds undergo regioselective alkyne cycloaddition reactions, thereby providing a general approach to 5-trifluoromethylpyrazoles. This method has been employed in a short formal synthesis of the herbicide fluazolate.

RCM-mediated synthesis of trifluoromethyl-containing nitrogen heterocycles.

A ring-closing metathesis mediated pathway to trifluoromethyl-containing piperidines is detailed. This involves the development of a synthetic route to a new (trifluoromethyl)allylating reagent via a Diels-Alder/retro-Diels-Alder strategy, its application in the synthesis of a series of trifluormethyl-substituted diolefin precursors for ring-closing metathesis, and eventually the successful cyclization of these precursor molecules into the corresponding functionalized piperidines.