**9. Conclusions/prognosis/summary/future developments**

This author has frequently concluded, about once per decade, that spin labeling has met its limits and should go into the category of the 'on the shelf' routine technique given all of its limitations. However, we have found one or two cases of a renaissance in the use of nitroxides, particularly the inception of the SDSL technique using MTSL labels which have given it a major rebirth. The future should involve marrying various techniques that can utilize paramagnetic materials, some of which that have already been mentioned earlier: NMR, fluorescence, dynamic nuclear polarization (DNP) and other technologies yet to be developed or discovered. Some examples are shown below.

Optical probes, e.g., absorb in the visible or are fluorescent, when coupled to a paramagnetic moiety, experience shifts or lifetime relaxation from a nearby free electron. One example below (Figure 18), is a nitroxide fluorophore, which exhibits significantly quenched fluorescence emission. Some applications are cartooned in Figures 19 and 20.

This nitrone spin trap depicted in Fig. 21 is one of several synthesized and tested by David Becker [32] at Florida International University. Upon reaction with reactive oxygen species, the absorption properties of the nitrone shifts are depicted below.

mechanism and detailed rate constants) [35-36]

TEMPONE, or for that matter TEMPOL, are rapidly converted to the hydroxylamine with an immediate loss of the paramagnetism. This occurs within a few minutes. The fivemembered ring species, however, have much longer half-life, i.e. of the order of 15 to 30 min., allowing one to study some aspects of the metabolism and perhaps the ability to image this paramagnetic material in a living species. The first experiments were done by the Brasch group where they were evaluating nitroxides as MRI contrast agents[33-34]. This was followed by a plethora of studies on animals, tissue samples, blood samples, etc. where we obtained a wealth of pharmacokinetic data (although no totally clear understanding of the

Suffice it to say, imaging by EPR methods is challenging, if not hopelessly low resolution, since most nitroxide labels have linewidths of, at best, 0.3-0.5G for a compound that is deuterium and N-15 enriched. It has only been with the trityl radicals mentioned early where any hope of imaging was possible. However, if one takes advantage of the power and high resolution of magnetic resonance imaging (MRI) and the fact that the contrast agents in this methodology are paramagnet, then organic radicals have a place. Therefore nitroxide spin label/spin probe analogs have been tested as MRI contras agents and have met with some success. One must overcome the problem of biological reduction, also a problem with the radical adducts of nitrone spin traps since the cellular/tissue milieu contain many reducing agents such as NADH, ascorbic acid, and mitochondrial reduction sources [33]. The real quest here is to produce a well protected, aminoxyl radical that is highly resistant to biological reduction yet can be incorporate into the tissue system of choice. A few examples have been reported to date, particularly where the tetramethyl groups that flank the N-O group are replaced by long aliphatic chains such as lipids or tertiary butyl chains or cages.

This author has frequently concluded, about once per decade, that spin labeling has met its limits and should go into the category of the 'on the shelf' routine technique given all of its limitations. However, we have found one or two cases of a renaissance in the use of nitroxides, particularly the inception of the SDSL technique using MTSL labels which have given it a major rebirth. The future should involve marrying various techniques that can utilize paramagnetic materials, some of which that have already been mentioned earlier: NMR, fluorescence, dynamic nuclear polarization (DNP) and other technologies yet to be

Optical probes, e.g., absorb in the visible or are fluorescent, when coupled to a paramagnetic moiety, experience shifts or lifetime relaxation from a nearby free electron. One example below (Figure 18), is a nitroxide fluorophore, which exhibits significantly quenched

This nitrone spin trap depicted in Fig. 21 is one of several synthesized and tested by David Becker [32] at Florida International University. Upon reaction with reactive oxygen species,

fluorescence emission. Some applications are cartooned in Figures 19 and 20.

the absorption properties of the nitrone shifts are depicted below.

**9. Conclusions/prognosis/summary/future developments** 

developed or discovered. Some examples are shown below.

**Figure 18.** ((2-Carboxy)phenyl)-5-hydroxy-1-((2,2,5,5-tetramethyl-1-oxypyrrolidin-3-yl)methyl)-3 phenyl-2-pyrrolin-4-one sodium salt [30]

**Figure 19.** Schematic of the spin label in Figure 18 , where the red nitroxide depicts the paramagnetic N-O group, while the weak fluorescence reflects quenching by the paramagnet.

**Figure 20.** Upon reduction of the spin label the corresponding hydroxylamine, e.g., in a biological system by NADH or ascorbic acid, the fluorescence emission is strong and the EPR spectrum from the spin label has disappeared.

One can take advantage of NMR/MRI by spin labeling a cell surface with multiple nitroxide labels. The highly labeled surface now acts as an excellent paramagnetic relaxation enhancement site for exchanging water molecules, enhancing contrast in MRI [33-34] and being uniquely sensitive to changes in conformation, permeability and flexibility of the cell membrane surface as depicted below.

**Figure 21.** A colorimetric nitrone spin trap.

**Figure 22.** Schematic of the reaction of a colorimetric or fluorescent nitrone spin trap with a radical

**Figure 23.** Schematic of a proton relaxation enhancement (PRE) spin labeled cell. Multiple paramagnetic labels are affixed to the cell surface by specific binding or covalent attachment. This results in a significantly enhanced PRE, which is detectable in MRI.

Hence future developments in several of these areas should show great promise for the future.
