Electron paramagnetic resonance (EPR) spectroscopy has emerged as an important technique for examining the structures and dynamics of biomolecules. The considerable applicability of this tool is attributed to its ability to function under biological conditions, independently of the size of the biomolecules, with high accuracy and sensitivity in detecting even small changes. EPR techniques typically require the presence of paramagnetic centres, which are not commonly found in the predominantly diamagnetic biopolymers. Therefore, paramagnetic probes can be incorporated into specific regions of the target molecule of interest, a strategy termed site-directed spin labelling (SDSL). Nitroxide-based probes have gained a unique position in this field, among other factors due to their small size and minimal impact on the biomolecular structure. In order for a nitroxide to be suitable and widely applicable as a spin label, particularly in an intracellular environment, it must possess sufficient aqueous solubility, efficient labelling reactivity (usually with the sulfhydryl group of a cysteine amino acid), be minimally perturbing to the protein structure, be resilient to reducing conditions, and have sufficiently long relaxation times (particularly for transverse relaxation, i.e. the phase memory time Tm). These properties are influenced by structural factors such as the nitroxide ring size and saturation, the nature of the α-substituents shielding the paramagnetic centre, or electronic effects of functional groups. Saturated five-membered pyrrolidinyl nitroxides exhibit greater kinetic stability towards reducing agents than their unsaturated pyrrolinyl or sixmembered piperidinyl counterparts. Increasing the bulk of the α-substituents, meanwhile, can be beneficial to reduction stability, but also detrimental for the relaxation parameters. For example, tetraethyl substituents around the nitroxide moiety can provide exceptional stability compared to the most widely used tetramethyl-substituted scaffold; however, as rotation of the CH3 of the ethyl groups leads to poor relaxation times at temperatures above 70 K, measurements require costly cryogenic conditions using liquid helium. Optimal relaxation parameters are exhibited by conformationally rigid nitroxides featuring spirocycles at the α-positions, which notably enable measurements at elevated temperatures. Our previous research revealed that the conformations adopted by these spirocyclic scaffolds is another important factor influencing nitroxide stability. Recently, we have also developed nitroxides based on a novel exo-methylene substituted spirocyclic scaffold and conducted extensive analyses of their physical properties. In this work, we build on these prior discoveries to develop a novel spin label based on an exo-methylene substituted spirocyclic nitroxide, and implement it in the examination of proteins through double electron-electron resonance (DEER; also known as pulsed electron double resonance, PELDOR) experiments. The synthesis of the new spin label began by the introduction of the spirocyclohexyl substituents by ketone exchange with 1,2,2,6,6-pentamethylpiperidin-4-one 1 and cyclohexanone, resulting in the spirocyclic piperidone 2 (Scheme 1). Bromination led to the quantitative formation of hydrobromide salt 3, which was subsequently subjected to a Favorskii rearrangement, resulting in the formation of spirocyclic pyrroline ester 4 in good yield. Oxidation of secondary amine 4 with m-chloroperbenzoic acid (m-CPBA) gave the nitroxide 5. As the further transformations required harsh conditions, the nitroxide moiety was at this point protected as the methoxyamine 6 using previously reported Fenton chemistry. The ester group in 6 was reduced with diisobutylaluminium hydride (DIBAL-H) to the alcohol 7 in very good yield. This allylic alcohol was then subjected to a two-step Overman rearrangement as the key step in our synthetic strategy. Alcohol 7 was first treated with trichloroacetonitrile in the presence of a base, yielding a trichloroacetimidate. This labile intermediate was immediately subjected to a basecatalysed, microwave-assisted [3,3]-sigmatropic rearrangement to form the exo-methylene substituted trichloroacetamide 8 in excellent yield over two steps. Deprotection of the methoxyamine by treatment with m-CPBA resulted in a Cope-type elimination to the nitroxide 9, albeit in moderate yield due to competing epoxidation of the exocyclic double bond. The crystalline trichloroacetamide was analysed using X-ray crystallography, which revealed that the nitroxide adopts a semi-open conformation of the spirocyclohexyl moieties in the solid state (Scheme 1). Previous reports suggest that such conformations may limit the accessibility of potential reducing agents to the nitroxide radical centre, thus enhancing its kinetic stability. Base-promoted hydrolysis of the trichloroacetamide moiety provided primary amine 10 in excellent yield, which was thereafter transformed to chloroacetamide 11 by acylation. Finally, a Finkelstein reaction provided the exo-methylene substituted spirocyclic pyrrolidinyl iodoacetamide 12, which was further used for protein spin labelling.