Neutral nitroxides

Neutral Nitroxide spin label — Reduced spin label... 

In marked contrast with RjNO molecules, the dianion, (OjS)2NO , has an isotropic hyperfine splitting that is remarkably insensitive to changes in solvation [18]. Also, the electronic spectrum is insensitive, in contrast with those for neutral nitroxides. This difference seems to be largely due to very strong hydrogen bonding to the two -SO J units in protic solvents, which dominates the overall solvation. 

Let us end this section with the synthesis of three relevant molecules TCNQ, Qf-nitronyl nitroxides and CuPc. The synthesis route that efficiently leads to the preparation of the strong acceptor TCNQ is depicted in Fig. 2.10. It is based on the ready condensation of malonitrile with 1,4-cyclohexadione. Further treatment with C5H5N and Br2 gives TCNQ in excellent yields (Acker Herder, 1962). The electronic band structure of neutral TCNQ will be discussed in Section 6.1. 

Since malonate (Structure I) is able to fulfill the role of the anion in transferrin, it seemed reasonable to see whether spin-labeled derivatives of malonate could serve as probes of the active sites. Two such spin-labled derivatives were prepared and tentatively identified as having structures II (N-4-(2,2,6,6-tetramethylpiperidin-l-oxyl)malonamide) and III (N-4-(2,2,6,6-tetramethylpiperidin-l-oxyl)malonate). Similar results were obtained with each (Figure 3). Upon mixing Fe(III), transferrin, and II at low pH, and then raising the pH to near-neutrality with C02-free ammonia, the characteristic orange-red color of the ternary Fe-transferrin-anion complex is promptly displayed. However, the anticipated EPR signal of the nitroxide spin-label is not observed, presumably because it is broadened beyond detectability by its proximity... 

Figure 4.3. Spin relaxation rate of charged molecules as a function of nitroxide spin probe concentration. Neutral, negative, and positive charged probes denoted are respectively denoted as 0, -, and + (Likhtenshtein et al., 1999). Reproduced with permission.

According to the experimental data on rate constants of spin exchange at encounters between heme groups and nitroxides presented in Table 4.2., the accessibility of the heme group of cytochrome c to the encounters with neutral spin-probes IV is -31-33 times lower than that observed for free hemin. At present, it is difficult to separate the effect of heme group immersion into the protein globular structure from that of association in the relatively concentrated solutions (2-10mM) utilized in these studies. 

TABLE 4.2. Apparent spin-exchange rate-constants (k+, k, and k°) of the positively charged (VI), negatively charged (V) and neutral (IV) nitroxide spin-probes with hemin, ferricyanide anion, and heme proteins in aqueous solutions and apparent local charges (ZH) in the vicinity of the heme groups3 (Likhtenshtein, 2000). Reproduced with permission. 

Neutral organic grouping Nitroxide radicals Lysozyme inhibitors ... 

Owing to the presence of acidic groups in melanins (carboxyls, phenolic groups) positively charged reagents react faster than anions or neutral species, especially in basic media. Thus, cationic nitroxides react much faster than anionic ones, and the reaction is twofold faster at pH 10 than pH 5. The slow reaction with Nitro Blue Tetrazolium is dramatically accelerated in the presence of a cationic detergent (92). 

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