Rapid and Bifunctional Chemoselective Metabolome Analysis of Liver Disease Plasma Using the Reagent 4-Nitrophenyl-2H-azirine.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts that has been associated with diverse metabolic carboxylic acids. Mass spectrometric techniques are the method of choice for their analysis. However, the broad investigation of this metabolite class remains challenging. Derivatization of carboxylic acids represents a strategy to overcome these limitations but available methods suffer from diverse analytical challenges. Herein, we have designed a novel strategy introducing 4-nitrophenyl-2H-azirine as a new chemoselective moiety for the first time for carboxylic acid metabolites. This moiety was selected as it rapidly forms a stable amide bond and also generates a new ketone, which can be analyzed by our recently developed quant-SCHEMA method specific for carbonyl metabolites. Optimization of this new method revealed a high reproducibility and robustness, which was utilized to validate 102 metabolic carboxylic acids using authentic synthetic standard conjugates in human plasma samples including nine metabolites that were newly detected. Using this sequential analysis of the carbonyl- and carboxylic acid-metabolomes revealed alterations of the ketogenesis pathway, which demonstrates the vast benefit of our unique methodology. We anticipate that the developed azirine moiety with rapid functional group transformation will find broad application in diverse chemical biology research fields.

Triethylamine-Promoted Oxidative Cyclodimerization of 2H-Azirine-2-carboxylates to Pyrimidine-4,6-dicarboxylates: Experimental and DFT Study.

An unprecedented oxidative cyclodimerization reaction of 2H-azirine-2-carboxylates to pyrimidine-4,6-dicarboxylates under heating with triethylamine in air is described. In this reaction, one azirine molecule undergoes formal cleavage across the C-C bond and another across the C=N bond. According to the experimental study and DFT calculations, the key steps of the reaction mechanism include nucleophilic addition of N,N-diethylhydroxylamine to an azirine to form an (aminooxy)aziridine, generation of an azomethine ylide, and its 1,3-dipolar cycloaddition to the second azirine molecule. The crucial condition for the synthesis of pyrimidines is generation of N,N-diethylhydroxylamine in the reaction mixture in a very low concentration, which is ensured by the slow oxidation of triethylamine with air oxygen. Addition of a radical initiator accelerated the reaction and resulted in higher yields of the pyrimidines. Under these conditions, the scope of the pyrimidine formation was elucidated, and a series of pyrimidines was synthesized.

α-Lactam Electrophiles for Covalent Chemical Biology.

Electrophilic groups are one of the key pillars of contemporary chemical biology and medicinal chemistry. For instance, 3-membered N-heterocyclic compounds-such as aziridines, azirines, and oxaziridines-possess unique electronic and structural properties which underlie their potential and applicability as covalent tools. The α-lactams are also members of this group of compounds, however, their utility within the field remains unexplored. Here, we demonstrate an α-lactam reagent (AM2) that is tolerant to aqueous buffers while being reactive towards biologically relevant nucleophiles. Interestingly, carboxylesterases 1 and 2 (CES1/2), both serine hydrolases with key roles in endo- and xenobiotic metabolism, were found as primary covalent targets for AM2 in HepG2 liver cancer cells. All in all, this study constitutes the starting point for the further development and exploration of α-lactam-based electrophilic probes in covalent chemical biology.

One-Pot Synthesis of Multifunctionalized 1-Pyrrolines from 2-Alkyl-2H-azirines and Diazocarbonyl Compounds.

A novel strategy for the synthesis of 1-pyrrolines based on formal [4 + 1] annulation of 2-alkyl-2H-azirines with diazocarbonyl compounds has been developed. This one-pot approach includes the Rh(II)-catalyzed formation of 4-alkyl-2-azabuta-1,3-dienes, followed by the DBU-promoted cyclization, and features a good substrate tolerance. The 1-pyrrolines containing an ester group at the C3 were prepared in a three-step one-pot procedure starting from 5-alkoxyisoxazoles. The cyclization of 2-azabutadienes to 1-pyrrolines most likely proceeds via the 6π electrocyclization of a conjugated NH-azomethine ylide.

Identification of small-molecule urea derivatives as PTPC modulators targeting the c subunit of F1/Fo-ATP synthase.

Maintaining a high percentage of living and functional cells in those pathologies in which excessive cell death occurs, such as neurodegenerative disorders and cardiovascular diseases, is one of the most intriguing challenges in the field of biochemical research for drug discovery. Here, mitochondrial permeability transition-driven regulated cell death is the main mechanism of mitochondrial impairment and cell fate; this pathway is still lacking of satisfying pharmacological treatments to counteract its becoming; for this reason, it needs continuous and intense research to find new compounds as modulator of the permeability transition pore complex (PTPC) activity. In this study, we report the identification of small-molecule urea derivatives able to inhibit PTPC opening following calcium overload and selected for future use in cytoprotection.

Synthesis of functionalized 1-aminoisoquinolines through cascade three-component reaction of ortho-alkynylbenzaldoximes, 2H-azirines, and electrophiles.

We have developed a new three-component approach using ortho-alkynylbenzaldoximes involving the formation of a cyclic nitrone in the presence of Br2 or ICl for the synthesis of 1-aminoisoquinolines via cascade 6-endo-cyclization, 1,3-dipolar cycloaddition reaction with 2H-azirines, and ring-opening reaction sequences. The broad range of structurally diverse products, good to high yields, high atom-economy and high bond-formation efficiency make this method an attractive alternative for the synthesis of 1-aminoisoquinolines.

Azirine-triazole hybrids: selective synthesis of 5-(2H-azirin-2-yl)-, 5-(1H-pyrrol-2-yl)-1H-1,2,3-triazoles and 2-(5-(2H-azirin-2-yl)-1H-1,2,3-triazol-1-yl)pyridines.

Azirine-triazole hybrids, 1-R-5-(3-aryl-2H-azirin-2-yl)-1H-1,2,3-triazoles, were selectively synthesized by reaction of 1-(3-aryl-2H-azirin-2-yl)-2-(triphenylphosphoranylidene)ethanones with tosyl and (E)-2-benzoylvinyl azides in high yields at rt. The reaction with 2-azidopyridine makes it possible to obtain azirine-triazole-pyridine hybrids, albeit in moderate yields, at 170 °C. The mechanism and selectivity of the reaction of α-carbonylphosphoranes with azides are discussed on the basis of DFT calculations. According to the calculation, the reaction of α-carbonylphosphoranes with model mesyl azide, leading to 1,5-disubstituted triazoles proceeds via a non-concerted cycloaddition, while the reaction leading to 1,4-disubstituted triazoles proceeds via a concerted azide cycloaddition, but through the transition state which has much higher energy. In contrast to the reaction of α-(triphenylphosphoranylidene)ketones with TsN3, the reaction with TfN3 yields the α-diazo ketones. Ni-Catalyzed reaction of azirinyl-1,2,3-triazoles with acetylacetone provides pyrrole-triazole and pyrrole-triazole-pyridine hybrids in good yields under mild conditions.

Non-Aziridination Approaches to 3-Arylaziridine-2-carboxylic Acid Derivatives and 3-Aryl-(aziridin-2-yl)ketones.

Highly functionalized aziridines, including compounds with aromatic moieties, are attractive substrates both in synthetic and medical areas of chemistry. There is a broad and interesting set of synthetic methods for reaching these compounds. Aziridination represents the most explored tool, but there are several other more specific, less well-known, but highly promising approaches. Therefore, the current review focuses on recently described or updated ways to obtain 3-arylated aziridines via different non-aziridination-based synthetic methods, reported mainly since 2000. The presented methods belong to two main directions of synthesis, namely, cyclization of open-chain substrates and rearrangement of other heterocycles. Cyclization of open-chain substrates includes the classic Gabriel-Cromwell type cyclization of halogenated substrates with amines, base-promoted cyclization of activated aminoalcohols (or its analogues), and the oxidative cyclization of ß-dicarbonyls. Rearrangements of other heterocycles are presented as the Baldwin rearrangement of 4-isoxazolines, the cycloaddition of 1.3-dipoles or dienes to 2H-azirines, and the addition of C- and N-nucleophiles to the double bond of azirines.

5-Chloroisoxazoles: A Versatile Starting Material for the Preparation of Amides, Anhydrides, Esters, and Thioesters of 2H-Azirine-2-carboxylic Acids.

Amides, anhydrides, esters, and thioesters of 2H-azirine-2-carboxylic acids were prepared by a rapid procedure at room temperature involving FeCl2-catalyzed isomerization of 5-chloroisoxazoles to 2H-azirine-2-carbonyl chlorides, followed by reaction with N-, O-, or S-nucleophiles mediated by an ortho-substituted pyridine. With readily available chloroisoxazoles and a nucleophile, 2-picoline can be used as an inexpensive base. When a high yield of the acylation product is important, the reagent 2-(trimethylsilyl)pyridine/ethyl chloroformate is more suitable for the acylation with 2H-azirine-2-carbonyl chlorides.

Copper(II)-Catalyzed (3+2) Cycloaddition of 2H-Azirines to Six-Membered Cyclic Enols as a Route to Pyrrolo[3,2-c]quinolone, Chromeno[3,4-b]pyrrole, and Naphtho[1,8-ef]indole Scaffolds.

A method for the [2+3] pyrroline annulation to the six-membered non-aromatic enols using 3-aryl-2H-azirines as annulation agents is developed in the current study. The reaction proceeds as a formal (3+2) cycloaddition via the N1-C2 azirine bond cleavage and is catalyzed by both Cu(II) and Cu(I) compounds. The new annulation method can be applied to prepare pyrrolo[3,2-c]quinoline, chromeno[3,4-b]pyrrole, and naphtho[1,8-ef]indole derivatives in good to excellent yields from enols of the quinolin-2-one, 2H-chromen-2-one, and 1H-phenalen-1-one series.

Anti-Inflammatory Dysidazirine Carboxylic Acid from the Marine Cyanobacterium Caldora sp. Collected from the Reefs of Fort Lauderdale, Florida.

Dysidazirine carboxylic acid (1) was isolated from the lipophilic extract of a collection of the benthic marine cyanobacterium Caldora sp. from reefs near Fort Lauderdale, Florida. The planar structure of this new compound was determined by spectroscopic methods and comparisons between HRMS and NMR data with its reported methyl ester. The absolute configuration of the single chiral center was determined by the conversion of 1 to the methyl ester and the comparison of its specific rotation data with the two known methyl ester isomers, 2 and 3. Molecular sequencing with 16S rDNA indicated that this cyanobacterium differs from Caldora penicillata (Oscillatoriales) and represents a previously undocumented and novel Caldora species. Dysidazirine (2) showed weak cytotoxicity against HCT116 colorectal cancer cells (IC50 9.1 µM), while dysidazirine carboxylic acid (1) was non-cytotoxic. Similar cell viability patterns were observed in RAW264.7 cells with dysidazirine only (2), displaying cytotoxicity at the highest concentration tested (50 µM). The non-cytotoxic dysidazirine carboxylic acid (1) demonstrated anti-inflammatory activity in RAW264.7 cells stimulated with LPS. After 24 h, 1 inhibited the production of NO by almost 50% at 50 µM, without inducing cytotoxicity. Compound 1 rapidly decreased gene expression of the pro-inflammatory gene iNOS after 3 h post-LPS treatment and in a dose-dependent manner (IC50 ~1 µM); the downregulation of iNOS persisted at least until 12 h.

Carbon Atom Insertion into Pyrroles and Indoles Promoted by Chlorodiazirines.

Herein, we report a reaction that selectively generates 3-arylpyridine and quinoline motifs by inserting aryl carbynyl cation equivalents into pyrrole and indole cores, respectively. By employing α-chlorodiazirines as thermal precursors to the corresponding chlorocarbenes, the traditional haloform-based protocol central to the parent Ciamician-Dennstedt rearrangement can be modified to directly afford 3-(hetero)arylpyridines and quinolines. Chlorodiazirines are conveniently prepared in a single step by oxidation of commercially available amidinium salts. Selectivity as a function of pyrrole substitution pattern was examined, and a predictive model based on steric effects is put forward, with DFT calculations supporting a selectivity-determining cyclopropanation step. Computations surprisingly indicate that the stereochemistry of cyclopropanation is of little consequence to the subsequent electrocyclic ring opening that forges the pyridine core, due to a compensatory homoaromatic stabilization that counterbalances orbital-controlled torquoselectivity effects. The utility of this skeletal transform is further demonstrated through the preparation of quinolinophanes and the skeletal editing of pharmaceutically relevant pyrroles.

2H-Azirine-Based Reagents for Chemoselective Bioconjugation at Carboxyl Residues Inside Live Cells.

Protein modification by chemical reagents has played an essential role in the treatment of human diseases. However, the reagents currently used are limited to the covalent modification of cysteine and lysine residues. It is thus desirable to develop novel methods that can covalently modify other residues. Despite the fact that the carboxyl residues are crucial for maintaining the protein function, few selective labeling reactions are currently available. Here, we describe a novel reactive probe, 3-phenyl-2H-azirine, that enables chemoselective modification of carboxyl groups in proteins under both in vitro and in situ conditions with excellent efficiency. Furthermore, proteome-wide profiling of reactive carboxyl residues was performed with a quantitative chemoproteomic platform.

Light-Activated, Bioadhesive, Poly(2-hydroxyethyl methacrylate) Brush Coatings.

Rapid adhesion between tissue and synthetic materials is relevant to accelerate wound healing and to facilitate the integration of implantable medical devices. Most frequently, tissue adhesives are applied as a gel or a liquid formulation. This manuscript presents an alternative approach to mediate adhesion between synthetic surfaces and tissue. The strategy presented here is based on the modification of the surface of interest with a thin polymer film that can be transformed on-demand, using UV-light as a trigger, from a nonadhesive into a reactive and tissue adhesive state. As a first proof-of-concept, the feasibility of two photoreactive, thin polymer film platforms has been explored. Both of these films, colloquially referred to as polymer brushes, have been prepared using surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-hydroxyethyl methacrylate (HEMA). In the first part of this study, it is shown that direct UV-light irradiation of PHEMA brushes generates tissue-reactive aldehyde groups and facilitates adhesion to meniscus tissue. While this strategy is very straightforward from an experimental point of view, a main drawback is that the generation of the tissue reactive aldehyde groups uses the 250 nm wavelength region of the UV spectrum, which simultaneously leads to extensive photodegradation of the polymer brush. The second part of this report outlines the synthesis of PHEMA brushes that are modified with 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acid (TFMDA) moieties. UV-irradiation of the TFMDA containing brushes transforms the diazirine moieties into reactive carbenes that can insert into C-H, N-H, and O-H bonds and mediate the formation of covalent bonds between the brush surface and meniscus tissue. The advantage of the TFMDA-modified polymer brushes is that these can be activated with 365 nm wavelength UV light, which does not cause photodegradation of the polymer films. While the work presented in this manuscript has used silicon wafers and fused silica substrates as a first proof-of-concept, the versatility of SI-ATRP should enable the application of this strategy to a broad range of biomedically relevant surfaces.

Synthesis of α-Aminophosphonic Acid Derivatives Through the Addition of O- and S-Nucleophiles to 2H-Azirines and Their Antiproliferative Effect on A549 Human Lung Adenocarcinoma Cells.

This work reports a straightforward regioselective synthetic methodology to prepare α-aminophosphine oxides and phosphonates through the addition of oxygen and sulfur nucleophiles to the C-N double bond of 2H-azirine derivatives. Determined by the nature of the nucleophile, different α-aminophosphorus compounds may be obtained. For instance, aliphatic alcohols such as methanol or ethanol afford α-aminophosphine oxide and phosphonate acetals after N-C3 ring opening of the intermediate aziridine. However, addition of 2,2,2-trifluoroethanol, phenols, substituted benzenthiols or ethanethiol to 2H-azirine phosphine oxides or phosphonates yields allylic α-aminophosphine oxides and phosphonates in good to high general yields. In some cases, the intermediate aziridine attained by the nucleophilic addition of O- or S-nucleophiles to the starting 2H-azirine may be isolated and characterized before ring opening. Additionally, the cytotoxic effect on cell lines derived from human lung adenocarcinoma (A549) and non-malignant cells (MCR-5) was also screened. Some α-aminophosphorus derivatives exhibited very good activity against the A549 cell line in vitro. Furthermore, selectivity towards cancer cell (A549) over non-malignant cells (MCR-5) has been detected in almost all compounds tested.

Terminal Diazirines Enable Reverse Polarization Transfer from 15 N2 Singlets.

Diazirine moieties are chemically stable and have been incorporated into biomolecules without impediment of biological activity. The 15 N2 labeled diazirines are appealing motifs for hyperpolarization supporting relaxation protected states with long-lived lifetimes. The (-CH15 N2 ) diazirine groups investigated here are analogues to methyl groups, which provides the opportunity to transfer polarization stored on a relaxation protected (-CH15 N2 ) moiety to 1 H, thus combining the advantages of long lifetimes of 15 N polarization with superior sensitivity of 1 H detection. Despite the proximity of 1 H to 15 N nuclei in the diazirine moiety, 15 N T1 times of up to (4.6±0.4) min and singlet lifetimes Ts of up to (17.5±3.8) min are observed. Furthermore, we found terminal diazirines to support hyperpolarized 1 H2 singlet states in CH2 groups of chiral molecules. The singlet lifetime of 1 H singlets is up to (9.2±1.8) min, thus exceeding 1 H T1 relaxation time (at 8.45 T) by a factor of ≈100.

Traceless Immobilization of Analytes for High-Throughput Experiments with SAMDI Mass Spectrometry.

Label-free assays, and particularly those based on the combination of mass spectroscopy with surface chemistries, enable high-throughput experiments of a broad range of reactions. However, these methods can still require the incorporation of functional groups that allow immobilization of reactants and products to surfaces prior to analysis. In this paper, we report a traceless method for attaching molecules to a self-assembled monolayer for matrix-assisted laser desorption and ionization (SAMDI) mass spectrometry. This method uses monolayers that are functionalized with a 3-trifluoromethyl-3-phenyl-diazirine group that liberates nitrogen when irradiated and gives a carbene that inserts into a wide range of bonds to covalently immobilize molecules. Analysis of the monolayer with SAMDI then reveals peaks for each of the adducts formed from molecules in the sample. This method is applied to characterize a P450 drug metabolizing enzyme and to monitor a Suzuki-Miyaura coupling chemical reaction and is important because modification of the substrates with a functional group would alter their activities. This method will be important for high-throughput experiments in many areas, including reaction discovery and optimization.

Molecular Targets of the Hydrophobic Block of Pluronics in Cells: a Photo Affinity Labelling Approach.

PURPOSE: Pluronics are known as inhibitors of multidrug resistance thus making tumor cells sensitive to therapeutic doses of drugs. The purpose of our study consists in revealing molecular targets of the hydrophobic poly(propylene oxide) block of pluronics in living cells and the dependence of the polymers chemosensitizing efficiency upon targeting. METHODS: A photo sensitive tracer was attached to the hydrophobic poly(propylene oxide) block of 3H-labeled tert-Bu-EO-PO copolymer. The conjugate was used for treatment cells in culture. We searched for its complexes with cellular lipids or proteins using RP TLC and SDS-electrophoresis, respectively. The chemosensitizing efficiency of pluronics was evaluated by their least concentrations sufficient for MDR reversion (CMDR). RESULTS: The poly(propylene oxide) block inserts in the lipid core of plasma membrane. No preferential binding of the conjugate with any cellular protein, particularly P-gp, has been detected. FITC-labeled pluronic L61 bound to alcohol insoluble cellular targets did not participate in MDR reversion. CMDR values of 13 block copolymers have been determined. These values inversely correlated with the polymers affinity toward lipids and the ability to accelerate flip-flop. CONCLUSION: Insertion of the hydrophobic poly(propylene oxide) block of amphiphiles in the lipid core of plasma membrane and acceleration of flip-flop of lipids underlie the mechanism of MDR reversion.

Cationic Amphiphilic Tris-Cyclometalated Iridium(III) Complexes Induce Cancer Cell Death via Interaction with Ca2+-Calmodulin Complex.

In our previous paper, we reported on the preparation of some cationic amphiphilic Ir complexes (2c, 2d) containing KKGG peptides that induce and detect cell death of Jurkat cells. Mechanistic studies suggest that 2c interacts with anionic molecules and/or membrane receptors on the cell surface to trigger an intracellular Ca2+ response, resulting in the induction of cell death, accompanied by membrane disruption. We have continued the studies of cell death of Jurkat cells induced by 2c and found that xestospongin C, a selective inhibitor of an inositol 1,4,5-trisphosphate receptor located on the endoplasmic reticulum (ER), reduces the cytotoxicity of 2c, suggesting that 2c triggers the release of Ca2+ from the ER, leading to an increase in the concentration of cytosolic Ca2+, thus inducing cell death. Moreover, we synthesized a series of new amphiphilic cationic Ir complexes 5a-c containing photoreactive 3-trifluoromethyl-3-phenyldiazirine (TFPD) groups, in an attempt to identify the target molecules of 2c. Interestingly, it was discovered that a TFPD group functions as a triplet quencher of Ir complexes. It was also found that 5b is useful as a turn-on phosphorescent probe of acidic proteins such as bovine serum albumin (BSA) (pI = 4.7) and their complexation was confirmed by luminescence titrations and SDS-PAGE of photochemical products between them. These successful results allowed us to carry out photoaffinity labeling of the target biomolecules of 5b (2c and analogues thereof) in Jurkat cells. A proteomic analysis of the products obtained by the photoirradiation of 5b with Jurkat cells suggests that the Ca2+-binding protein "calmodulin (CaM)" is one of target proteins of the Ir complexes. Indeed, 5b was found to interact with the Ca2+-CaM complex, as evidenced by luminescence titrations and the results of photochemical reactions of 5b with CaM in the presence of Ca2+ (SDS-PAGE). A plausible mechanism for cell death induced by a cationic amphiphilic Ir complex is discussed on the basis of our results.

Synthesis and preliminary biological evaluation of radiolabeled 5-BDBD analogs as new candidate PET radioligands for P2X4 receptor.

P2X4 receptor has become an interesting molecular target for treatment and PET imaging of neuroinflammation and associated brain diseases such as Alzheimer's disease. This study reports the first design, synthesis, radiolabeling and biological evaluation of new candidate PET P2X4 receptor radioligands using 5-BDBD, a specific P2X4 receptor antagonist, as a scaffold. 5-(3-Hydroxyphenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD analog, [11C]9) and 5-(3-Bromophenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD, [11C]8c) were prepared from their corresponding desmethylated precursors with [11C]CH3OTf through N-[11C]methylation and isolated by HPLC combined with SPE in 30-50% decay corrected radiochemical yields with 370-1110GBq/µmol specific activity at EOB. 5-(3-[18F]Fluorophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]F-5-BDBD, [18F]5a) and 5-(3-(2-[18F]fluoroethoxy)phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]FE-5-BDBD, [18F]11) were prepared from their corresponding nitro- and tosylated precursors by nucleophilic substitution with K[18F]F/Kryptofix 2.2.2 and isolated by HPLC-SPE in 5-25% decay corrected radiochemical yields with 111-740GBq/µmol specific activity at EOB. The preliminary biological evaluation of radiolabeled 5-BDBD analogs indicated these new radioligands have similar biological activity with their parent compound 5-BDBD.