**1. Introduction**

The perspective of this chapter is very much historical. The author was fortunate enough to have begun his graduate studies at the very inception of the technique of spin labeling. Mind you, the topic of this book is nitroxides a.k.a. aminoxyl radicals which in fact preceded the spin labeling method and its inception. Hence the chapter will cover a history of the synthetic developments with nitroxides, the history of the development of spin labels, and the use of nitroxides and will provide an overview to the future of its applications. The intent is to cover the very beginning and then discuss some of the key areas (always dominated by synthetic organic chemistry) that allowed this technique to blossom more and more. Needless to say, while the definition of spin labeling is the incorporation of a stable, free radical into a macromolecular system of choice, we have yet to find anything (aside from the trityl radicals) that will fulfill this purpose. And the trityl radicals, which may be covered briefly in this chapter, give no dynamic or structural information whatsoever as they yield a single narrow line spectrum. The author feels confident to discuss these areas since he was deeply involved in the synthetic organic chemistry of these compounds, frequently repeating the syntheses of the basic starting compounds that were either commercially unavailable or quite expensive at the time. This includes the synthesis of phorone/triacetonamine from ammonia, acetone and calcium, in a 1898 synthesis[1], which was the basis for the nitroxide TEMPONE(2,2,6,6-tetramethylpiperidinone 1-oxyl).

Perhaps one of the earliest papers describing 'nitroxides' was from the American Cyanamid Company laboratories about the reaction of t-nitrobutane with metallic sodium[2]. They found a g value of 2.0065 and a single line linewidth of 8.5G (probably because they observed the compound in neat form where exchange and dipolar broadening were predominant). A follow-up publication produced a plethora of compounds derived from

© 2012 Berliner, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Berliner, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

phenyl derivatives. They were able to measure a hyperfine coupling constant for DTBN of 15.25G [3]. We should recall that the earliest example of these radicals was the famous Fremy's salt, used to calibrate EPR machines to this day. This long-lived free radical, shown below, was discovered in 1845 by Edmond Frémy [4].

**Figure 1.** (left) di-t- butylnitroxide[2];(right) Fremy's salt [1]

In the 1960s, stable paramagnetic compounds were developed extensively in the USSR Academy of Sciences, Institute of Chemical Physics, that contained aminoxyl (or iminoxyl or nitroxyl or nitroxide) 'reporter' groups. Until these compounds became commercially available, one was obligated to prepare them homemade, but their syntheses were fairly straightforward (starting with the either phorone or triacetoneamine) [1]. The Russian group was led by organic chemists M.B. Neiman and E.G. Rozantsev and the group expanded these syntheses into a broad range of compounds, some of which could be applied as protein modification reagents [5-6].

**Figure 2.** Piperidine, pyrrolidine and pyrroline nitroxides.

Let us not overlook the tremendous advantages of nitroxides that contribute to their versatility in the study of (biological) macromolecules i.e., they are very stable in most solvents over a wide range of pH values. The paramagnetic N-O bond moiety is quite tolerant to various synthetic conditions, specifically those in the tetramethyl flanked piperidine, pyrroline or pyrrolidine rings. Freezing, thawing, distilling or boiling usually impart no adverse affects on their stability, ie, paramagnetism is retained. Since EPR does not require optical transparency, and is not sensitive to magnetic susceptibility effects (which plagues NMR), one can work in opaque solutions, solids or mixtures. And the EPR sensitivity is 600- 700 times higher per spin compared with a proton in NMR. Thus, with very narrow linewidth spectra, one could detect nitroxide spectra in solution down to nanomolar levels with high sensitivity cavities. The EPR spectral lineshape reflects nitroxide tumbling motion, hence one can distinguish freely tumbling, 'unattached' or unreacted label in a sample with other bound species. The only real drawback of spin labels is their susceptibility to reduction to the corresponding diamagnetic hydroxylamine in the presence of organic or biological reducing agents, which will be addressed under in-vivo studies. Yet, where some synthetic recipes may utilize e.g., NaBH4 which reduces the N-O moiety, the radical is easily regenerated in mild H2O2 or exposure to O2.

4 Nitroxides – Theory, Experiment and Applications

below, was discovered in 1845 by Edmond Frémy [4].

**Figure 1.** (left) di-t- butylnitroxide[2];(right) Fremy's salt [1]

**Figure 2.** Piperidine, pyrrolidine and pyrroline nitroxides.

protein modification reagents [5-6].

phenyl derivatives. They were able to measure a hyperfine coupling constant for DTBN of 15.25G [3]. We should recall that the earliest example of these radicals was the famous Fremy's salt, used to calibrate EPR machines to this day. This long-lived free radical, shown

In the 1960s, stable paramagnetic compounds were developed extensively in the USSR Academy of Sciences, Institute of Chemical Physics, that contained aminoxyl (or iminoxyl or nitroxyl or nitroxide) 'reporter' groups. Until these compounds became commercially available, one was obligated to prepare them homemade, but their syntheses were fairly straightforward (starting with the either phorone or triacetoneamine) [1]. The Russian group was led by organic chemists M.B. Neiman and E.G. Rozantsev and the group expanded these syntheses into a broad range of compounds, some of which could be applied as

Let us not overlook the tremendous advantages of nitroxides that contribute to their versatility in the study of (biological) macromolecules i.e., they are very stable in most solvents over a wide range of pH values. The paramagnetic N-O bond moiety is quite tolerant to various synthetic conditions, specifically those in the tetramethyl flanked piperidine, pyrroline or pyrrolidine rings. Freezing, thawing, distilling or boiling usually impart no adverse affects on their stability, ie, paramagnetism is retained. Since EPR does not require optical transparency, and is not sensitive to magnetic susceptibility effects (which plagues NMR), one can work in opaque solutions, solids or mixtures. And the EPR sensitivity is 600- 700 times higher per spin compared with a proton in NMR. Thus, with very narrow linewidth spectra, one could detect nitroxide spectra in solution down to

**Figure 3.** First meeting of L. J. Berliner with E. G. Rozantsev, USSR Institute of Chemical Physics, 1979
