Sterically shielded spin labels for in-cell EPR spectroscopy: Analysis of stability in reducing environment

Abstract

Electron paramagnetic resonance (EPR) spectroscopy is a powerful and widely used technique for studying structure and dynamics of biomolecules under bio-orthogonal conditions. In-cell EPR is an emerging area in this field; however, it is hampered by the reducing environment present in cells, which reduces most nitroxide spin labels to their corresponding diamagnetic N-hydroxyl derivatives. To determine which radicals are best suited for in-cell EPR studies, we systematically studied the effects of substitution on radical stability using five different classes of radicals, specifically piperidine-, imidazolidine-, pyrrolidine-, and isoindoline-based nitroxides as well as the Finland trityl radical. Thermodynamic parameters of nitroxide reduction were determined by cyclic voltammetry; the rate of reduction in the presence of ascorbate, cellular extracts, and after injection into oocytes was measured by continuous-wave EPR spectroscopy. Our study revealed that tetraethyl-substituted nitroxides are good candidates for in-cell EPR studies, in particular pyrrolidine derivatives, which are slightly more stable than the trityl radical.

Reduction kinetics and electrochemistry of tetracarboxylate nitroxides

“…5-Membered ring nitroxides with gem -diethyl substituent groups, such as pyrrolidine nitroxide 1 (Figure 1), were found to undergo reduction with ascorbate at rates that are at least one order of magnitude slower than the corresponding gem -dimethyl nitroxides such as 2 [9,14]; similar findings were reported for 1 in cell extracts and in live oocyte cells [11]. Notably, gem -diethyl pyrroline nitroxides, such as 3 , are reduced by ascorbate at only slightly faster (up to 20%) rates than 1 [10].…”

“…In the EPR kinetic studies, rates of reduction for pyrroline nitroxides 4 – 8 are determined under pseudo-first-order conditions using a 10-20 fold excess of ascorbate in pH 7.4 PBS buffer at 295 K. For comparison, the rates of reduction of for nitroxides 1 – 3 are measured under similar conditions [9-11,14]. Second-order rate constants, k , are obtained by monitoring the decay of the low-field EPR peak height of nitroxides at 295 K (Figure 3, top panels and Tables 2 and 3).…”

Synthesis, Characterization, and Nanoencapsulation of Tetrathiatriarylmethyl and Tetrachlorotriarylmethyl (Trityl) Radical Derivatives—A Study To Advance Their Applicability as in Vivo EPR Oxygen Sensors

“…Tris (4-ethoxy-carbonyl-2,3,5,6-tetrachlorophenyl)methyl radical (18). 29 Tris (4-tert-butoxycarbonyl-2,3,5,6-tetrachlorophenyl)methyl radical (19). 44 The procedure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 37 1687, 1573, 1457, 1394, 1370, 1334, 1288, 1240, 1159, …”

“…12,13 These aspects in mind, we intend to shed more light on EPR oximetry using soluble spin probes, in particular trityl radicals. Their main limitations might be overcome by appropriate formulations: By encapsulation, their oxygen sensitivities can be improved, 14-17 a 5 defined microenvironment is created, which ensures specificity of the sensors to oxygen, and the capsule shell might provide protection against oxidoreductants, [17][18][19] and prevent biocompatibility concerns.…”

Cellular accumulation and antioxidant activity of acetoxymethoxycarbonyl pyrrolidine nitroxides

“…The main challenges in biological applications of nitroxides are related to their fast bioreduction to electron paramagnetic resonance (EPR)-silent hydroxylamines (>N-OH) and rapid excretion via kidney or via hepatobiliary system [3]. Recently, much attention has been drawn to sterically shielded nitroxides which demonstrate very high stability in model systems and in living cells [4]. Broad application of these nitroxides is, however, complicated due to complex multistep synthesis [5–7].…”