Synthesis of 2,5-Bis(spirocyclohexane)-Substituted Nitroxides of Pyrroline and Pyrrolidine Series, Including Thiol-Specific Spin Label: An Analogue of MTSSL with Long Relaxation Time Igor A. Kirilyuk,* ,† Yuliya F. Polienko, † Olesya A. Krumkacheva, ‡ Rodion K. Strizhakov, ‡ Yurii V. Gatilov, † Igor A. Grigor’ev, † and Elena G. Bagryanskaya †,‡ † N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry Sb RAS, Academician Lavrentjev Ave. 9, Novosibirsk, 630090, Russia ‡ International Tomography Center SB RAS, Institutskaya str. 3a, 630090, Novosibirsk, Russia * S Supporting Information ABSTRACT: The nitroxides of 7-azadispiro[5.1.5.2]- pentadecane and 7-azadispiro[5.1.5.2]pentadeca-14-ene series have been prepared, including thiol-specific methane thiosul- fonate spin label for site-directed spin labeling. The effect of spirocyclohexane moieties on chemical and spectral properties has been studied. The obtained temperature dependencies of electron spin relaxation parameters demonstrate that new nitroxides may be suitable for PELDOR distance measurements at 80-120 K. Moreover, the new nitroxides demonstrated much higher stability toward reduction by ascorbate than spirocyclohexane-substituted nitroxides of piperidine series and showed 1.3- 3.14 times lower reduction rates compared to corresponding 2,2,5,5-tetramethyl nitroxides. ■ INTRODUCTION Stable nitroxyl radicals (nitroxides) have found broad applications in various fields of science and technology. Numerous recent reviews demonstrate increasing importance of nitroxides in synthetic polymer chemistry, 1 magnetic materials, 2 reagents for selective oxidation, 1,3 biophysics, 4 structural and molecular dynamics studies, 5 therapeutic agents and biomedical research, 5,6 electrode active and charge storage materials in rechargeable batteries, 7 etc. Most of the widely used nitroxides have two pairs of methyl groups at α-carbon atoms of nitroxide moiety. However, it is known that introduction of spirocyclic moieties instead of the geminal methyl groups can improve properties of nitroxide with respect to nitroxide-mediated polymerization (NMP). 8 Moreover, an increase in steric requirements of substituents at α-carbon atoms produced by spirocylic moieties adjacent to nitroxide group may retard reduction of nitroxides with low-molecular biogenic reductants and enzymatic systems. 9-13 It has been found recently that piperidine nitroxides with spirocyclic moieties at α-carbons of nitroxide group may have serious advantages over 2,2,6,6-tetramethyl analogues in structural studies using pulse electron double resonance (PELDOR). 14,15 These studies imply application of site-directed spin labeling (SDSL) technique. Meanwhile, plenty of spin labels, e.g., MTSSL and TPA (Chart 1), which are often used in SDSL, belong to pyrroline nitroxides family. It is also well-known that pyrrolidine nitroxides demonstrate much higher stability to reduction compared to piperidine derivatives. 16 Here we describe preparation of 7- azadispiro[5.1.5.2]pentadecane and 7-azadispiro[5.1.5.2]- pentadeca-14-ene nitroxides, including the spin label MTSSL analogue, from 7-azadispiro[5.1.5.3]hexadecan-15-one 1 (Scheme 1). The reduction rate constants of nitroxides with ascorbate have been measured, and EPR parameters and temperature dependence of electron spin relaxation times of the new nitroxides are described. ■ RESULTS AND DISCUSSION Synthesis. A classic pathway to 2,2,5,5-tetramethyl-3- pyrroline and 2,2,5,5-tetramethylpyrrolidine derivatives implies Favorskii rearrangement of brominated 2,2,6,6-tetramethylpi- Received: June 18, 2012 Published: August 23, 2012 Chart 1 Scheme 1 Article pubs.acs.org/joc © 2012 American Chemical Society 8016 dx.doi.org/10.1021/jo301235j | J. Org. Chem. 2012, 77, 8016-8027