Baldwin and Whitehead's Manzamine Alkaloids Biosynthesis Hypothesis Involves a Finely Tuned Reactivity of Acrolein: Automated Extraction of Reactivity Patterns from LC-MS2 Data.

Inspired by the multicomponent reaction-type scenario involving fatty dialdehydes, a nitrogen source, and acrolein, as a key C3 unit, put forward by Baldwin and Whitehead to explain the formation of manzamine-type alkaloids, 96 multicomponent reactions were designed, and their analytical readouts were deconvoluted using a herein-provided chemoinformatic workflow. This strategy pinpointed relevant conditions tuning the reactivity of acrolein to fulfill Baldwin and Whitehead's manzamine alkaloids biosynthetic hypothesis. This strategy can become part of a general method for the high-content analysis of multicomponent reactions applied to a natural product biosynthetic scenario.

Metal-free synthesis of γ-ketosulfones through Brønsted acid-promoted conjugate addition of sulfinamides.

A straightforward and general metal-free method has been developed to add sufinamide-derived sulfone units on Michael acceptors under mild conditions. This reaction enables the preparation of a large variety of original γ-ketosulfones, of which only a few synthetic methods have been reported. The mild reaction conditions used tolerate a wide diversity of functional groups and empower the implementation of a late-stage functionalisation strategy.

Carbon Nucleophile-Initiated Rauhut-Currier Reaction: An Atom-Economical Synthesis of Highly Functionalized Carbocycles.

A Rauhut-Currier reaction cascade is achieved in the presence of carbon nucleophiles under mild conditions. This original atom-economical transformation enables an efficient one-pot synthesis of densely substituted carbocycles from readily accessible substrates. The key promoter role of the cesium cation in the cascade process was demonstrated.

Characterization of extensive 2-fluorodeschloroketamine metabolism in pooled human liver microsomes, urine and hair from an addicted patient using high-resolution accurate mass spectrometry.

2-Fluorodeschloroketamine (2F-DCK) is a ketamine derivative involved in acute intoxications and deaths. The aim of this study is to investigate its metabolism using pooled human liver microsomes (pHLMs) and to apply it to authentic samples (urine, hair and seized materials) from a drug user. 2F-DCK (100 µM) incubates with pHLMs were analyzed by liquid chromatography-high-resolution accurate mass (LC-HRAM; Q-Exactive, Thermo Fisher Scientific®) according to a previously published protocol. Spectra annotation was performed using Compound Discoverer® software and the metabolic scheme was drawn using ChemDraw software. Urine (200 µL) and hair (previously decontaminated using dichloromethane and segmented into three segments: A, 0-3 cm; B, 3-6 cm; C, 6-9 cm) were extracted with a mixture of hexane:ethyl acetate (1:1) and chloroform:isopropanol (4:1). About 10 µL of both reconstituted residues were analyzed by LC-HRAM. Hair was also analyzed by LC-MS-MS (TSQ Vantage, Thermo Fisher Scientific®) for 2F-DCK and deschloroketamine (DCK) quantification. The two presumed 2F-DCK crystals consumed by the patient were dissolved in methanol (1 mg/mL) and 10 µL were analyzed by LC-MS-MS (Quantum Access Max, Thermo Fisher Scientific®). Twenty-six putative 2F-DCK metabolites were identified, 15 being reported for the first time. Thirteen metabolites were detected in pHLMs, 10 confirmed in both the patient's urine and hair and all were found in at least one of the two samples. Twenty-three metabolites were detected in urine and 20 in hair. Our research confirms the reliability of nor-2F-DCK as a target analyte and suggests OH-dihydro-nor-2F-DCK and dehydro-nor-2F-DCK as new target analytes in urine and hair, respectively. This is the first study to report DCK as a 2F-DCK metabolite using pHLMs and to determine its concentrations in hair (A/B/C, 885/1,500/1,850 pg/mg) following chronic use. Finally, the two seized crystals contained 2F-DCK at 67% and 96% with traces of DCK (0.4% and 0.6%) related to cross-contamination by container exchange.

Fumarate as positive modulator of allosteric transitions in the pentameric ligand-gated ion channel GLIC: requirement of an intact vestibular pocket.

Gloeobacter violaceus ligand-gated ion channel (GLIC) is a prokaryotic orthologue of brain pentameric neurotransmitter receptors. Using whole-cell patch-clamp electrophysiology in a host cell line, we show that short-chain dicarboxylate compounds are positive modulators of pHo 5-evoked GLIC activity, with a rank order of action fumarate > succinate > malonate > glutarate. Potentiation by fumarate depends on intracellular pH, mainly as a result of a strong decrease of the pHo 5-evoked current when intracellular pH decreases. The modulating effect of fumarate also depends on extracellular pH, as fumarate is a weak inhibitor at pHo 6 and shows no agonist action at neutral pHo. A mutational analysis of residue dependency for succinate and fumarate effects, based on two carboxylate-binding pockets previously identified by crystallography (Fourati et al., 2020), shows that positive modulation involves both the inter-subunit pocket, homologous to the neurotransmitter-binding orthotopic site, and the intra-subunit (also called vestibular) pocket. An almost similar pattern of mutational impact is observed for the effect of caffeate, a known negative modulator. We propose, for both dicarboxylate compounds and caffeate, a model where the inter-subunit pocket is the actual binding site, and the region corresponding to the vestibular pocket is required either for inter-subunit binding itself, or for binding-to-gating coupling during the allosteric transitions involved in pore-gating modulation. KEY POINTS: Using a bacterial orthologue of brain pentameric neurotransmitter receptors, we show that the orthotopic/orthosteric agonist site and the adjacent vestibular region are functionally interdependent in mediating compound-elicited modulation. We propose that the two sites in the extracellular domain are involved 'in series', a mechanism which may have relevance for eukaryote receptors. We show that short-chain dicarboxylate compounds are positive modulators of the Gloeobacter violaceus ligand-gated ion channel (GLIC). The most potent compound identified is fumarate, known to occupy the orthotopic/orthosteric site in previously published crystal structures. We show that intracellular pH modulates GLIC allosteric transitions, as previously known for extracellular pH. We report a caesium to sodium permeability ratio (PCs /PNa ) of 0.54 for GLIC ion pore.

Characterization of 3-Hydroxyeticyclidine (3-HO-PCE) Metabolism in Human Liver Microsomes and Biological Samples Using High-Resolution Mass Spectrometry.

3-Hydroxyeticyclidine (3-HO-PCE) is a ketamine derivative that produces dissociative, hallucinogenic, and euphoric effects when consumed, but little is known about its pharmacological properties, metabolism, and toxicity compared to other designer ketamine analogs. To address this gap in knowledge, this study explored for the first time the metabolism of 3-HO-PCE. Based on this investigation, it is hypothesized that combining the use of Human Liver Microsomes (HLM) as an In vitro model with urine and hair samples from drug users may enable the identification of key analytes that can extend the detection window of 3-HO-PCE, particularly in cases of overdose. The analysis identified 15 putative metabolites, 12 of which are produced through phase I metabolism involving N-dealkylation, deamination, and oxidation, and 3 through phase II O-glucuronidation. The metabolism of 3-HO-PCE is similar to that of O-PCE, another designer ketamine of the eticyclidine family. The study identified M2a and hydroxy-PCA as reliable biomarkers for untargeted screening of the eticyclidine family in urine and hair, respectively. For targeted screening of 3-HO-PCE, M10 is recommended as the target analyte in urine, and M5 shows promise for long-term monitoring of 3-HO-PCE using hair analysis.

Metabolic profiling of deschloro-N-ethyl-ketamine and identification of new target metabolites in urine and hair using human liver microsomes and high-resolution accurate mass spectrometry.

The aim of this study was to identify new markers of deschloro-N-ethyl-ketamine (O-PCE), a ketamine analogue that has been involved in acute intoxications with severe outcomes including death and whose metabolism has never been studied before. In vitro study after 2-h incubation with pooled human liver microsomes (HLMs) cross-checked by the analysis of urine and hair from a 43-year-old O-PCE user (male) were performed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Acquired data were processed by the Compound Discoverer® software, and a full metabolic profile of O-PCE was proposed. In total, 15 metabolites were identified, 10 were detected in vitro (HLMs) and confirmed in vivo (urine and/or hair), two were present only in HLMs, and the remaining three metabolites were identified only in biological specimens. While O-PCE was no longer detected in urine, nine metabolites were identified allowing to increase its detection window. In descending order of metabolites abundance, we suggest using 2-en-PCA-N-Glu (34%, first), M3 (16%, second), O-PCA-N-Glu (15.4%, third), OH-O-PCE (15%, fourth) and OH-PCE (11.9%, fifth) as target metabolites to increase the detection window of O-PCE in urine. In hair, nine metabolites were identified. OH-PCA was the major compound (78%) with a relevant metabolite to parent drug ratio (=6) showing its good integration into hair and making it the best marker for long-term monitoring of O-PCE exposure.

Identification and characterization of a naïve CD8+ T cell repertoire for benzylpenicillin.

BACKGROUND: Beta-lactams allergy is the most commonly reported drug allergy and constitutes an important health problem. We previously showed the pre-existence of a naïve CD4+ T cell repertoire for benzylpenicillin (BP) coupled to human serum albumin (HSA) but little is known about the naïve CD8+ T cell repertoire specific for BP. OBJECTIVE: The purpose of this work was to identify naïve CD8+ T cells specific for BP and to explore mechanisms dictating their activation. METHODS: Co-cultures were established with naïve CD8+ T cells and autologous dendritic cells (DCs) loaded with HSA-BP or free BP. T cells were restimulated once a week with autologous DCs loaded with HSA-BP or BP. The specific CD8+ T cell response was measured using an IFN-γ ELISpot assay. RESULTS: When using free BP, we were able to detect a naïve CD8+ T cell repertoire for BP in the 6 out of 7 tested healthy donors. However, our results showed that HSA-BP was recognized by naïve CD8+ T cells in only one donor out of five tested healthy donors. Using free BP, we evidenced its binding to cellular proteins in DCs that was concentration dependent and was correlated with BP-specific CD8+ T cell activation. Moreover, the BP-specific CD8+ cell response was MHC class I-dependent and required intracellular processing and proteasome activity. CONCLUSION AND CLINICAL RELEVANCE: This work showed the existence of a naïve CD8+ T cell repertoire for BP when DCs were treated with free BP suggesting that patients could be immunized by haptenated peptides from cellular proteins generated in antigen-presenting cells.

Identification of a pre-active conformation of a pentameric channel receptor.

Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical signaling through global allosteric transitions. Despite the existence of several high-resolution structures of pLGICs, their dynamical properties remain elusive. Using the proton-gated channel GLIC, we engineered multiple fluorescent reporters, each incorporating a bimane and a tryptophan/tyrosine, whose close distance causes fluorescence quenching. We show that proton application causes a global compaction of the extracellular subunit interface, coupled to an outward motion of the M2-M3 loop near the channel gate. These movements are highly similar in lipid vesicles and detergent micelles. These reorganizations are essentially completed within 2 ms and occur without channel opening at low proton concentration, indicating that they report a pre-active intermediate state in the transition pathway toward activation. This provides a template to investigate the gating of eukaryotic neurotransmitter receptors, for which intermediate states also participate in activation.

Barbiturates Bind in the GLIC Ion Channel Pore and Cause Inhibition by Stabilizing a Closed State.

Barbiturates induce anesthesia by modulating the activity of anionic and cationic pentameric ligand-gated ion channels (pLGICs). Despite more than a century of use in clinical practice, the prototypic binding site for this class of drugs within pLGICs is yet to be described. In this study, we present the first X-ray structures of barbiturates bound to GLIC, a cationic prokaryotic pLGIC with excellent structural homology to other relevant channels sensitive to general anesthetics and, as shown here, to barbiturates, at clinically relevant concentrations. Several derivatives of barbiturates containing anomalous scatterers were synthesized, and these derivatives helped us unambiguously identify a unique barbiturate binding site within the central ion channel pore in a closed conformation. In addition, docking calculations around the observed binding site for all three states of the receptor, including a model of the desensitized state, showed that barbiturates preferentially stabilize the closed state. The identification of this pore binding site sheds light on the mechanism of barbiturate inhibition of cationic pLGICs and allows the rationalization of several structural and functional features previously observed for barbiturates.

Bioinspired Design and Oriented Synthesis of Immunogenic Site-Specifically Penicilloylated Peptides.

ß-Lactam antibiotics allergy is recognized as a public health concern. By covalently binding to serum proteins, penicillins are known to form immunogenic complexes. The latter are recognized and digested by antigen-presenting cells into drug-hapten peptides leading to the immunization of treated persons and IgE-mediated hypersensitivity reactions encompassing anaphylaxis. If type I allergic reactions to drugs are often unpredictable, they are known to be dependent on CD4+ T-cells. This fundamental study revisits the chemical basis of the benzylpenicillin (BP) allergy with the aim of identifying immunologically relevant biomimetic benzylpenicilloylated peptides through the analysis of BP-conjugated human serum albumin (BP-HSA) profile and the evaluation of the naïve CD4+ T-cell responses to candidate BP-HSA-derived peptides. The chemical structures of BP-HSA bioconjugates synthesized in vitro at both physiological and basic pH were investigated by mass spectrometry. From the ten most representative lysine residues grafted by BP-hapten, HSA-bioinspired 15-mer peptide sequences were designed and the potential T-cell epitope profile of each peptide was predicted using two complementary in silico approaches, i.e., HLA class II binding prediction tools from the Immune Epitope Database and Analysis Resource (IEDB) and computational alanine scanning mutagenesis. Twelve structurally diversified benzylpenicilloylated peptides (BP-Ps) were selected and synthesized with the aid of a flexible synthesis pathway using an original benzylpenicilloylated lysine monomer as common precursor. In order to corroborate their predicted "epitope" profile, the naïve CD4+ T-cell response specific to BP was evaluated through a coculture approach. To our knowledge, this study showed for the first time the ability of bioinspired peptides structurally stemming from BP-HSA to be recognized by naïve CD4+ T-cells thus identifying a pre-existing T-cell repertoire for penicillin molecules bound to proteins. It also established a promising model approach expandable to other most frequently used penicillin classes of antibiotics to reveal biomimetic drug-modified antigenic peptides relevant for qualitative and quantitative drug allergy studies.

Emergence of diversity and stereochemical outcomes in the biosynthetic pathways of cyclobutane-centered marine alkaloid dimers.

Covering: up to 2016Dictazoles and sceptrins are singular metabolites of marine origin. The present dichotomic case study provides a comprehensive perspective on these cyclobutane-centered alkaloids and their respective families. Indeed, their upstream and downstream chemistry are both treated herein. Relevant isolation reports and bio-inspired total syntheses are used to decipher the currently admitted biosynthetic hypotheses as well as the emergence of diversity in the two series. This review proposes a transversal vision of the topic, where most aspects of natural product chemistry have a critical importance.

Palladium-Catalyzed Oxidative Synthesis of Unsymmetrical Azophenols.

A straightforward palladium-catalyzed oxidative hydroxylation of azobenzenes is reported. The developed methodology tolerates various functional groups and allows the synthesis of diverse unsymmetrical azophenols under mild conditions in good to excellent yields. A complementary procedure was also investigated by in situ generation of PIFA. This study represents the first general method for the synthesis of o-hydroxyazobenzenes starting from simple azoarenes.

Access to Highly Functionalized Sulfonated Cyclopentanes by Acid-Promoted Rauhut-Currier Reaction with Sulfinamides.

An unexpected acid-mediated cascade reaction induced by conjugate addition of sulfinamides to dienediones has been developed. This highly efficient Rauhut-Currier reaction enables the rapid, high-yielding construction of sulfonated cyclopentanes with three contiguous stereogenic centers in a single operation starting from simple sulfinamides. This process constitutes the first example of sulfinamide-promoted cycloisomerization.

Two-Component Domino Reactions Initiated from Ketenes: Serendipitous Synthesis of Quinolizidinones Analogous to Chelated Lobeline's Conformation.

An original and efficient synthesis of quinolizidinones through a one-pot two-component cascade reaction of norlobelanine with in situ generated ketenes is reported. Functionalized fused azabicyclic scaffolds bearing multiple stereogenic centers were prepared with excellent diastereoselectivities. Mild optimized conditions involving a key "shuttle base" deprotonation strategy was applied to the synthesis, in a short sequence, of a constrained mimetic of the privileged H-bonded conformation of (-)-lobeline.

A unified bioinspired "aplysinopsin cascade": total synthesis of (±)-tubastrindole B and related biosynthetic congeners.

Applying a biomimetic approach, the first total synthesis of (±)-tubastrindole B is reported herein. This work features a ring-expansion cascade of a dictazole-type precursor into cycloaplysinopsin-type congeners. Moreover, the isolation of a transient biogenetic intermediate represents a milestone in the biosynthetic understanding of this family of marine alkaloids.

Spontaneous biomimetic formation of (±)-dictazole B under irradiation with artificial sunlight.

Guided by biosynthetic considerations, the total synthesis of dictazole B is reported for the first time. Experimental evidence for an easy access to challenging cyclobutane alkaloids of marine origin, which are often postulated to be biosynthetic precursors of more complex structures, is provided.

Scavenging iron: a novel mechanism of plant immunity activation by microbial siderophores.

Siderophores are specific ferric iron chelators synthesized by virtually all microorganisms in response to iron deficiency. We have previously shown that they promote infection by the phytopathogenic enterobacteria Dickeya dadantii and Erwinia amylovora. Siderophores also have the ability to activate plant immunity. We have used complete Arabidopsis transcriptome microarrays to investigate the global transcriptional modifications in roots and leaves of Arabidopsis (Arabidopsis thaliana) plants after leaf treatment with the siderophore deferrioxamine (DFO). Physiological relevance of these transcriptional modifications was validated experimentally. Immunity and heavy-metal homeostasis were the major processes affected by DFO. These two physiological responses could be activated by a synthetic iron chelator ethylenediamine-di(o-hydroxyphenylacetic) acid, indicating that siderophores eliciting activities rely on their strong iron-chelating capacity. DFO was able to protect Arabidopsis against the pathogenic bacterium Pseudomonas syringae pv tomato DC3000. Siderophore treatment caused local modifications of iron distribution in leaf cells visible by ferrocyanide and diaminobenzidine-H2O2 staining. Metal quantifications showed that DFO causes a transient iron and zinc uptake at the root level, which is presumably mediated by the metal transporter iron regulated transporter1 (IRT1). Defense gene expression and callose deposition in response to DFO were compromised in an irt1 mutant. Consistently, plant susceptibility to D. dadantii was increased in the irt1 mutant. Our work shows that iron scavenging is a unique mechanism of immunity activation in plants. It highlights the strong relationship between heavy-metal homeostasis and immunity.

Recent contributions from the asymmetric aza-Michael reaction to alkaloids total synthesis.

This review focuses on recent applications of the aza-Michael reaction in alkaloids total synthesis with a special emphasis on stereoselectivity. The report highlights achievements and challenges over the past five years and describes stereoselective intra- and inter-molecular conjugate addition of nitrogen-containing nucleophiles, including tandem and cascade processes. Total asymmetric syntheses of natural scaffolds, such as pyrrolidine, piperidine and "izidine" families, are depicted. Multi-step syntheses of highly challenging natural products are further detailed, assessing the scope of the stereocontrolled aza-Michael reaction as a powerful tool in alkaloid chemistry.

Identification of cinnamic acid derivatives as novel antagonists of the prokaryotic proton-gated ion channel GLIC.

Pentameric ligand gated ion channels (pLGICs) mediate signal transduction. The binding of an extracellular ligand is coupled to the transmembrane channel opening. So far, all known agonists bind at the interface between subunits in a topologically conserved "orthosteric site" whose amino acid composition defines the pharmacological specificity of pLGIC subtypes. A striking exception is the bacterial proton-activated GLIC protein, exhibiting an uncommon orthosteric binding site in terms of sequence and local architecture. Among a library of Gloeobacter violaceus metabolites, we identified a series of cinnamic acid derivatives, which antagonize the GLIC proton-elicited response. Structure-activity analysis shows a key contribution of the carboxylate moiety to GLIC inhibition. Molecular docking coupled to site-directed mutagenesis support that the binding pocket is located below the classical orthosteric site. These antagonists provide new tools to modulate conformation of GLIC, currently used as a prototypic pLGIC, and opens new avenues to study the signal transduction mechanism.