2,5-Di-tert-butyl-2,5-diethylpyrrolidine-1-oxyls: Where Is a Reasonable Limit of Sterical Loading for Higher Resistance to Reduction?

The pyrrolidine nitroxides with four bulky alkyl substituents adjacent to the N-O∙ group demonstrate very high resistance to reduction with biogenic antioxidants and enzymatic systems. This makes them valuable molecular tools for studying the structure and functions of biomolecules directly in a living cell and for functional EPR and NMR tomography in vivo. The first example of highly strained pyrrolidine nitroxides with both ethyl and tert-butyl groups at each of the α-carbon atoms of the nitroxide moiety with cis-configuration of the tert-butyl groups was prepared using a three-component domino reaction of tert-leucine and 2,2-dimethylpentan-3-one with dimethyl fumarate with subsequent conversion of the resulting strained pyrrolidine into 1-pyrroline-1-oxide and addition of EtLi. The nitroxide has demonstrated unexpectedly fast reduction with ascorbate, the rate constant k2 = (2.0 ± 0.1) × 10-3 M-1s-1. This effect was explained by destabilization of the planar nitroxide moiety due to repulsion with the two neighboring tert-butyl groups cis to each other.

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.

Origin of Long-Range Hyperfine Couplings in the EPR Spectra of 2,2,5,5-Tetraethylpyrrolidine-1-oxyls.

Cyclic nitroxides with several bulky alkyl substituents adjacent to the nitroxide group are known to demonstrate a much higher stability to bioreduction than their tetramethyl analogues. Among these so-called "sterically shielded" nitroxides, the pyrrolidine derivatives are the most stable. The EPR spectra of some sterically shielded pyrrolidine-1-oxyls were reported to show one or two large additional doublet splittings with a hyperfine coupling (hfc) constant (ca. 0.2-0.4 mT). To determine the origin of these hfc, a series of 2-R-2,5,5-triethyl-3,4-bis(hydroxymethyl)-pyrrolidine-1-oxyls with methylene groups stereospecifically enriched with deuterium were prepared, and their CW EPR spectra were studied. In addition, these nitroxides were investigated using quantum chemical calculations on the UB3LYP/def2-TZVP level and NBO analysis. The apparent constants were assigned to hfc with γ-hydrogen in the side chain, with the contribution of the NBO orbital ßπ*(N-O) to the natural localized molecular orbital ßσ(C-H) playing the major role. This interaction is efficient if the ethyl substituent is in the pseudoaxial position of the ring and the CH2-CH3 bond is codirected with (parallel to) N-O. The apparent constant aH increases with the Boltzmann population of this conformation.

An EPR Study on Highly Stable Nitroxyl-Nitroxyl Biradicals for Dynamic Nuclear Polarization Applications at High Magnetic Fields.

Nitroxide biradicals are efficient polarizing agents in dynamic nuclear polarization (DNP) solid-state nuclear magnetic resonance. Many recently reported radicals possess substantial DNP efficiency in organic solvents but have poor solubility in water media which is unfavorable for biological applications. In this paper, we report DNP efficiency at a high magnetic field for two water-soluble biradicals resistant to reducing media. Water solubility was achieved by obtaining the radicals in the form of quaternary ammonium salts. Parameters of hyperfine interaction and exchange interaction were quantified by EPR spectroscopy, and their influence on the DNP effect was determined. The resistance of the biradicals to strongly reducing media was characterized. High stability was achieved using tetraethyl substituents and pyrrolidine moieties.

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.

Stability of ZIF-8 Nanoparticles in Most Common Cell Culture Media.

Zeolite imidazolate framework-8 (ZIF-8) is a promising platform for drug delivery, and information regarding the stability of ZIF-8 nanoparticles in cell culture media is essential for proper interpretation of in vitro experimental results. In this work, we report a quantitative investigation of the ZIF-8 nanoparticle's stability in most common cell culture media. To this purpose, ZIF-8 nanoparticles containing sterically shielded nitroxide probes with high resistance to reduction were synthesized and studied using electron paramagnetic resonance (EPR). The degradation of ZIF-8 in cell media was monitored by tracking the cargo leakage. It was shown that nanoparticles degrade at least partially in all studied media, although the degree of cargo leakage varies widely. We found a strong correlation between the amount of escaped cargo and total concentration of amino acids in the environment. We also established the role of individual amino acids in ZIF-8 degradation. Finally, 2-methylimidazole preliminary dissolved in cell culture media partially inhibits the degradation of ZIF-8 nanoparticles. The guidelines for choosing the proper cell culture medium for the in vitro study of ZIF-8 nanoparticles have been formulated.

A Simple Method of Synthesis of 3-Carboxy-2,2,5,5-Tetraethylpyrrolidine-1-oxyl and Preparation of Reduction-Resistant Spin Labels and Probes of Pyrrolidine Series.

Stable free radicals are widely used as molecular probes and labels in various biophysical and biomedical research applications of magnetic resonance spectroscopy and imaging. Among these radicals, sterically shielded nitroxides of pyrrolidine series demonstrate the highest stability in biological systems. Here, we suggest new convenient procedure for preparation of 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl, a reduction-resistant analog of widely used carboxy-Proxyl, from cheap commercially available reagents with the yield exceeding the most optimistic literature data. Several new spin labels and probes of 2,2,5,5-tetraethylpyrrolidine-1-oxyl series were prepared and reduction of these radicals in ascorbate solutions, mice blood and tissue homogenates was studied.

The effects of nitroxide structure upon 1H Overhauser dynamic nuclear polarization efficacy at ultralow-field.

The efficacy in 1H Overhauser dynamic nuclear polarization in liquids at ultralow magnetic field (ULF, B0 = 92 ± 0.8 µT) and polarization field (Bp = 1-10 mT) was studied for a broad variety of 26 different spin probes. Among others, piperidine, pyrrolidine, and pyrroline radicals specifically synthesized for this study, along with some well-established commercially available nitroxides, were investigated. Isotope-substituted variants, some sterically shielded reduction-resistant nitroxides, and some biradicals were included in the measurements. The maximal achievable enhancement, Emax, and the radio frequency power, P1/2, needed for reaching Emax/2 were measured. Physico-chemical features such as molecular weight, spectral linewidth, heterocyclic structure, different types of substituents, deuteration, and 15N-labeling as well as the difference between monoradicals and biradicals were investigated. For the unmodified nitroxide radicals, the Emax values correlate with the molecular weight. The P1/2 values correlate with the spectral linewidth and are additionally influenced by the type of substituents neighboring the nitroxide group. The nitroxide biradicals with high intramolecular spin-spin coupling show low performance. Nitroxides enriched with 15N and/or 2H afford significantly higher |Emax| and require lower power to do so, compared to their unmodified counterparts containing at natural abundance predominantly 14N and 1H. The results allow for a correlation of chemical features with physical hyperpolarization-related properties and indicate that small nitroxides with narrow spectral lines have clear advantages for the use in Overhauser dynamic nuclear polarization experiments. Perdeuteration and 15N-labeling can be used to additionally boost the spin probe performance.

The Reactions of 6-(Hydroxymethyl)-2,2-dimethyl-1-azaspiro[4.4]nonanes with Methanesulfonyl Chloride or PPh3-CBr4.

Activation of a hydroxyl group towards nucleophilic substitution via reaction with methanesulfonyl chloride or PPh3-CBr4 system is a commonly used pathway to various functional derivatives. The reactions of (5R(S),6R(S))-1-X-6-(hydroxymethyl)-2,2-dimethyl- 1-azaspiro[4.4]nonanes 1a-d (X = O·; H; OBn, OBz) with MsCl/NR3 or PPh3-CBr4 were studied. Depending on substituent X, the reaction afforded hexahydro-1H,6H-cyclopenta[c]pyrrolo[1,2-b]isoxazole (2) (for X = O), a mixture of 2 and octahydrocyclopenta[c]azepines (4-6) (for X = OBn, OBz), or perhydro-cyclopenta[2,3]azeto[1,2-a]pyrrol (3) (for X = H) derivatives. Alkylation of the latter with MeI with subsequent Hofmann elimination afforded 2,3,3-trimethyl-1,2,3,4,5,7,8,8a-octahydrocyclopenta[c]azepine with 56% yield.

Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy.

RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3-16 µM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.

Unexpected one-pot formation of the 1H-6a,8a-epiminotricyclopenta[a,c,e][8]annulene system from cyclopentanone, ammonia and dimethyl fumarate. Synthesis of highly strained polycyclic nitroxide and EPR study.

The unexpected formation of a highly strained polycyclic amine was observed in a one-pot synthesis from cyclopentanone, dimethyl fumarate and ammonium acetate. This multistep reaction includes 1,3-dipolar cycloaddition of dimethyl fumarate to the cyclic azomethine ylide formed in situ from cyclopentanone and ammonia. The polycyclic amine product was easily converted into a sterically shielded polycyclic nitroxide. The EPR spectra and spin relaxation behavior of the nitroxide were studied in solution. The spin relaxation seems well suited for the use as a biological spin label and are comparable with those of cyclic nitroxides with two spirocyclic moieties adjacent to the N-O · group.

Synthesis of 3,4-Bis(hydroxymethyl)-2,2,5,5-tetraethylpyrrolidin-1-oxyl via 1,3-Dipolar Cycloaddition of Azomethine Ylide to Activated Alkene.

A simple method for the synthesis of sterically shielded pyrrolidine nitroxides, including the title compound, has been suggested. The key procedure implies assembling the pyrrolidine ring from α-amino acid, ketone, and activated alkene in a three-component domino process, followed by oxidation to nitrone and Grignard reagent addition. The new nitroxides demonstrate very high stability against reduction with ascorbate.