Enantioselective Carbon Isotope Exchange.

The synthesis of isotopically labeled organic molecules is vital for drug and agrochemical discovery and development. Carbon isotope exchange is emerging as a leading method to generate carbon-labeled targets, which are sought over hydrogen-based labels due to their enhanced stability in biological systems. While many bioactive small molecules bear carbon-containing stereocenters, direct enantioselective carbon isotope exchange reactions have not been established. We describe the first example of an enantioselective carbon isotope exchange reaction, where (radio)labeled α-amino acids can be generated from their unlabeled precursors using a stoichiometric chiral aldehyde receptor with isotopically labeled CO2 followed by imine hydrolysis. Many proteinogenic and non-natural derivatives undergo enantioselective labeling, including the late-stage radiolabeling of complex drug targets.

Verticillins: fungal epipolythiodioxopiperazine alkaloids with chemotherapeutic potential.

Covering: 1970 through June of 2023Verticillins are epipolythiodioxopiperazine (ETP) alkaloids, many of which possess potent, nanomolar-level cytotoxicity against a variety of cancer cell lines. Over the last decade, their in vivo activity and mode of action have been explored in detail. Notably, recent studies have indicated that these compounds may be selective inhibitors of histone methyltransferases (HMTases) that alter the epigenome and modify targets that play a crucial role in apoptosis, altering immune cell recognition, and generating reactive oxygen species. Verticillin A (1) was the first of 27 analogues reported from fungal cultures since 1970. Subsequent genome sequencing identified the biosynthetic gene cluster responsible for producing verticillins, allowing a putative pathway to be proposed. Further, molecular sequencing played a pivotal role in clarifying the taxonomic characterization of verticillin-producing fungi, suggesting that most producing strains belong to the genus Clonostachys (i.e., Bionectria), Bionectriaceae. Recent studies have explored the total synthesis of these molecules and the generation of analogues via both semisynthetic and precursor-directed biosynthetic approaches. In addition, nanoparticles have been used to deliver these molecules, which, like many natural products, possess challenging solubility profiles. This review summarizes over 50 years of chemical and biological research on this class of fungal metabolites and offers insights and suggestions on future opportunities to push these compounds into pre-clinical and clinical development.

Somatic variants as a cause of drug-resistant epilepsy including mesial temporal lobe epilepsy with hippocampal sclerosis.

OBJECTIVE: The contribution of somatic variants to epilepsy has recently been demonstrated, particularly in the etiology of malformations of cortical development. The aim of this study was to determine the diagnostic yield of somatic variants in genes that have been previously associated with a somatic or germline epilepsy model, ascertained from resected brain tissue from patients with multidrug-resistant focal epilepsy. METHODS: Forty-two patients were recruited across three categories: (1) malformations of cortical development, (2) mesial temporal lobe epilepsy with hippocampal sclerosis, and (3) nonlesional focal epilepsy. Participants were subdivided based on histopathology of the resected brain. Paired blood- and brain-derived DNA samples were sequenced using high-coverage targeted next generation sequencing to high depth (585× and 1360×, respectively). Variants were identified using Genome Analysis ToolKit (GATK4) MuTect-2 and confirmed using high-coverage Amplicon-EZ sequencing. RESULTS: Sequence data on 41 patients passed quality control. Four somatic variants were validated following amplicon sequencing: within CBL, ALG13, MTOR, and FLNA. The diagnostic yield across 41 patients was 10%, 9% in mesial temporal lobe epilepsy with hippocampal sclerosis and 20% in malformations of cortical development. SIGNIFICANCE: This study provides novel insights into the etiology of mesial temporal lobe epilepsy with hippocampal sclerosis, highlighting a potential pathogenic role of somatic variants in CBL and ALG13. We also report candidate diagnostic somatic variants in FLNA in focal cortical dysplasia, while providing further insight into the importance of MTOR and related genes in focal cortical dysplasia. This work demonstrates the potential molecular diagnostic value of variants in both germline and somatic epilepsy genes.

Impacts of Gestational FireMaster 550 Exposure on the Neonatal Cortex Are Sex Specific and Largely Attributable to the Organophosphate Esters.

INTRODUCTION: Flame retardants (FRs) are common bodily and environmental pollutants, creating concern about their potential toxicity. We and others have found that the commercial mixture FireMaster® 550 (FM 550) or its individual brominated (BFR) and organophosphate ester (OPFR) components are potential developmental neurotoxicants. Using Wistar rats, we previously reported that developmental exposure to FM 550 or its component classes produced sex- and compound-specific effects on adult socioemotional behaviors. The underlying mechanisms driving the behavioral phenotypes are unknown. METHODS: To further mechanistic understanding, here we conducted transcriptomics in parallel with a novel lipidomics approach using cortical tissues from newborn siblings of the rats in the published behavioral study. Inclusion of lipid composition is significant because it is rarely examined in developmental neurotoxicity studies. Pups were gestationally exposed via oral dosing to the dam to FM 550 or the BFR or OPFR components at environmentally relevant doses. RESULTS: The neonatal cortex was highly sexually dimorphic in lipid and transcriptome composition, and males were more significantly impacted by FR exposure. Multiple adverse modes of action for the BFRs and OPFRs on neurodevelopment were identified, with the OPFRs being more disruptive than the BFRs via multiple mechanisms including dysregulation of mitochondrial function and disruption of cholinergic and glutamatergic systems. Disrupted mitochondrial function by environmental factors has been linked to a higher risk of autism spectrum disorders and neurodegenerative disorders. Impacted lipid classes included ceramides, sphingomyelins, and triacylglycerides. Robust ceramide upregulation in the OPFR females could suggest a heightened risk of brain metabolic disease. CONCLUSIONS: This study reveals multiple mechanisms by which the components of a common FR mixture are developmentally neurotoxic and that the OPFRs may be the compounds of greatest concern.

Postictal Psychosis in Epilepsy: A Clinicogenetic Study.

OBJECTIVE: Psychoses affecting people with epilepsy increase disease burden and diminish quality of life. We characterized postictal psychosis, which comprises about one quarter of epilepsy-related psychoses, and has unknown causation. METHODS: We conducted a case-control cohort study including patients diagnosed with postictal psychosis, confirmed by psychiatric assessment, with available data regarding epilepsy, treatment, psychiatric history, psychosis profile, and outcomes. After screening 3,288 epilepsy patients, we identified 83 with psychosis; 49 had postictal psychosis. Controls were 98 adults, matched by age and epilepsy type, with no history of psychosis. Logistic regression was used to investigate clinical factors associated with postictal psychosis; univariate associations with a p value < 0.20 were used to build a multivariate model. Polygenic risk scores for schizophrenia were calculated. RESULTS: Cases were more likely to have seizure clustering (odds ratio [OR] = 7.59, p < 0.001), seizures with a recollected aura (OR = 2.49, p = 0.013), and a family history of psychiatric disease (OR = 5.17, p = 0.022). Cases showed predominance of right temporal epileptiform discharges (OR = 4.87, p = 0.007). There was no difference in epilepsy duration, neuroimaging findings, or antiseizure treatment between cases and controls. Polygenic risk scores for schizophrenia in an extended cohort of postictal psychosis cases (n = 58) were significantly higher than in 1,366 epilepsy controls (R2  = 3%, p = 6 × 10-3 ), but not significantly different from 945 independent patients with schizophrenia (R2  = 0.1%, p = 0.775). INTERPRETATION: Postictal psychosis occurs under particular circumstances in people with epilepsy with a heightened genetic predisposition to schizophrenia, illustrating how disease biology (seizures) and trait susceptibility (schizophrenia) may interact to produce particular outcomes (postictal psychosis) in a common disease. ANN NEUROL 2021;90:464-476.

Enantioselective Tertiary Electrophile (Hetero)Benzylation: Pd-Catalyzed Substitution of Isoprene Monoxide with Arylacetates*.

The enantioselective generation of quaternary carbon centers remains challenging but is of growing importance for the preparation of functional molecules. Metal catalyzed allylic alkylations of tertiary electrophiles can provide access to these substructures but remain generally incompatible with organometallic benzyl nucleophiles. Here we demonstrate that electron-deficient arylacetates can serve as benzyl nucleophile surrogates to generate enantioenriched acyclic molecules containing a quaternary carbon center via a two-step substitution-decarboxylation process using isoprene monoxide. Products are often obtained in >90 % ee using a commercially available catalyst. An array of electron-withdrawing functional groups on the arylacetate moiety are tolerated. The lactone generated by the initial substitution reaction can be used in further stereoselective transformations to prepare molecules with acyclic vicinal quaternary stereocenters.

Multiplexed azido-group isotopic capture (MAGIC) beads: Selective analysis of azido compounds using a propargyl-based cleavable linker, a proof of concept.

RATIONALE: A cleavable linker is designed and synthesized for the selective capture of azide-containing compounds. This article presents a proof of concept methodology involving the use of peptide-functionalized aminopropyl silica, on which the peptide is constructed by solid-phase peptide synthesis. METHODS: The peptide linker has L-propargylglycine (Pra) at one terminal end to allow the conjugation of azide-containing molecules by copper assisted azide alkyne cycloaddition, also known as click reaction. L-Arginine (Arg) is placed just before Pra to permit the release of the captured product by tryptic cleavage. Three glycine (Gly) residues, as part of the linker, are appended to the silica bead to present a spacer section that allows efficient tryptic cleavage devoid of steric hindrance imposed by the bulky bead. The bead composition is Si-O-propyl-NH-Gly-Gly-Gly-Arg-Pra. RESULTS: This solid-phase material can be used to capture and release azide-functionalized compounds. The beads are first tested on three azido compounds, 2-azido-2-deoxyglucose (ADG), BOC-p-azido-Phe-OH (BAzPhe), where BOC = tert-butoxycarbonyl, and tetraacetylated-N-azidomannosamine (Ac4 ManNAz). Copper-mediated click reaction conditions are used and released products are characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS (MS/MS). CONCLUSIONS: This method allows easy identification of captured compounds based on mass and fragmentation analysis. Moreover, it is useful for the analysis of small azide-containing compounds by MALDI-TOF-MS which may not be possible otherwise due to matrix interferences. The insertion of isotopically labeled Arg residues provides the possibility of multiplex analysis, from which the beads have been called MAGIC (for Multiplexed Azido-Group Isotopic Capture). Copyright © 2016 John Wiley & Sons, Ltd.

A practical guide for the preparation of C1-labeled α-amino acids using aldehyde catalysis with isotopically labeled CO2.

Isotopically carbon-labeled α-amino acids are valuable synthetic targets that are increasingly needed in pharmacology and medical imaging. Existing preparations rely on early stage introduction of the isotopic label, which leads to prohibitive synthetic costs and time-intensive preparations. Here we describe a protocol for the preparation of C1-labeled α-amino acids using simple aldehyde catalysts in conjunction with [*C]CO2 (* = 14, 13, 11). This late-stage labeling strategy is enabled by the one-pot carboxylate exchange of unprotected α-amino acids with [*C]CO2. The protocol consists of three separate procedures, describing the syntheses of (±)-[1-13C]phenylalanine, (±)-[1-11C]phenylalanine and (±)-[1-14C]phenylalanine from unlabeled phenylalanine. Although the delivery of [*C]CO2 is operationally distinct for each experiment, each procedure relies on the same fundamental chemistry and can be executed by heating the reaction components at 50-90 °C under basic conditions in dimethylsulfoxide. Performed on scales of up to 0.5 mmol, this methodology is amenable to C1-labeling of many proteinogenic α-amino acids and nonnatural derivatives, which is a breakthrough from existing methods. The synthesis of (±)-[1-13C]phenylalanine requires ~2 d, with product typically obtained in a 60-80% isolated yield (n = 3, µ = 71, σ = 8.3) with an isotopic incorporation of 70-88% (n = 18, µ = 72, σ = 9.0). Starting from the preformed imino acid (~3 h preparation time), rapid synthesis of (±)-[1-11C]phenylalanine can be completed in ~1 h with an isolated radiochemical yield of 13%. Finally, (±)-[1-14C]phenylalanine can be accessed in ~2 d with a 51% isolated yield and 11% radiochemical yield.

Assessing the Fate of Dissolved Organic Compounds in Landfill Leachate and Wastewater Treatment Systems.

Landfill leachate and municipal wastewater are major sources of chemical pollutants that contaminate our drinking water sources. Evaluating the dissolved organic chemical composition in wastewater treatment plants is therefore essential to understand how the discharge impacts the environment, wildlife, and human health. In this study, we utilized a nontargeted analysis method coupling liquid chromatography and tandem mass spectrometry (LC-MS/MS) to analyze chemical features at different points along two landfill leachate treatment plants (LLTPs) and two municipal wastewater treatment plants (WWTPs) in the Southeastern United States. Significant feature differences were observed for the WWTPs where activated sludge clarification was employed versus the LLTPs utilizing reverse osmosis. Specifically, even though both LLTPs had the largest number of features in their influent water, their effluent following reverse osmosis yielded a lower number of features than the WWTPs. Additionally, the clarification processes of each WWTP exhibited different efficiencies as chemical disinfection removed more features than UV disinfection. Feature identification was then made using the LC, MS, and MS/MS information. Analysis of the identified molecules showed that lipids were the most effectively removed from all plants, while alkaloid and organic nitrogen compounds were the most recalcitrant.

Aldehyde-catalysed carboxylate exchange in α-amino acids with isotopically labelled CO2.

The isotopic labelling of small molecules is integral to drug development and for understanding biochemical processes. The preparation of carbon-labelled α-amino acids remains difficult and time consuming, with established methods involving label incorporation at an early stage of synthesis. This explains the high cost and scarcity of C-labelled products and presents a major challenge in 11C applications (11C t1/2 = 20 min). Here we report that aldehydes catalyse the isotopic carboxylate exchange of native α-amino acids with *CO2 (* = 14, 13, 11). Proteinogenic α-amino acids and many non-natural variants containing diverse functional groups undergo labelling. The reaction probably proceeds via the trapping of *CO2 by imine-carboxylate intermediates to generate iminomalonates that are prone to monodecarboxylation. Tempering catalyst electrophilicity was key to preventing irreversible aldehyde consumption. The pre-generation of the imine carboxylate intermediate allows for the rapid and late-stage 11C-radiolabelling of α-amino acids in the presence of [11C]CO2.

Spectrum of Phenotypic, Genetic, and Functional Characteristics in Patients With Epilepsy With KCNC2 Pathogenic Variants.

BACKGROUND AND OBJECTIVES: KCNC2 encodes Kv3.2, a member of the Shaw-related (Kv3) voltage-gated potassium channel subfamily, which is important for sustained high-frequency firing and optimized energy efficiency of action potentials in the brain. The objective of this study was to analyze the clinical phenotype, genetic background, and biophysical function of disease-associated Kv3.2 variants. METHODS: Individuals with KCNC2 variants detected by exome sequencing were selected for clinical, further genetic, and functional analysis. Cases were referred through clinical and research collaborations. Selected de novo variants were examined electrophysiologically in Xenopus laevis oocytes. RESULTS: We identified novel KCNC2 variants in 18 patients with various forms of epilepsy, including genetic generalized epilepsy (GGE), developmental and epileptic encephalopathy (DEE) including early-onset absence epilepsy, focal epilepsy, and myoclonic-atonic epilepsy. Of the 18 variants, 10 were de novo and 8 were classified as modifying variants. Eight drug-responsive patients became seizure-free using valproic acid as monotherapy or in combination, including severe DEE cases. Functional analysis of 4 variants demonstrated gain of function in 3 severely affected DEE cases and loss of function in 1 case with a milder phenotype (GGE) as the underlying pathomechanisms. DISCUSSION: These findings implicate KCNC2 as a novel causative gene for epilepsy and emphasize the critical role of KV3.2 in the regulation of brain excitability.

Oxidative cross-coupling processes inspired by the Chan-Lam reaction.

The Cu-catalyzed oxidative cross-coupling of N- and O-nucleophiles with aryl boronic acids (the Chan-Lam reaction) remains among the most useful approaches to prepare aniline and phenol derivatives. The combination of high chemoselectivity, mild reaction conditions, and the ability to use simple Cu-salts as catalysts makes this process a valuable alternative to aromatic substitutions and Pd-catalyzed reactions of aryl electrophiles (Buchwald-Hartwig coupling). Despite the widespread use of Chan-Lam reactions in synthesis, the analogous carbon-carbon bond forming variant of this process had not been developed prior to our work. This feature article describes our discovery and application of Cu-catalyzed oxidative coupling reactions of activated methylene derivatives or carboxylic acids with nucleophiles including aryl boronic esters and amines.

Semisynthetic Derivatives of the Verticillin Class of Natural Products through Acylation of the C11 Hydroxy Group.

The verticillins, a class of epipolythiodioxopiperazine alkaloids (ETPs) first described 50 years ago with the discovery of verticillin A (1), have gained attention due to their potent activity against cancer cells, noted both in vitro and in vivo. In this study, the complex scaffold afforded through optimized fermentation was used as a feedstock for semisynthetic efforts designed to explore the reactivity of the C11 and C11' hydroxy substituents. Functionality introduced at these positions would be expected to impact not only the potency but also the pharmacokinetic properties of the resulting compound. With this in mind, verticillin H (2) was used as a starting material to generate nine semisynthetic analogues (4-12) containing a variety of ester, carbonate, carbamate, and sulfonate moieties. Likewise, verticillin A succinate (13) was synthesized from 1 to demonstrate the successful application of this strategy to other ETPs. The synthesized compounds and their corresponding starting materials (i.e., 1 and 2) were screened for activity against a panel of melanoma, breast, and ovarian cancer cell lines: MDA-MB-435, MDA-MB-231, and OVCAR3. All analogues retained IC50 values in the nanomolar range, comparable to, and in some cases more potent than, the parent compounds.