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.

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 -butyl groups at each of the α-carbon atoms of the nitroxide moiety with -configuration of the -butyl groups was prepared using a three-component domino reaction of -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.

Synthesis and Properties of (1(),5(),7(),8())-1,8-Bis(hydroxymethyl)-6-azadispiro[4.1.4.2]tridecane-6-oxyl: Reduction-Resistant Spin Labels with High Spin Relaxation Times.

Site-directed spin labeling followed by investigation using Electron Paramagnetic Resonance spectroscopy is a rapidly expanding powerful biophysical technique to study structure, local dynamics and functions of biomolecules using pulsed EPR techniques and nitroxides are the most widely used spin labels. Modern trends of this method include measurements directly inside a living cell, as well as measurements without deep freezing (below 70 K), which provide information that is more consistent with the behavior of the molecules under study in natural conditions. Such studies require nitroxides, which are resistant to the action of biogenic reductants and have high spin relaxation (dephasing) times, T.

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 4-imidazole-3-oxide with alkynylmagnesium bromide wereused. The reaction gave corresponding nitroxides with an alkynyl group adjacent to the N-O moiety.

3,4-Unsubstituted 2--Butyl-pyrrolidine-1-oxyls with Hydrophilic Functional Groups in the Side Chains.

Pyrrolidine nitroxides with four bulky alkyl substituents adjacent to N-O group are known for their high resistance to bioreduction. The 3,4-unsubstituted 2--butyl-2-ethylpyrrolidine-1-oxyls were prepared from the corresponding 2--butyl-1-pyrroline-1-oxides via either the addition of ethinylmagnesium bromide with subsequent hydrogenation or via treatment with ethyllithium. The new nitroxides showed excellent stability to reduction with ascorbate with no evidence for additional large hyperfine couplings in the EPR spectra.

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.