Cyclopropanation and aziridination catalyzed by non-heme iron and 2-oxoglutarate dependent enzymes.

Cyclopropane and azacyclopropane, also known as aziridine, moieties are found in natural products. These moieties serve as pivotal components that lead to a broad spectrum of biological activities. While diverse strategies involving various classes of enzymes are utilized to catalyze formation of these strained three-membered rings, how non-heme iron and 2-oxoglutarate (Fe/2OG) dependent enzymes enable regio- and stereo-selective C-C and C-N ring closure has only been reported very recently. Herein, we present detailed experimental protocols for mechanistically studying Fe/2OG enzymes that catalyze cyclopropanation and aziridination reactions. These protocols include protein purification, in vitro assays, biophysical spectroscopies, and isotope-tracer experiments. We also report how to use in silico approaches to look for Fe/2OG aziridinases. Furthermore, our current mechanistic understanding of three-membered ring formation is discussed. These results not only shed light on the reaction mechanisms of Fe/2OG enzymes-catalyzed cyclopropanation and aziridination, but also open avenues for expanding the reaction repertoire of the Fe/2OG enzyme superfamily.

Total Synthesis and Biological Profiling of Putative (±)-Marinoaziridine B and (±)-N-Methyl Marinoaziridine A.

The total synthesis of two new marine natural products, (±)-marinoaziridine B 7 and (±)-N-methyl marinoaziridine A 8, was accomplished. The (±)-marinoaziridine 7 was prepared in a six-step linear sequence with a 2% overall yield. The key steps in our strategy were the preparation of the chiral epoxide (±)-5 using the Johnson Corey Chaykovsky reaction, followed by the ring-opening reaction and the Staudinger reaction. The N,N-dimethylation of compound (±)-7 gives (±)-N-methyl marinoaziridine A 8. The NMR spectra of synthetized (±)-marinoaziridine B 7 and isolated natural product did not match. The compounds are biologically characterized using relevant in silico, in vitro and in vivo methods. In silico ADMET and bioactivity profiling predicted toxic and neuromodulatory effects. In vitro screening by MTT assay on three cell lines (MCF-7, H-460, HEK293T) showed that both compounds exhibited moderate to strong antiproliferative and cytotoxic effects. Antimicrobial tests on bacterial cultures of Escherichia coli and Staphylococcus aureus demonstrated the dose-dependent inhibition of the growth of both bacteria. In vivo toxicological tests were performed on zebrafish Danio rerio and showed a significant reduction of zebrafish mortality due to N-methylation in (±)-8.

Structural Evaluation of a Nitroreductase Engineered for Improved Activation of the 5-Nitroimidazole PET Probe SN33623.

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.

Recent Developments in the Biosynthesis of Aziridines.

Only 0.016 % of all known natural products contain an aziridine ring, but this unique structural feature imparts high reactivity and cytotoxicity to the compounds in which it is found. Until 2021, no naturally occurring aziridine-forming enzymes had been identified. Since 2021, the biosynthetic enzymes for ~10 % of known aziridine containing natural products have been identified and characterized. This article describes the recent advances in our understanding of enzyme-catalyzed aziridine formation in the context of historical methods for aziridine formation through synthetic chemistry.

Lactic Acid Bacteria-Derived Postbiotics as Adjunctive Agents in Breast Cancer Treatment to Boost the Antineoplastic Effect of a Conventional Therapeutic Comprising Tamoxifen and a New Drug Candidate: An Aziridine-Hydrazide Hydrazone Derivative.

Breast cancer is associated with high mortality and morbidity rates. As about 20-30% of patients exhibiting ER-positive phenotype are resistant to hormonal treatment with the standard drug tamoxifen, finding new therapies is a necessity. Postbiotics, metabolites, and macromolecules isolated from probiotic bacteria cultures have been proven to have sufficient bioactivity to exert prohealth and anticancer effects, making them viable adjunctive agents for the treatment of various neoplasms, including breast cancer. In the current study, postbiotics derived from L. plantarum and L. rhamnosus cultures were assessed on an in vitro breast cancer model as potential adjunctive agents to therapy utilizing tamoxifen and a candidate aziridine-hydrazide hydrazone derivative drug. Cell viability and cell death processes, including apoptosis, were analyzed for neoplastic MCF-7 cells treated with postbiotics and synthetic compounds. Cell cycle progression and proliferation were analyzed by PI-based flow cytometry and Ki-67 immunostaining. Postbiotics decreased viability and triggered apoptosis in MCF-7, modestly affecting the cell cycle and showing a lack of negative impact on normal cell viability. Moreover, they enhanced the cytotoxic effect of tamoxifen and the new candidate drug toward MCF-7, accelerating apoptosis and the inhibition of proliferation. This illustrates postbiotics' potential as natural adjunctive agents supporting anticancer therapy based on synthetic drugs.

[Fe(NCMe)6](BF4)2 is a bifunctional catalyst for styrene aziridination by nitrene transfer and heterocycle expansion by subsequent dipolar insertion.

The solvated iron(II) salt [Fe(NCMe)6](BF4)2 (Me = methyl) is shown to be a bifunctional catalyst with respect to aziridination of styrene. The salt serves as an active catalyst for nitrene transfer from PhINTs to styrene to form 2-phenyl-N-tosylaziridine (Ph = phenyl; Ts = tosyl, -S{O}2-p-C6H4Me). The iron(II) salt also acts as a Lewis acid in non-coordinating CH2Cl2 solution, to catalyze heterolytic CN bond cleavage of the aziridine and insertion of dipolarophiles. The 1,3-zwitterionic intermediate is presumably supported by interaction of the metal dication with the anion, and by resonance stabilization of the carbocation. Nucleophilic dipolarophiles then insert to give a five-membered heterocyclic ring. The result is a two-step cycloaddition, formally [2 + 1 + 2], that is typically regiospecific, but not stereospecific. This reaction mechanism was confirmed by conducting a series of one-step, [3 + 2] additions of unsaturated molecules into pre-formed 2-phenyl-N-tosylaziridine, also catalyzed by [Fe(NCMe)6](BF4)2. Relevant substrates include styrenes, carbonyl compounds and alkynes. These yield five-membered heterocylic rings, including pyrrolidines, oxazolidines and dihydropyrroles, respectively. The reaction scope appears limited only by the barrier to formation of the dipolar intermediate, and by the nucleophilicity of the captured dipolarophile. The bifunctionality of an inexpensive, earth-abundant and non-toxic catalyst suggests a general strategy for one-pot construction of heterocyclic rings, as demonstrated specifically for pyrrolidine ring formation.

Aza-Prilezhaev Aziridination-Enabled Multidimensional Analysis of Isomeric Lipids via High-Resolution U-Shaped Mobility Analyzer-Mass Spectrometry.

Unsaturated lipids constitute a significant portion of the lipidome, serving as players of multifaceted functions involving cellular signaling, membrane structure, and bioenergetics. While derivatization-assisted liquid chromatography tandem mass spectrometry (LC-MS/MS) remains the gold standard technique in lipidome, it mainly faces challenges in efficiently labeling the carbon-carbon double bond (C═C) and differentiating isomeric lipids in full dimension. This presents a need for new orthogonal methodologies. Herein, a metal- and additive-free aza-Prilezhaev aziridination (APA)-enabled ion mobility mass spectrometric method is developed for probing multiple levels of unsaturated lipid isomerization with high sensitivity. Both unsaturated polar and nonpolar lipids can be efficiently labeled in the form of N-H aziridine without significant side reactions. The signal intensity can be increased by up to 3 orders of magnitude, achieving the nM detection limit. Abundant site-specific fragmentation ions indicate C═C location and sn-position in MS/MS spectra. Better yet, a stable monoaziridination product is dominant, simplifying the spectrum for lipids with multiple double bonds. Coupled with a U-shaped mobility analyzer, identification of geometric isomers and separation of different lipid classes can be achieved. Additionally, a unique pseudo MS3 mode with UMA-QTOF MS boosts the sensitivity for generating diagnostic fragments. Overall, the current method provides a comprehensive solution for deep-profiling lipidomics, which is valuable for lipid marker discovery in disease monitoring and diagnosis.

ß-l-Arabinofurano-cyclitol Aziridines Are Covalent Broad-Spectrum Inhibitors and Activity-Based Probes for Retaining ß-l-Arabinofuranosidases.

GH127 and GH146 microorganismal retaining ß-l-arabinofuranosidases, expressed by human gut microbiomes, feature an atypical catalytic domain and an unusual mechanism of action. We recently reported that both Bacteroides thetaiotaomicron BtGH146 and Bifidobacterium longum HypBA1 are inhibited by ß-l-arabinofuranosyl cyclophellitol epoxide, supporting the action of a zinc-coordinated cysteine as a catalytic nucleophile, where in most retaining GH families, an aspartate or glutamate is employed. This work presents a panel of ß-l-arabinofuranosyl cyclophellitol epoxides and aziridines as mechanism-based BtGH146/HypBA1 inhibitors and activity-based probes. The ß-l-arabinofuranosyl cyclophellitol aziridines both inhibit and label ß-l-arabinofuranosidase efficiently (however with different activities), whereas the epoxide-derived probes favor BtGH146 over HypBA1. These findings are accompanied by X-ray structural analysis of the unmodified ß-l-arabinofuranosyl cyclophellitol aziridine in complex with both isozymes, which were shown to react by nucleophilic opening of the aziridine, at the pseudoanomeric carbon, by the active site cysteine nucleophile to form a stable thioether bond. Altogether, our activity-based probes may serve as chemical tools for the detection and identification of low-abundance ß-l-arabinofuranosidases in complex biological samples.

Recent advances in the accessibility, synthetic utility, and biological applications of aziridines.

Compounds featuring aziridine moieties are widely known and extensively reported in the literature. Due to their great potential from both synthetic and pharmacological points of view, many researchers have focused their efforts on the development of new methodologies for the preparation and transformation of these interesting compounds. Over the years, more and more ways to obtain molecules bearing these three-membered functional groups, which are challenging due to their inherent reactivity, have been described. Among them, several are more sustainable. In this review, we report the recent advances in the biological and chemical evolution of aziridine derivatives, in particular, the variety of methodologies described for the synthesis of aziridines and their chemical transformations leading to the formation of interesting derivatives, such as 4-7 membered heterocycles of pharmaceutical interest due to their promising biological activities.

Sulfenate Anion Catalyzed Diastereoselective Synthesis of Aziridines.

Aziridines are highly valued synthetic targets in organic and medicinal chemistry. The organocatalytic synthesis of such structures with broad substrate scope and good diastereoselectivity, however, is rare. Herein, we report a broadly applicable and diastereoselective synthetic method for the synthesis of trans-aziridines from imines and benzylic or alkyl halides utilizing sulfenate anions (PhSO- ) as the catalyst. Substrates bearing heterocyclic aromatic groups, alkyl, and electron-rich and electron-poor aryl groups were shown to be compatible with this method (33 examples), giving good yields and high diastereoselectivities (trans : cis >20 : 1). Further functionalization of aziridines containing cyclopropyl or cyclobutyl groups was achieved through ring-opening reactions, with a cyclobutyl-substituted norephedrine derivative obtained through a four-step synthesis. We offer a mechanistic proposal involving reversible addition of the deprotonated benzyl sulfoxide to the imine to explain the high trans-diastereoselectivity.

Synthesis of Optically Active Organofluorides by Ring Opening of Oxazolidinone-Fused Aziridines.

A general method for synthesizing optically active, primary, secondary, and tertiary organofluorides was developed. This chiral pool synthesis utilized the skeleton of arabinose to generate diastereomerically pure 2-oxazolidinone-fused aziridines, which underwent ring opening with a fluoride anion. The adducts, polyoxygenated organofluorides, were useful precursors to various fluorinated compounds, such as fluorinated amino acids.

Catalytic enantioselective desymmetrization of meso-aziridines.

As a type of readily available small strained-ring heterocycle, meso-aziridines may undergo catalytic desymmetrizing transformations to empower the rapid construction of diverse nitrogen-containing structures bearing contiguous stereocenters, which have great relevance in natural product synthesis, drug development and the design and synthesis of chiral catalysts/ligands for asymmetric catalysis. This review outlines the advances achieved in the catalytic asymmetric desymmetrization of meso aziridines and highlights some promising avenues for further work in this realm.

Novel Class of Proteasome Inhibitors: In Silico and In Vitro Evaluation of Diverse Chloro(trifluoromethyl)aziridines.

The ubiquitin-proteasome pathway (UPP) is the major proteolytic system in the cytosol and nucleus of all eukaryotic cells. The role of proteasome inhibitors (PIs) as critical agents for regulating cancer cell death has been established. Aziridine derivatives are well-known alkylating agents employed against cancer. However, to the best of our knowledge, aziridine derivatives showing inhibitory activity towards proteasome have never been described before. Herein we report a new class of selective and nonPIs bearing an aziridine ring as a core structure. In vitro cell-based assays (two leukemia cell lines) also displayed anti-proliferative activity for some compounds. In silico studies indicated non-covalent binding mode and drug-likeness for these derivatives. Taken together, these results are promising for developing more potent PIs.

Rhodium(II)-Catalyzed Enantioselective Intermolecular Aziridination of Alkenes.

C4-Symmetrical dirhodium(II) tetracarboxylates are highly efficient catalysts for the asymmetric intermolecular aziridination of substituted alkenes with sulfamates. The reaction proceeds with high levels of efficiency and chemoselectivity to afford aziridines with excellent yields of up to 95% and enantiomeric excesses of up to 99%. The scope of the alkene aziridination includes mono-, di-, and trisubstituted olefins as well as the late-stage functionalization of complex substrates. The reaction can be performed on a gram-scale with a catalyst loading of 0.1 mol %. Our DFT study led us to propose a two-spin-state mechanism, involving a triplet Rh-nitrene species as key intermediate to drive the stereocontrolled approach and activation of the substrate.

Molecular Basis for Enzymatic Aziridine Formation via Sulfate Elimination.

Natural products containing an aziridine ring, such as mitomycin C and azinomycin B, exhibit antitumor activities by alkylating DNA via their aziridine rings; however, the biosynthetic mechanisms underlying the formation of these rings have not yet been elucidated. We herein investigated the biosynthesis of vazabitide A, the structure of which is similar to that of azinomycin B, and demonstrated that Vzb10/11, with no similarities to known enzymes, catalyzed the formation of the aziridine ring via sulfate elimination. To elucidate the detailed reaction mechanism, crystallization of Vzb10/11 and the homologous enzyme, AziU3/U2, in the biosynthesis of azinomycin B was attempted, and the structure of AziU3/U2, which had a new protein fold overall, was successfully determined. The structural analysis revealed that these enzymes adjusted the dihedral angle between the amino group and the adjacent sulfate group of the substrate to almost 180° and enhanced the nucleophilicity of the C6-amino group temporarily, facilitating the SN2-like reaction to form the aziridine ring. The present study reports for the first time the molecular basis for aziridine ring formation.

Re-Evaluation of Product Outcomes in the Rh-Catalyzed Ring Expansion of Aziridines with N-Sulfonyl-1,2,3-Triazoles.

N-heterocycles are prevalent in pharmaceuticals and natural products, but traditional methods often do not introduce significant stereochemical complexity into the ring. We previously reported a Rh-catalyzed ring expansion of aziridines and N-sulfonyl-1,2,3-triazoles to furnish dehydropiperazines with excellent diastereocontrol. However, later studies employing ketone-containing carbene precursors showed that [3,9]-bicyclic aziridine formation competes with production of the desired heterocyclic scaffolds. In light of these surprising results, our initial findings were re-examined both experimentally and computationally to reveal how noncovalent interactions and restricted bond rotation in the aziridinium ylide intermediate promote this unexpected reaction pathway.

Rational Tuning of the Reactivity of Three-Membered Heterocycle Ring Openings via SN 2 Reactions.

The development of small-molecule covalent inhibitors and probes continuously pushes the rapidly evolving field of chemical biology forward. A key element in these molecular tool compounds is the "electrophilic trap" that allows a covalent linkage with the target enzyme. The reactivity of this entity needs to be well balanced to effectively trap the desired enzyme, while not being attacked by off-target nucleophiles. Here we investigate the intrinsic reactivity of substrates containing a class of widely used electrophilic traps, the three-membered heterocycles with a nitrogen (aziridine), phosphorus (phosphirane), oxygen (epoxide) or sulfur atom (thiirane) as heteroatom. Using quantum chemical approaches, we studied the conformational flexibility and nucleophilic ring opening of a series of model substrates, in which these electrophilic traps are mounted on a cyclohexene scaffold (C6 H10 Y with Y=NH, PH, O, S). It was revealed that the activation energy of the ring opening does not necessarily follow the trend that is expected from C-Y leaving-group bond strength, but steeply decreases from Y=NH, to PH, to O, to S. We illustrate that the HOMONu -LUMOSubstrate interaction is an all-important factor for the observed reactivity. In addition, we show that the activation energy of aziridines and phosphiranes can be tuned far below that of the corresponding epoxides and thiiranes by the addition of proper electron-withdrawing ring substituents. Our results provide mechanistic insights to rationally tune the reactivity of this class of popular electrophilic traps and can guide the experimental design of covalent inhibitors and probes for enzymatic activity.

The Synthesis of (2R)-Aziridine-2-carboxylic Acid Containing Dipeptides.

Optimized conditions for the synthesis of fully deprotected (2R)-aziridine containing dipeptides are described. Preparation of fully protected N- and C- terminal aziridine containing dipeptides was found to be straightforward and high yielding for the majority of compounds, whereas their full deprotection was possible only for C-terminal analogs. Deprotection of N-terminal derivatives using standard procedures of peptide chemistry was found difficult providing only mixtures of unidentifiable products. The described molecules have potential as building blocks in synthetic chemistry, in the chemical biology arena, as covalent modifiers, and as biomarkers.

N-Aminopyridinium reagents as traceless activating groups in the synthesis of N-Aryl aziridines.

N-functionalized aziridines, which are both useful intermediates and important synthetic targets, can be envisioned as arising from the addition of nitrenes (i.e., NR fragments) to olefinic substrates. The exceptional reactivity of most nitrenes, in particular with respect to unimolecular decomposition, prevents general application of nitrene-transfer to the synthesis of N-functionalized aziridines. Here we demonstrate N-aryl aziridine synthesis via 1) olefin aziridination with N-aminopyridinium reagents to afford N-pyridinium aziridines followed by 2) Ni-catalyzed C-N cross-coupling of the N-pyridinium aziridines with aryl boronic acids. The N-pyridinium aziridine intermediates also participate in ring-opening chemistry with a variety of nucleophiles to afford 1,2-aminofunctionalization products. Mechanistic investigations indicate aziridine cross-coupling proceeds via a noncanonical mechanism involving initial aziridine opening promoted by the bromide counterion of the Ni catalyst, C-N cross-coupling, and finally aziridine reclosure. Together, these results provide new opportunities to achieve selective incorporation of generic aryl nitrene equivalents in organic molecules.

Aza-Quasi-Favorskii Reaction: Construction of Highly Substituted Aziridines through a Concerted Multibond Rearrangement Process.

A new molecular rearrangement, the aza-Quasi-Favorskii rearrangement, has been developed for the construction of highly substituted aziridines. Electron-deficient O-sulfonyl oximes react readily with α,α-disubstituted acetophenone-derived enolates to furnish highly substituted aziridines via this unprecedented domino process. In-depth computational studies reveal an asynchronous yet concerted nitrenoid-type rearrangement pathway.