Tuning the properties of nitroxide spin labels for use in electron paramagnetic resonance spectroscopy through chemical modification of the nitroxide framework

Marius M. Haugland, Edward A. Anderson and Janet E. Lovett

DOI: 10.1039/9781782629436-00001

Spin labels containing nitroxyl radicals possess many properties that render them useful for electron paramagnetic resonance (EPR) spectroscopy. This review describes the relationships between the structure and properties of nitroxide spin labels, methods for their synthesis, advances in methods for their incorporation into biomolecules, and selected examples of applications in biomolecule structural investigations.

1 Introduction

Within the field of electron paramagnetic resonance (EPR) spectroscopy, 'spin labelling' describes the attachment of a radical or paramagnetic centre (i.e. a molecule containing at least one unpaired electron spin) onto a material of interest, which enables its investigation using paramagnetic resonance spectroscopy. For such applications, spin labels should ideally fulfil several criteria: the framework of the label must stabilise the radical against redox processes; the radical must possess desirable properties for the magnetic resonance experiment (such as chemical stability and spin coherence persistence); and, the label must be readily (and site-specifically) attached without structural distortion of the system under study.

By far the largest family of spin labels are those based on the nitroxyl (N-O•) radical, which are called nitroxide spin labels. These are typically five- or six-membered heterocyclic derivatives of piperidine, pyrrolidine, isoindoline, and other heterocycles containing two heteroatoms; importantly, the nitroxyl radical is flanked by two quaternary carbon atoms. The 'classic' nitroxide is the piperidine-based 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO, 1, Fig. 1), which has found use in many chemical and materials applications. This radical, in which the unpaired electron is located mainly on the nitrogen and oxygen atoms, is stabilised by the steric screening imparted by its four adjacent methyl groups, which protect the radical from reduction or other processes. The lack of a-protons also prevents the decomposition of the nitroxyl to the corresponding nitrone. Some other examples of common nitroxide families (2-7) are illustrated.

A 'spin label' can be defined as a derivative of the parent nitroxide in which the core ring system (or its substituents) is modified to enable its incorporation into a larger framework, and thus to be used as a probe. The framework of interest may be a polymer, a surface, or a biomolecule such as a protein, nucleic acid, sugar or lipid.

Spin labels are commonly used to measure interspin distances (i.e. the distance between two stable free radicals) using continuous wave (CW) or pulsed techniques, which for nitroxides have an effective range of 0.5 to >10 nm. They can also be used to probe the local environment of the label, such as its accessibility and dynamic mobility. Nitroxides are also employed as paramagnetic relaxation enhancers in nuclear magnetic resonance (NMR) spectroscopy, and as polarisation/contrast agents in dynamic nuclear polarisation (DNP) or magnetic resonance imaging (MRI) experiments.

Modification of the basic structure of the nitroxide can lead to dramatic changes in the properties of the spin label, and it is for this reason that a myriad of spin labels have been designed. Essential considerations centre on the structure of the nitroxide around the nitroxyl radical itself, and the functionality used to enable spin labelling. This chapter discusses these aspects, along with recent advances in the synthesis and applications of nitroxide spin labels in EPR spectroscopy.

2 The nitroxide spin label as a probe in EPR spectroscopy

2.1 Magnetic properties

The Zeeman splitting for the nitroxide spin labels is anisotropic and typically gxx > gyy > gzz with gxx and gyy close in value and greater than the free electron g-value. The gzz axis is roughly coincident with the p orbital, approximated as a linear combination of the 2p orbitals of oxygen and nitrogen. Therefore, in planar systems such as pyrrolinoxyl spin labels, the gzz is perpendicular to the plane of the ring. The x-axis is taken as coincident with the NO bond.

The unpaired electron is considered to reside in the p orbital. The spin density is on the nitrogen and oxygen with almost no delocalisation over the rest of the framework (for adjacent alkyl groups). The hyperfine coupling constant, Aiso, for nitroxide spin labels is typically in the region of 40 to 47 MHz. Due to the relative spin localisation the hyperfine splitting of the Zeeman levels is dominated by the nitrogen of the NO group (16O nuclei have zero spin and the predominant isotope of nitrogen is 14N with a nuclear spin, I, of 1). The hyperfine axes approximately follow the g-tensor principal axes with Axx˜Ayy where Azz is typically about 100 MHz. Both g and Aiso are weakly sensitive to solvent polarity and proticity, with Aiso increasing and g-values decreasing in increasingly polar/protic solvents. This property has been used to map membrane protein channels, and to probe changes in solvent behaviour associated with the glass-transition temperature in water/ glycerol mixtures.

Coupling of proximal nuclei (I ? 0) to the electron gives rise to the characteristic appearance of an EPR spectrum. Usually, the greatest splitting is caused by coupling to the nitroxyl nitrogen atom, with much smaller splitting from other ring substituents. However, this super-hyperfine splitting can afford additional information, such as the extent of protonation of imidazolinyl and imidazolidinyl spin labels. The nature of the substituents flanking the nitroxyl radical can also significantly affect signal linewidth. Substituent effects on the ring conformation can be influential: the faster dynamic averaging of the hyperfine interaction of the nitroxyl with the methyl protons in 4-oxo-TEMPO (8, Fig. 2) compared to 4-hydroxy-TEMPO (9) can be explained by a higher barrier to conformational ring flip in the latter, where the ring framework is fully sp3-hybridised (aside from the nitroxyl). Isotopic labelling such as perdeuteration or 15N-substitution, can also lead to line narrowing, and therefore improve the sensitivity of the spin label. This property has been used to improve the precision of measurement of tumbling rates, and...