The complexation of two diastereoisomers of a β-phosphorylated cyclic nitroxide radical (2,5-dimethyl-5-hydroxymethylene-2-diethoxyphosphonyl-pyrrolidin-N-oxyl) with cyclodextrins (β-CD and γ-CD) was investigated using electron paramagnetic resonance (EPR) spectroscopy and cyclic voltammetry. The two isomers have different EPR signatures due to the differences in their phosphorus and nitrogen hyperfine splitting constants. The cis stereoisomer (1c˙) promotes intramolecular hydrogen bonding, involving the HO group attached to the methylene group and O atom from the phosphorus moiety, while in the case of the trans stereoisomer (1t˙), this intramolecular bonding is not possible. The EPR spectra indicate the higher affinity of the 1t˙ isomer for γ-CD, highlighting the different EPR parameters of the free radical and complexed radical. The binding constants determined based on the EPR and cyclic voltammetry data show stronger affinity for γ-CD compared with β-CD, particularly for 1t˙. An increasing potassium chloride concentration enhances the stability of the complex linearly by modulating the solvation and electrostatic interactions, as indicated by the thermodynamic parameters obtained from EPR measurements. Electrochemical measurements demonstrate decreased diffusion coefficients and anodic peak shifts upon complexation, supporting the spectroscopic results. These findings highlight the important role of molecular stereochemistry and ionic strength in modulating host-guest interactions between cyclodextrins and nitroxides.

This study reports the synthesis of six new nitroxides with a paramagnetic moiety attached to a linear alkyl chain, their analyses by electron paramagnetic resonance spectroscopy, and their interaction with two classes of host molecules: cyclodextrins (α- and β-CD) and cucurbiturils (CB[n], n = 6-8). The series of new nitroxides consists of two PROXYL radicals, two TEMPO radicals and two β-phosphorylated nitroxides. As evidenced by changes in the hyperfine coupling constants, the PROXYL and TEMPO spin probes form complexes with CDs by threading of the alkyl chain and/or by inclusion of the paramagnetic moiety. The binding constants of the host-guest complexes, evaluated from the variation in the rotational correlation time with the concentration of the host, fall in the range of 102-103 M-1. The β-phosphorylated nitroxides present low water solubility but are solubilised by interaction with CDs at high concentrations, while complexation with CB[n] has not been evidenced for either probe. The interaction of the cationic nitroxides CAT1 and CAT16 with CDs and CB[n] was also considered in order to elucidate the role played by charged species in the complexation. Both CAT1 and CAT16 form complexes with CB[n]. In the particular case of CAT1/CB[6], the initial association of the complexes at high concentrations of CB[6] was evidenced by the appearance of the spin-spin interaction, followed by oxidation of iodine and separation of red crystals characterized by X-ray diffraction.

The rise of drug-resistant pathogens and the dose-limiting side effects of current therapies highlight the urgent need for innovative treatment strategies. We recently demonstrated that hybrid alkoxyamine-peptide conjugates, upon enzymatic cleavage by specific peptidases, undergo C-O bond homolysis to generate highly reactive radical species capable of inducing broad-spectrum damage to pathogenic organisms (cancerous cells, parasites, fungi, etc.). Relying on this concept, we now report on the design and synthesis of a new class of enzyme-triggered, releasing radical prodrugs with a carbohydrate structure as a leaving group. These molecules offer a versatile strategy for the development of next-generation therapeutics that couple enzymatic specificity using glucosidase with controlled radical release to fight resistant and difficult-to-treat infections.

Sterilization of biopharmaceutical products has commonly employed gamma radiation from cobalt-60, a radioisotope with associated security and supply chain risks. Increased use of non-radioisotope alternative ionizing energy technologies, such as electron beam (e-beam) and X-rays, is promising but has been hindered by lack of thorough evaluation of the compatibility of these techniques with product materials. This study investigates radical generation in a polyethylene (PE)/ethylene-vinyl alcohol copolymer (EVOH)/PE multilayer film product (Sartorius Flexsafe (R) with the S80 film) under varying e-beam and X-ray irradiation conditions to address data gaps for these materials and technologies. Utilizing a full factorial design of experiments (DoE), we examined the effects of irradiation technology, dose level, dose rate, processing temperature, and ambient oxygen concentration on radical formation, as quantified by electron spin resonance (ESR). Our results indicate that the type of ionizing technology (e-beam vs. X-ray) does not significantly affect radical concentration generation in the materials. However, higher doses and lower temperatures were found to correlate with increased radical concentrations, while elevated oxygen levels effectively suppressed radicals through a presumed scavenging mechanism. For e-beam processing, a higher dose rate reduced radical accumulation, while the opposite trend was found for X-ray irradiation. Complementary Fourier-transform infrared spectroscopy (FTIR) and principal component analysis (PCA) revealed subtle oxidation differences of PE under specific irradiation conditions.

Enzyme-induced cytotoxicity can be considered the main challenge in modern cancer therapy that provides unique opportunities for targeted apoptosis. To address this challenge, we implemented enzyme-triggered activation of alkoxyamine-galactose conjugates with the formation of active carbon-centered radical species for the apoptosis of cancer cells. The comparative analysis of two alkoxyamine derivatives, bearing stable and self-immolative linkers between the radical precursor and galactose moiety, revealed the crucial role of molecular structure in anticancer activity. The study of cytotoxicity and induced oxidative stress of free amine and two galactosides revealed the enzyme-dependent nature of the activity of alkoxyamines having a self-immolative linker against distinct cancer cell lines such as PC-3 (prostate adenocarcinoma), SKOV-3 (ovarian adenocarcinoma), MCF-7 (breast adenocarcinoma), A-431 (epidermoid carcinoma), and Jurkat cells (human T-lymphoblastic leukemia). The collected data prove the applicability of enzyme-triggered glycosylated alkoxyamines as a new family of targeted prodrugs against cancer.

Nitroreductase-mediated alkoxyamine activation: a selective approach for prodrugs.

The study investigates structural and mechanical changes in polyoxymethylene (POM) resulting from gamma, X-ray, and e-beam irradiation used for sterilization. Techniques such as Electron Spin Resonance (ESR), Differential Scanning Calorimetry (DSC), and tensile testing were employed to examine irradiation-induced effects. As the irradiation dose increases, the radical concentration consistently rises for all radiation technologies, although slight variations are observed depending on the specific irradiation technology. Mechanical properties, including yield strength and elongation at break, progressively decline with increasing dose, whereas the melting temperature remains nearly constant, indicating the preservation of the crystalline phase. FTIR analysis confirms the onset of oxidative degradation at higher doses. Overall, the findings demonstrate that irradiation can effectively sterilize POM, provided that the dose is carefully controlled to prevent detrimental alterations to the material's performance.

This study investigates the impact of gamma rays, X-rays, and electron beam irradiation on PE/EVOH/PE multilayer films focusing on how dose rate influences polymer modifications and the formation of reactive species like peroxides and methionine sulfoxide from methionine solution, which can affect protein stability. Using advanced analytical techniques such as electron spin resonance (ESR) and high-performance liquid chromatography (HPLC), the study measures radical formation and methionine oxidation. Results indicate that post-irradiation ageing and contact time with methionine significantly affect methionine sulfoxide levels, while the impact of dose rate varies by irradiation technology. Oxidation of methionine solution in contact with irradiated film remains similar whatever gamma, X-ray and e-beam technologies are used to irradiate the film.

This study investigates the effects of gamma rays, X-rays, and electron beams on ethylene vinyl acetate (EVA) multilayer films, commonly used in biotechnological applications. Electron spin resonance (ESR) analysis showed that irradiation generates unstable hydroxyalkyl radicals, quantifiable one day post-exposure, with concentrations decreasing within nine days, and with similar kinetics observed across all three irradiation technologies. The research focuses on dose rate impacts, which significantly influence polymer properties. Reactive species like hydrogen peroxide and hydroxyl radicals, generated during irradiation, can affect protein function through methionine oxidation. Advanced analytical techniques reveal that the dose rate significantly impacted the levels of reactive species, impacting the film's structural integrity and chemical stability comparably. Gamma irradiation generates more oxidative species. The study concludes that dose rate is crucial in methionine sulfoxide generation, with longer exposure leading to increased concentrations, particularly in gamma irradiation. These findings underscore the importance of considering dose rate and irradiation technology to optimize the stability and performance of multilayer films.

In this study, electron paramagnetic resonance (EPR) spectroscopy was used to investigate the formation of host-guest complexes of gamma- cyclodextrin and 2-hydroxy-propyl-gamma-cyclodextrin with nitroxides linked to long aliphatic chains and beta-phosphorylated nitroxides. The host-guest complexation of nitroxides is often accompanied by changes in EPR parameters reflecting the change in polarity around the nitroxide group, as well as changes in nitroxide dynamics. The EPR spectra of the solutions of the two series of paramagnetic compounds in the presence of gamma-cyclodextrin or 2-hydroxypropyl-gamma-cyclodextrin showed significant changes in the hyperfine splitting constants (aN and aP) proving the formation of host-guest complexes. Simulation of the experimental EPR spectra allowed to estimate the association constants for each radical/cyclodextrin pair. The values for these constants were lower for the functionalized cyclodextrin.

The emergence of antifungal-resistant Aspergillus fumigatus (A. fumigatus) became a serious public health concern, underscoring the need for new effective antifungal agents. Here, we present a strategy based on the in situ generation of radical species that are toxic to the pathogen. The synthesis of an alkoxyamine linked to a peptide substrate recognized by A. fumigatus-secreted dipeptidyl peptidase is described. Kinetic experiments show a stable prodrug prior to enzymatic activation. Ensuing peptide cleavage and spontaneous homolysis resulted in the generation of a stable nitroxide and a reactive alkyl radical moiety. Next, the exposure of A. fumigatus spores to the prodrug lead to pathogen growth inhibition in a compound concentration-dependent fashion (e.g., 42% inhibition at 10 µg/L). Importantly, the designed alkoxyamine inhibited not only the growth of a clinical voriconazole-susceptible A. fumigatus strain, but also the growth of a strain resistant to this azole. To determine the antifungal importance of the reactive alkyl radical, its substitution with a non-radical structure did not prevent A. fumigatus growth. Furthermore, the introduction of succinic group in the peptide substrate resulted in the loss of alkoxyamine antifungal properties. Our work reports a novel chemical strategy for antifungal therapy against A. fumigatus based on the pathogen enzyme-mediated generation of toxic radicals. Significantly, these findings are timely since they could overcome the emerged resistance to conventional drugs that are known to target defined pathogen biologic mechanisms such as ergosterol synthesis.

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This research investigated the impact of gamma, e-beam, and X-ray irradiation on medical-grade polypropylene (PP). A comprehensive array of analytical methods was employed to assess the chemical and physical changes in irradiated PP. The main findings revealed that irradiation did not significantly affect the mechanical properties, as tensile strength and elongation at break remained unaltered across all irradiation types and doses. Thermal analysis using DSC indicated a slight reduction in melting temperature at higher doses. ESR identified peroxyl radicals, which decayed similarly regardless of dose or irradiation type. Colorimetric analysis revealed yellowing in PP samples, particularly with gamma and X-ray irradiation, likely associated with specific additives. HPLC analysis showed that the oxidation potential was more influenced by the PP formulation than by the irradiation technology.

The progress in plasmonic chemistry requires research on energy transfer, mechanisms, and materials discovery. In this pursuit, there are >3000 papers applying the azo coupling of 4-nitrothiophenol (PNTP) as a model reaction. Here, we challenge the status of this reaction as a model due to experimental evidence of thiol desorption during plasmon excitation using laser irradiation monitored by X-ray photoelectron spectroscopy (XPS) as an analytic technique. The azo coupling was performed on commonly used Au nanoparticles (NPs) coated with PNTP and confirmed by Raman spectroscopy and XPS. Changes in the N 1s and S 2p spectral regions indicated the cleavage of the Au-S bond, accompanied by thiol oxidation. Based on XPS data, we hypothesized a chemical pathway and a kinetic model that surpasses previously used simple models in complexity, making it challenging to draw reliable conclusions. The dissociation of the Au-S bond is triggered by plasmonic heating, supported by experimentally and theoretically determined local temperatures exceeding the thiol desorption temperature. The azo coupling reaction does not fit within the requirements of the model one, which should be simple and proceed with structurally evidenced products. As one of the alternative reactions, we suggest alkoxyamine homolysis tracked by electron paramagnetic resonance spectroscopy because of known products and the simple kinetic model. Applications of suitable model reactions accelerate discoveries in plasmon catalysis.

The impacts of e-beam and X-ray irradiation on medical-grade high-density polyethylene (HDPE) are compared with that of gamma irradiation to evaluate their potential effects on pharmaceutical applications. An extensive suite of analytical techniques was employed to evaluate the chemical and physical transformations of irradiated HDPE. Key findings indicated that irradiation did not significantly alter the mechanical properties, as tensile strength and elongation at break remained stable across all irradiation types and doses. Thermal analysis via Differential Scanning Calorimetry (DSC) revealed a slight decrease in melting temperature at higher doses, with an equivalent melting temperature peak observed for all three irradiation technologies. Electron Spin Resonance (ESR) detected alkyl and allyl radicals, which decreased over time, showing no significant differences attributable to irradiation type or dose. Colorimetric analysis indicated yellowing in the samples, linked to specific additives. The quantification of methionine oxidized byproducts by High-Performance Liquid Chromatography (HPLC) demonstrated that the oxidation potential was equivalent for the three irradiation technologies for HDPE. The study concluded that there was a lack of significant impact of irradiation technologies on several physical, chemical and mechanical properties of HDPE.

Dynamic covalent bonding (DCB) has been a rising concept for the past several years in materials sciences. This article describes how the bond lability involved in DCB is applied to develop drugs against tropical parasitic diseases such as malaria and bilharziasis. Recently, we showed that some alkoxyamines (typical molecules exhibiting DCB) exhibit in vitro activities against S. mansoni (for A8L, 100% worm mortality in 48 hours at 10 μg ml-1) and P. falciparum (for A8L, IC50 = 270 nM). Here, the combination of enzymatic-physical (solvent effect) activation or of enzymatic-chemical (acetal hydrolysis) activation is used to develop alkoxyamines that show activity against both parasites. The enzymatic step controls the specificity of the drug.

We report an investigation of a controlled radical release produced by iron oxide nanoparticles (IONPs) of ca. 25 nm covalently grafted through phosphonic groups with a thermosensitive alkoxyamine, (6-(4-(1-((di-tert-butylamino)oxy)ethyl)benzamido)hexyl)phosphonate, having a relatively low homolysis temperature (kd = 6.4 x 10-4 s-1 at 77 degrees C, Ea = 117.8 kJ mol-1). Action of an alternating current magnetic field (AMF) or light irradiation at 808 nm produces a rapid heating of the nanoparticles' surface, which induces the homolysis of the C-ON bond of alkoxyamines facilitating the efficient formation of free radicals. We demonstrated based on homolysis kinetics investigated by electron paramagnetic resonance (EPR) spectroscopy that light irradiation at 808 nm (2.6 W cm-2) enables efficient radical release from grafted nanoparticles at 44 degrees C (t1/2 = 23.6 min), whereas the free molecules required 20 h to show the same release at this temperature. AMF exposure accelerates the homolysis of alkoxyamine-grafted nanoparticles (16 kA m-1, 2.9 mg mL-1) twofold compared to the free alkoxyamine at 77 degrees C (t1/2 = 7.9 min vs. 18 min). These findings underscore the critical importance of localized nanoscale effects, demonstrating that the homolysis rate on the nanoparticle surface under external stimuli is significantly higher compared to that under external solution heating, with this enhancement being even more pronounced under light irradiation.

The interaction between a new spin probe, prepared by spin labelling 4-phenoxyaniline with the TEMPO paramagnetic moiety, and bovine serum albumin was studied for solutions in water and in trehalose. At the same time, the interaction of this spin probe with cyclodextrin provided information on the binding affinity and on the geometry of the host-guest complex. In order to obtain conclusive information on these interactions, the following physicochemical methods were used in this study: electron paramagnetic resonance, UV-Vis spectroscopy, circular dichroism, along with microcalorimetry and molecular docking. The experimental data showed that trehalose influences the effect of the spin probe binding to albumin on the secondary structure of the protein. The competitive experiment with cyclodextrin indicated its ability to extract the spin probe from the binding pocket of the albumin. The EPR spectroscopy data, together with UV-Vis data, provided insights into how the formation of the inclusion complex occurs, specifically via the aromatic fragment.

In search of better specificity and lower chances of resistance, protease-activatable alkoxyamine prodrugs to fight cancer have been proposed. These molecules are made of a peptide linked to an alkoxyamine. Proteolysis of the peptide converts the stable prodrug at 37 °C to a metastable alkoxyamine that spontaneously homolyzes into two free radicals: a stable nitroxide and a very reactive alkyl radical. The alkyl radical induces apoptosis in the surrounding cells by inducing random chemical alterations. Here, we show that varying the peptide moiety from succinyl-Ala-Ala-Pro-Val- to PyroGlu-Gly-Arg- or PyroGlu-Gly-Lys- is effective in switching the activating enzyme from elastase to urokinase. Furthermore, these prodrugs induce the death of HT-1080 cells, a cell line that secretes several active proteases in culture. This cytotoxic activity can be suppressed by protease inhibitors and does not affect cell lines devoid of active urokinase. We thus provide examples of alkoxyamine prodrugs that are efficiently activated by the limited intrinsic protease activity and that succeed in the destruction of cancer cell lines and cancer cells from tumor explants.

The present study investigated the effects of gamma-ray, electron beam, and X-ray radiation on polymers in EVA/ EVOH/EVA multilayer films. Spectroscopic methods, including FTIR and UV-Vis, were employed in conjunction with chemometric treatments such as PCA, SIMPLISMA treatments, and PLS models. The feasibility of dose prediction on multilayer film, given a known dose, is attainable across various irradiation technologies. Both gamma ray and X-ray exhibit comparable effects on the multilayer film, and it is feasible to anticipate the dose released on the film by utilizing UV-Vis spectra or infrared spectra for gamma ray or X-ray of a predetermined dose.