Hydrophilic Reduction-Resistant Spin Labels of Pyrrolidine and Pyrroline Series from 3,4-Bis-hydroxymethyl-2,2,5,5-tetraethylpyrrolidine-1-oxyl.

Highly resistant to reduction nitroxides open new opportunities for structural studies of biological macromolecules in their native environment inside living cells and for functional imaging of pH and thiols, enzymatic activity and redox status in living animals. 3,4-Disubstituted nitroxides of 2,2,5,5-tetraethylpyrrolidine and pyrroline series with a functional group for binding to biomolecules and a polar moiety for higher solubility in water and for more rigid attachment via additional coordination to polar sites were designed and synthesized. The EPR spectra, lipophilicities, kinetics of the reduction in ascorbate-containing systems and the decay rates in liver homogenates were measured. The EPR spectra of all 3,4-disubstituted pyrrolidine nitroxides showed additional large splitting on methylene hydrogens of the ethyl groups, while the spectra of similar pyrroline nitroxides were represented with a simple triplet with narrow lines and hyperfine structure of the nitrogen manifolds resolved in oxygen-free conditions. Both pyrrolidine and pyrroline nitroxides demonstrated low rates of reduction with ascorbate, pyrrolidines being a bit more stable than similar pyrrolines. The decay of positively charged nitroxides in the rat liver homogenate was faster than that of neutral and negatively charged radicals, with lipophilicity, rate of reduction with ascorbate and the ring type playing minor role. The EPR spectra of N,N-dimethyl-3,4-bis-(aminomethyl)-2,2,5,5-tetraethylpyrrolidine-1-oxyl showed dependence on pH with pKa = 3, ΔaN = 0.055 mT and ΔaH = 0.075 mT.

Enhancement of the production of TEMPO-mediated oxidation cellulose nanofibrils by kneading.

The industrial use of TEMPO-mediated oxidation (TMO) reaction to produce highly fibrillated cellulose nanofibrils has been hindered by high catalyst costs, long reaction times and high reaction volumes. The hypothesis that cellulose concentration during TMO process is key to increase the process of efficiency has been confirmed. The novelty of this research is the proof-of-concept for a significant enhancement of the TMO reaction by kneading the cellulose to work in concentrations above 120 g/L. Results show that the increase of the cellulose concentration in the TMO reaction, from the traditional 10 g/L to 120 g/L, increase not only the production for the same reaction volume (1200 %) but also the pulp recovery (up to 94 %). Moreover, the oxidation time can be reduced from 42 min to only 4 min while properties of both the oxidized pulps and the final nanocellulose are similar. On the other hand, the use of buffers in the TMO reaction allows us to keep the pH constant without using NaOH, and to improve the selectivity of the carboxyl groups production. The proposed process also minimizes the final environmental impact.

DNA-Compatible Copper/TEMPO Oxidation for DNA-Encoded Libraries.

Aldehydes are important synthons for DNA-encoded library (DEL) construction, but the development of a DNA-compatible method for the oxidation of alcohols to aldehydes remains a significant challenge in the field of DEL chemistry. We report that a copper/TEMPO catalyst system enables the solution-phase DNA-compatible oxidation of DNA-linked primary activated alcohols to aldehydes. The semiaqueous, room-temperature reaction conditions afford oxidation of benzylic, heterobenzylic, and allylic alcohols in high yield, with DNA compatibility verified by mass spectrometry, qPCR, Sanger sequencing, and ligation assays. Subsequent transformations of the resulting aldehydes demonstrate the potential of this method for robust library diversification.

Formation of free radicals in Chi-aroma Baijiu during aging process with fat pork.

Soaking aged fat pork is a special aging process in the production of Chi-aroma Baijiu considered to involve the formation of free radicals. This study aimed to investigate the free radicals' formation pathway in Chi-aroma Baijiu during aged fat pork soaking by using electron paramagnetic resonance (EPR) and spin trapping with 5,5-dimethyl-1-pyrrolin-n-oxide (DMPO). The alkyl radical adducts (DMPO-R) and hydroxyl radical adducts (DMPO-OH) were detected in Baijiu after soaking the fat pork for aging. During the preparation process of aged fat pork, alkoxy radicals adduct (DMPO-RO) were mainly detected since lipid oxidation. Oleic acid and linoleic acid, the two main unsaturated fatty acids in fat pork, produced alkoxy radicals in the oxidation process. The total amounts of spins in linoleic acid and oleic acid after 4-month oxidation treatment increased by 248.07 ± 26.65% and 34.17 ± 0.72% than 0-month. It indicated that the free radicals in aged Chi-aroma Baijiu were mainly derived from the two main unsaturated fatty acids in aged fat pork and linoleic acid had a stronger ability to produce free radicals than oleic acid. Alkoxy radicals (RO·) from fat pork reacted with ethanol in Baijiu to form alkyl radicals (R·). The peroxide bond of hydroperoxides from the oxidation of unsaturated fatty acid was cleaved to form hydroxyl radicals (·OH) that were transferred to Baijiu. The results provide theoretical guidance for the subsequent work of free radicals scavenging.

Determination of antibiotics by amperometry using nortropine N-oxyl, a highly active nitroxyl radical.

Nitroxyl radical compounds oxidize hydroxy groups and some amino groups upon application of an electric potential. The resulting anodic current depends on the concentration of these functional groups in solution. Thus, it is possible to quantify compounds containing these functional groups by electrochemical methods. Cyclic voltammetry has been used to evaluate the catalytic activity of nitroxyl radicals, and the ability of such radicals to sense biological and other compounds. In this study, we evaluated a method for quantifying compounds using constant-potential electrolysis (amperometry) of nitroxyl radicals for application in flow injection analysis and high-performance liquid chromatography as an electrochemical detector. When amperometry was performed using 2,2,6,6-tetramethylpiperidine 1-oxyl, a common nitroxyl radical compound, little change was observed even with 100 mM glucose due to its low reactivity in neutral aqueous solutions. In contrast, 2-azaadamantane N-oxyl and nortropine N-oxyl, which are highly active nitroxyl radicals, showed a concentration-dependent response in neutral aqueous solution. Responses of 33.8 and 125.9 µA, respectively, were observed. By recognition of hydroxy and amino groups, we have succeeded in the electrochemical detection of some drugs by amperometry. Streptomycin, an aminoglycoside antibiotic, was quantifiable in the range of 30-1000 µM.

One-pot and one-step preparation of "living" cellulose nanofiber hydrogel with active double-bond via chemical vapor deposition.

Due to the existence of water, it is still a challenge to conduct chemical modification on cellulose nanofiber (CNF) hydrogels with active double bonds. A simple one-pot and one-step method for constructing "living" CNF hydrogel with double bond was created at room temperature. The chemical vapor deposition (CVD) of methacryloyl chloride (MACl) was used to introduce physical-trapped, chemical-anchored and functional double bonds into TEMPO-oxidized cellulose nanofiber (TOCN) hydrogels. TOCN hydrogel could be fabricated within just 0.5 h, the minimum dosage of MACl could be reduced to 3.22 mg/g (MACl/TOCN hydrogel). Furthermore, the CVD methods showed high efficiency for mass production and recyclability. Moreover, the chemical "living" reactivity of the introduced double bonds were verified by the freezing and UV crosslinking, radical polymerization and thiol-ene click reaction. Compared with pure TOCN hydrogel, the obtained functionalized TOCN hydrogel exhibited remarkable improvements in mechanical properties, with enhancements of 12.34 times and 2.04 times, as well as an increase in hydrophobicity by 2.14 times and a fluorescence performance improvement of 2.93 times.

Six weeks effect of different nanocellulose on blood lipid level and small intestinal morphology in mice.

AIMS: Cellulose nanofibrils (CNF, or NFC), cellulose nanocrystals (CNC, or NCC), and Tempo (2,2,6,6-tetramethylpiperidine-1-oxyl radical) oxidized CNF (Tempo-CNF) were compared for the short-term effect on mice fed with a high-fat and high-sugar (Western diet, WD) to investigate their effect when combined with a sub-optimal diet. SCOPE: Thirty C57B/C female mice (10 weeks old; 5-6 mice/group) were given water, cellulose, or three types of nanocellulose once daily in a dose of 30 mg/kg body weight by oral gavage. After six weeks, weight changes, fecal output, glucose homeostasis, and gut permeability showed no significant among groups. Serum analysis including triglycerides, cholesterol and total bile acids and small intestinal morphology including villus length, villus width, crypt depth, goblet cell count and goblet cell density were no difference for all groups. Only CNC group had higher excretion of bile acids in the feces. CONCLUSIONS: These results suggest that current treated dose using three types of nanocellulose had no detrimental effects on blood lipid level and small intestinal morphology.

Tuning of water resistance and protein adsorption capacity of porous cellulose nanofiber particles prepared by spray drying with cross-linking reaction.

Porous particles composed of 2,2,6,6-tetramethylpiperidinyl-1-oxyl-oxidized cellulose nanofiber (TOCN) as building block, i.e., porous TOCN particles, are attracting attention due to their environmental friendliness, superior properties, such as easy handling, large surface area, and high adsorption capacity. However, the instability of TOCNs in aqueous environments limits their applications. An effective solution to improve water resistance of TOCN particles is to reduce the hydrophilicity of TOCNs by forming chemical bonds with a cross-linker. In this study, Carbodilite, a common, easy-to-use, commercially available cross-linker with carbodiimide groups, was used to investigate a chemical cross-linking strategy for porous TOCN particles prepared by spray drying. The water resistance of cross-linked TOCN particles was evaluated through morphological observation by SEM images. The presence of polycarbodiimide significantly increased water resistance of cross-linked TOCN particles up to 24 h. This study demonstrates the trade-off between water resistance and adsorption efficiency according to cross-linker concentrations. These data are useful for interface science of TOCNs in liquids, assisting in controlling specific properties of porous TOCN particles for particular applications in adsorption and separation.

Non-thermal plasma elicits ferrous chloride-catalyzed DMPO-OH.

Non-thermal plasma (NTP) induces the generation of reactive oxygen species (ROS) and reactive nitrogen species, such as hydroxyl radicals (•OH), hydrogen peroxide (H2O2), singlet oxygen, superoxide, ozone, and nitric oxide, at near-physiological temperatures. These molecules promote blood coagulation, wound healing, disinfection, and selective cancer cell death. Based on these evidences, clinical trials of NTP have been conducted for treating chronic wounds and head and neck cancers. Although clinical applications have progressed, the stoichiometric quantification of NTP-induced ROS remains unclear in the liquid phase in the presence of FeCl2 or FeCl3 in combination with biocompatible reducing agents, which may modulate the final biological effects of NTP. In this study, we employed electron paramagnetic resonance spectroscopy to quantify ROS using spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and H2O2, using luminescent probe in the presence of FeCl2 or FeCl3. NTP-induced DMPO-OH levels were elevated 10-100 µM FeCl2 or 500 and 1000 µM FeCl3. NTP-induced DMPO-OH with 10 µM FeCl2 or FeCl3 was significantly scavenged by ascorbate, α-tocopherol, dithiothreitol, reduced glutathione, or oxidized glutathione, whereas dehydroascorbate was ineffective in 2 mM DMPO. NTP-induced H2O2 was significantly degraded by 100 µM FeCl2 and FeCl3 in an iron-dependent manner. Meanwhile, decomposition of H2O2 by catalase decayed DMPO-OH efficiently in the presence of iron, indicating iron causes DMPO-OH production and degradation simultaneously. These results suggest that NTP-induced DMPO-OH is generated by the H2O2-consuming, iron-dependent Fenton reaction and ferryl intermediates. The potential iron-mediated ROS production by NTP is also discussed to clarify the interaction between NTP-induced ROS and biomolecules.

Synthesis of Sterically Shielded Nitroxides Using the Reaction of Nitrones with Alkynylmagnesium Bromides.

Sterically shielded nitroxides, which demonstrate high resistance to bioreduction, are the spin labels of choice for structural studies inside living cells using pulsed EPR and functional MRI and EPRI in vivo. To prepare new sterically shielded nitroxides, a reaction of cyclic nitrones, including various 1-pyrroline-1-oxides, 2,5-dihydroimidazole-3-oxide and 4H-imidazole-3-oxide with alkynylmagnesium bromide wereused. The reaction gave corresponding nitroxides with an alkynyl group adjacent to the N-O moiety. The hydrogenation of resulting 2-ethynyl-substituted nitroxides with subsequent re-oxidation of the N-OH group produced the corresponding sterically shielded tetraalkylnitroxides of pyrrolidine, imidazolidine and 2,5-dihydroimidazole series. EPR studies revealed large additional couplings up to 4 G in the spectra of pyrrolidine and imidazolidine nitroxides with substituents in 3- and/or 4-positions of the ring.

Real-Time Detection of Hydroxyl Radical Generated at Operating Electrodes via Redox-Active Adduct Formation Using Scanning Electrochemical Microscopy.

The hydroxyl radical (•OH) is one of the most attractive reactive oxygen species due to its high oxidation power and its clean (photo)(electro)generation from water, leaving no residues and creating new prospects for efficient wastewater treatment and electrosynthesis. Unfortunately, in situ detection of •OH is challenging due to its short lifetime (few ns). Using lifetime-extending spin traps, such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to generate the [DMPO-OH]• adduct in combination with electron spin resonance (ESR), allows unambiguous determination of its presence in solution. However, this method is cumbersome and lacks the necessary sensitivity and versatility to explore and quantify •OH generation dynamics at electrode surfaces in real time. Here, we identify that [DMPO-OH]• is redox-active with E0 = 0.85 V vs Ag|AgCl and can be conveniently detected on Au and C ultramicroelectrodes. Using scanning electrochemical microscopy (SECM), a four-electrode technique capable of collecting the freshly generated [DMPO-OH]• from near the electrode surface, we detected its generation in real time from operating electrodes. We also generated images of [DMPO-OH]• production and estimated and compared its generation efficiency at various electrodes (boron-doped diamond, tin oxide, titanium foil, glassy carbon, platinum, and lead oxide). Density functional calculations, ESR measurements, and bulk calibration using the Fenton reaction helped us unambiguously identify [DMPO-OH]• as the source of redox activity. We hope these findings will encourage the rapid, inexpensive, and quantitative detection of •OH for conducting informed explorations of its role in mediated oxidation processes at electrode surfaces for energy, environmental, and synthetic applications.

The regularity of heat-induced free radicals generation and transition of camellia oil.

In this study, the radicals formed in camellia oil upon a heating process were identified and quantified using Electron Spin Resonance (ESR) spectroscopy coupled with spin-trapping technique. PBN and DMPO were served as spin traps. The total amounts of free radicals of heated camellia oil showed an increasing trend with the extension of heating time at 140 °C, 150 °C and 160 °C. In accordance with hyperfine splitting constants (aN, aH) â”€ the crucial parameter for identifying free radical species â”€ of free radical, it was definitely confirmed that alkyl, alkoxyl, oxygen-centered and DMPO oxidate free radicals were present in heated camellia oil. A free radical transition pathway was proposed that alkyl free radical is initially generated, then, alkyl peroxy free radical is subsequently generated in the presence of oxygen which eventually shifts into alkoxyl free radical by means of an intermediate formed from H-capture reaction of alkyl peroxy free radical.

Formation of a Specific DMPO-OH Adduct Signal Related to Hydroxy Radical Generation in a Long Term Stored Improved Iodide Formulation.

A specific DMPO-OH adduct signal (1:2:2:1)related to hydroxyl radical generation in a longterm stored improved iodide formulation, tentatively designated as the distilled KMT reagent which prepared from a pH 0.3 solution containing FeCl3, EDTA, KI and ethanol termed the KMT reagent, was detected by electron spin resonance (ESR) spectroscopy.Although the color intensities of N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) and N,N-diethyl-p-phenylenediamine (DPD) differ, the mixture of the long-term stored distilled KMT reagent and TMPD exhibited a purple color similar to Wurster's blue, and the mixture of the long-term stored distilled KMT reagent and DPD exhibited a pink color similar to Wurster's red. There is a possibility that the long-term stored distilled KMT reagent may possess with the ability to generate a hydroxyl radical.

Recyclable nanocomposites of well-dispersed 2D layered silicates in cellulose nanofibril (CNF) matrix.

Nanocomposites based on components from nature, which can be recycled are of great interest in new materials for sustainable development. The range of properties of nacre-inspired hybrids of 1D cellulose and 2D clay platelets are investigated in nanocomposites with improved nanoparticle dispersion in the starting hydrocolloid mixture. Films with a wide range of compositions are prepared by capillary force assisted physical assembly (vacuum-assisted filtration) of TEMPO-oxidized cellulose nanofibers (TOCN) reinforced by exfoliated nanoclays of three different aspect ratios: saponite, montmorillonite and mica. X-ray diffraction and transmission electron micrographs show almost monolayer dispersion of saponite and montmorillonite and high orientation parallel to the film surface. Films exhibit ultimate strength up to 573 MPa. Young's modulus exceeds 38 GPa even at high MTM contents (40-80 vol%). Optical transmittance, UV-shielding, thermal shielding and fire-retardant properties are measured, found to be very good and are sensitive to the 2D nanoplatelet dispersion.

TEMPO-Assisted Free-Radical-Initiated Peptide Sequencing Mass Spectrometry for Ubiquitin Ions: An Insight on the Gas-Phase Conformations.

TEMPO ((2,2,6,6-tetramethylpiperidine-1-yl)oxyl)-assisted free-radical-initiated peptide sequencing mass spectrometry (FRIPS MS) is applied to the top-down tandem mass spectrometry of guanidinated ubiquitin (UB(Gu)) ions, i.e., p-TEMPO-Bn-Sc-guanidinated ubiquitin (UBT(Gu)), to shed a light on gas-phase ubiquitin conformations. Thermal activation of UBT(Gu) ions produced protein backbone fragments of radical character, i.e., a-/x- and c-/z-type fragments. It is in contrast to the collision-induced dissociation (CID) results for UB(Gu), which dominantly showed the specific charge-remote CID fragments of b-/y-type at the C-terminal side of glutamic acid (E) and aspartic acid (D). The transfer of a radical "through space" was mainly observed for the +5 and +6 UBT(Gu) ions. This provides the information about folding/unfolding and structural proximity between the positions of the incipient benzyl radical site and fragmented sites. The analysis of FRIPS MS results for the +5 charge state ubiquitin ions shows that the +5 charge state ubiquitin ions bear a conformational resemblance to the native ubiquitin (X-ray crystallography structure), particularly in the central sequence region, whereas some deviations were observed in the unstable second structure region (ß2) close to the N-terminus. The ion mobility spectrometry results also corroborate the FRIPS MS results in terms of their conformations (or structures). The experimental results obtained in this study clearly demonstrate a potential of the TEMPO-assisted FRIPS MS as one of the methods for the elucidation of the overall gas-phase protein structures.

Mycobacterium abscessus cell wall and plasma membrane characterization by EPR spectroscopy and effects of amphotericin B, miltefosine and nerolidol.

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to characterize the components of the Mycobacterium abscessus massiliense cell envelope and their interactions with amphotericin B (AmB), miltefosine (MIL), and nerolidol (NER). Spin labels analogous to stearic acid and phosphatidylcholine (PC) were distributed on an envelope layer with fluidity comparable to other biological membranes, probably the mycobacterial cell wall, because after treatment with AmB a highly rigid spectral component was evident in the EPR spectra. Methyl stearate analogue spin labels found a much more fluid membrane and did not detect the presence of AmB, except for at very high drug concentrations. Unlike other spin-labeled PCs, the TEMPO-PC spin probe, with the nitroxide moiety attached to the choline of the PC headgroup, also did not detect the presence of AmB. On the other hand, the steroid spin labels were not distributed across the membranes of M. abscessus and, instead, were concentrated in some other location of the cell envelope. Both MIL and NER compounds at 10 µM caused increased fluidity in the cell wall and plasma membrane. Furthermore, NER was shown to have a remarkable ability to extract lipids from the mycobacterial cell wall. The EPR results suggest that the resistance of mycobacteria to the action of AmB must be related to the fact that this drug does not reach the bacterial plasma membrane.

Electrocatalytic oxidative upgrading of biomass platform chemicals: from the aspect of reaction mechanism.

Oxidation reactions provide a wide range of important chemicals in industry; however, most of these chemicals are produced from fossil feedstocks. As a candidate of oxygen evolution reaction (OER), the electrooxidation of biomass platform chemicals instead of a petroleum source offers a sustainable and atom-economic avenue toward organic oxygenates, with additional benefits when coupled with renewable electricity driven processes. This highlight article describes the representative examples in this nascent area, including oxidative dehydrogenation, coupling, and cleavage. We classify the examples into inner-sphere and outer-sphere electrode reactions based on the classical electrocatalysis concept for better understanding of the reaction mechanism. Moreover, we highlight the recent progress in oxidative biomass electrorefining via inner-sphere anodic reactions, which are strongly dependent on the nature of the electrode material. Particularly, the understanding of the formation of reactive oxygen species, adsorption of substrates, and reconstruction of anode materials is presented. Finally, the existing challenges and perspectives are discussed.

Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates.

The intracellular concentration of reduced glutathione (GSH) lies in the range of 1-10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).

Improvement of O/W emulsion performance by adjusting the interaction between gelatin and bacterial cellulose nanofibrils.

This study was designed to improve the stability of medium internal phase emulsion by adjusting the electrostatic interaction between gelatin (GLT) and TEMPO-oxidized bacterial cellulose nanofibrils (TOBC). The influences of polysaccharide-protein ratio (1:10, 1:5, and 1:2.5) and pH (3.0, 4.7, 7.0, and 11.0) on the emulsion properties were investigated. The droplet size of TOBC/GLT-stabilized emulsion was increased with the TOBC proportion increasing at pH 3.0-11.0. Additionally, emulsion had a larger droplet size at pH 4.7 (the electrical equivalence point pH of mixtures). However, the addition of TOBC significantly improved the emulsion stability. The emulsions prepared with TOBC/GLT mixtures (mixing ratio of 1:2.5) at pH 3.0-7.0 were stable without creaming during the storage. It was because the formation of nanofibrils network impeded the droplet mobility, and the emulsion viscosity and viscoelastic modulus were increased with the addition of TOBC. These findings were meaningful to modulate the physical properties of emulsions.

DNA-Scaffolded Synergistic Catalysis.

We report DNA-scaffolded synergistic catalysis, a concept that combines the diverse reaction scope of synergistic catalysis with the ability of DNA to precisely preorganize abiotic groups and undergo stimuli-triggered conformational changes. As an initial demonstration of this concept, we focus on Cu-TEMPO-catalyzed aerobic alcohol oxidation, using DNA as a scaffold to hold a copper cocatalyst and an organic radical cocatalyst (TEMPO) in proximity. The DNA-scaffolded catalyst maintained a high turnover number upon dilution and exhibited 190-fold improvement in catalyst turnover number relative to the unscaffolded cocatalysts. By incorporating the cocatalysts into a DNA hairpin-containing scaffold, we demonstrate that the rate of the synergistic catalytic reaction can be controlled through a reversible DNA conformational change that alters the distance between the cocatalysts. This work demonstrates the compatibility of synergistic catalytic reactions with DNA scaffolding, opening future avenues in reaction discovery, sensing, responsive materials, and chemical biology.