2-Butyl-2-tert-butyl-5,5-diethylpyrrolidine-1-oxyls: Synthesis and Properties

Abstract

Nitroxides are broadly used as molecular probes and labels in biophysics, structural biology, and biomedical research. Resistance of a nitroxide group bearing an unpaired electron to chemical reduction with low-molecular-weight antioxidants and enzymatic systems is of critical importance for these applications. The redox properties of nitroxides are known to depend on the ring size (for cyclic nitroxides) and electronic and steric effects of the substituents. Here, two highly strained nitroxides, 5-(tert-butyl)-5-butyl-2,2-diethyl-3-hydroxypyrrolidin-1-oxyl (4) and 2-(tert-butyl)-2-butyl-5,5-diethyl-3,4-bis(hydroxymethyl)pyrrolidin-1-oxyl (5), were prepared via a reaction of the corresponding 2-tert-butyl-1-pyrroline 1-oxides with butyllithium. Thermal stability and kinetics of reduction of the new nitroxides by ascorbic acid were studied. Nitroxide 5 showed the highest resistance to reduction.

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

“…The EPR spectra of all the nitroxides are represented by broadened triplet of doublets with additional large hyperfine splitting constant (hfc) of 0.26–0.3 mT on one of methylene hydrogens ( Figure 1 , cf. [ 14 , 15 , 44 , 45 ]).…”

“…The EPR spectra of all the nitroxides are represented by broadened triplet of doublets with additional large hyperfine splitting constant (hfc) of 0.26-0.3 mT on one of methylene hydrogens (Figure 1, cf. [14,15,44,45]). Moderately basic amino groups can also provide higher retention/intracellular accumulation due to transmembrane potential or ion-trapping effect [42].…”

“…The EPR spectra of all the nitroxides are represented by broadened triplet of doublets with additional large hyperfine splitting constant (hfc) of 0.26-0.3 mT on one of methylene hydrogens (Figure 1, cf. [14,15,44,45]). Partition coefficients of the spin probes, defined as a ratio of concentrations in octanol to water Kp, vary from 5 to 70, indicating that the nitroxides may be capable of permeating cells.…”

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

“…The advantage of these reduction-resistant radicals over conventional tetramethyl-substituted nitroxides is especially obvious when they are used for EPR measurements inside living cells [ 7 , 8 ], or in vivo for functional imaging using MRI or EPRI techniques [ 9 , 10 ]. Pyrrolidine nitroxides with four ethyl groups (or three ethyl and one tert -butyl group) in the nearest environment of the nitroxide moiety demonstrated the highest resistance to reduction [ 4 , 5 , 11 , 12 , 13 , 14 ]. However, nitroxides with multiple bulky alkyl substituents are lipophilic or even insoluble in water, therefore their use in biophysics and structural biology is complicated [ 14 ].…”

“…Pyrrolidine nitroxides with four ethyl groups (or three ethyl and one tert -butyl group) in the nearest environment of the nitroxide moiety demonstrated the highest resistance to reduction [ 4 , 5 , 11 , 12 , 13 , 14 ]. However, nitroxides with multiple bulky alkyl substituents are lipophilic or even insoluble in water, therefore their use in biophysics and structural biology is complicated [ 14 ]. Moreover, the EPR spectra of these nitroxides may reveal large additional hyperfine couplings (ca.…”