**2.3. Detailed studies with** *p***-nitrostilbene-***tert***-butyl-nitrone, 1**

Spin-trapping of mainly superoxide anion radicals formed in mitochondria under various conditions was followed in HeLa cells as previously (Hauck et al., 2009) and as well for better comparison with data employing **2**. Fig. 9 shows the fluorescence decay after 100 sec in the absence of any inhibitor. All subsequent data are based on this control (Figs. 10 - 15). Results are summarized in Fig. 16.

The inhibitors of complexes I, rotenone, and complex III, antimycin A, have the strongest effect, particularly in combination. KCN is known to inhibit complex IV (Leavesley et al., 2010) and oligomycin blocks ATP synthesis by binding to the oligomycin sensitivity conferring protein, OSCP, of the F0 moiety of F1F0-ATP synthase (Xu et al., 2000). The latter leads to an increased membrane potential and drives the respiratory chain backwards. In the absence of sufficient substrates for NADH this is known to produce superoxide anion radicals in complex I as in complex III via reaction of oxygen with the coenzyme Q semiquinone radical (Muller et al., 2004).

Rather interesting is the effect of the protonophore CCCP (carbonyl-cyanide *m*-chlorophenyl hydrazone) which reduces the membrane potential (Nieminen et al., 1994) and thus, apparently also reduces ROS formation. The half-life of the fluorescence in its presence is almost doubled as compared to the control corroborating the opposite effect of oligomycin.

**Figure 9.** Fluorescence decay of **1** in HeLa cells within 100 sec in the absence of any inhibitor of respiration

## **2.4. 4-Pyrrolidine-1,8-naphthylimido-methylphenyl-***tert***-butyl-nitrone, 3**

In order to allow for excitation at longer wavelengths a third compound was synthesized according to the scheme in Fig. 17, again a *tert*-butyl-nitrone, but with 4-pyrrolidine-1,8 naphthylimido-benzylidene as fluorescent moiety. 4-(1,3-Dioxacyclopent-2-yl)benzonitrile **B** was synthesized according to Ouari et al., 1998 in good yield and in the last step the formation of the nitrone **3** was carried out according to Kim et al., 2007.

Fluorescent Nitrones for the Study of ROS Formation with Subcellular Resolution 355

354 Nitroxides – Theory, Experiment and Applications

Results are summarized in Fig. 16.

semiquinone radical (Muller et al., 2004).

respiration

**2.3. Detailed studies with** *p***-nitrostilbene-***tert***-butyl-nitrone, 1** 

Spin-trapping of mainly superoxide anion radicals formed in mitochondria under various conditions was followed in HeLa cells as previously (Hauck et al., 2009) and as well for better comparison with data employing **2**. Fig. 9 shows the fluorescence decay after 100 sec in the absence of any inhibitor. All subsequent data are based on this control (Figs. 10 - 15).

The inhibitors of complexes I, rotenone, and complex III, antimycin A, have the strongest effect, particularly in combination. KCN is known to inhibit complex IV (Leavesley et al., 2010) and oligomycin blocks ATP synthesis by binding to the oligomycin sensitivity conferring protein, OSCP, of the F0 moiety of F1F0-ATP synthase (Xu et al., 2000). The latter leads to an increased membrane potential and drives the respiratory chain backwards. In the absence of sufficient substrates for NADH this is known to produce superoxide anion radicals in complex I as in complex III via reaction of oxygen with the coenzyme Q

Rather interesting is the effect of the protonophore CCCP (carbonyl-cyanide *m*-chlorophenyl hydrazone) which reduces the membrane potential (Nieminen et al., 1994) and thus, apparently also reduces ROS formation. The half-life of the fluorescence in its presence is almost doubled as compared to the control corroborating the opposite effect of oligomycin.

**Figure 9.** Fluorescence decay of **1** in HeLa cells within 100 sec in the absence of any inhibitor of

**2.4. 4-Pyrrolidine-1,8-naphthylimido-methylphenyl-***tert***-butyl-nitrone, 3** 

formation of the nitrone **3** was carried out according to Kim et al., 2007.

In order to allow for excitation at longer wavelengths a third compound was synthesized according to the scheme in Fig. 17, again a *tert*-butyl-nitrone, but with 4-pyrrolidine-1,8 naphthylimido-benzylidene as fluorescent moiety. 4-(1,3-Dioxacyclopent-2-yl)benzonitrile **B** was synthesized according to Ouari et al., 1998 in good yield and in the last step the

**Figure 10.** Fluorescence decay of **1** in HeLa cells within 90 sec after addition of rotenone with t½ = 32 sec, control 176 sec assuming exponential decay (dotted lines)

**Figure 11.** Fluorescence decay of **1** in HeLa cells within 90 sec after addition of antimycin A with t½ = 20 sec, control 176 sec assuming exponential decay (dotted lines)

**Figure 12.** Fluorescence decay of **1** in HeLa cells within 90 sec after addition of rotenone and antimycin A with t½ = 16 sec, control 176 sec assuming exponential decay (dotted lines)

**Figure 13.** Fluorescence decay of **1** in HeLa cells within 90 sec after addition KCN with t½ = 40 sec, control 176 sec assuming exponential decay (dotted lines)

Fluorescent Nitrones for the Study of ROS Formation with Subcellular Resolution 357

356 Nitroxides – Theory, Experiment and Applications

**Figure 12.** Fluorescence decay of **1** in HeLa cells within 90 sec after addition of rotenone and antimycin

**Figure 13.** Fluorescence decay of **1** in HeLa cells within 90 sec after addition KCN with t½ = 40 sec,

control 176 sec assuming exponential decay (dotted lines)

A with t½ = 16 sec, control 176 sec assuming exponential decay (dotted lines)

**Figure 14.** Fluorescence decay of **1** in HeLa cells within 90 sec after addition of oligomycin with t½ = 23 sec, control 176 sec assuming exponential decay (dotted lines)

**Figure 15.** Fluorescence decay of **1** in HeLa cells within 90 sec in the presence of CCCP with t½ = 336 sec, control 176 sec (red) assuming exponential decay (dotted lines)

**Figure 16.** Effect of inhibitors of mitochondrial respiration on ROS formation as monitored by **1** (**A**) and **2** (**B**)

**Figure 17.** Synthetic scheme for the synthesis of 4-pyrrolidine-1,8-naphthylimido-methylphenyl-*tert*butyl-nitrone, **3** 

Absorbance and fluorescence emission spectra are shown in Fig. 18. Fig. 18 A shows the fluorescence spectrum (red) with a maximum at 522 nm upon excitation at 488 nm and the UV/VIS spectrum with a maximum at 457 nm (black). Fig. 18 B illustrates the fluorescence spectrum (red) with a maximum at 514 nm upon excitation at 405 nm. Excitation at 488 nm is well in the range of standard confocal laser microscopes and certainly, not likely to lead to radiation damage in biological systems. Within the concentration range feasible due to limitations in solubility, **3** was non-toxic as studied so far in MCF-7 cells up to 5 µM (data not shown).

358 Nitroxides – Theory, Experiment and Applications

**A B**

N

O

pyrrolidine EGME *p*-TsOH

OH OH toluene

**A + B** EtOH THF

O

O

**2** (**B**)

O

Br

butyl-nitrone, **3** 

**Figure 16.** Effect of inhibitors of mitochondrial respiration on ROS formation as monitored by **1** (**A**) and

**A**

N

O O

O

<sup>R</sup> <sup>O</sup> <sup>R</sup>

HCl

*<sup>p</sup>*-TsOH LiAlH4

N

O

N

Et2O

N

O

NH CH3 CH3

OH CH3 HCl

O

CH2Cl2 NEt3

**3**

NH2 O

**B**

O

N+ O- CH3 CH3

<sup>3</sup>CH

O

O

O

**Figure 17.** Synthetic scheme for the synthesis of 4-pyrrolidine-1,8-naphthylimido-methylphenyl-*tert*-

**Figure 18.** Absorption and fluorescence emission spectra of **3**. Excitation at 488 nm (**A**) or 405 nm (**B**), respectively

Preliminary spin trapping experiments with **3** were carried out with a regular confocal microscope with excitation at 488 nm. Fig. 19 shows the control in the absence of inhibitors. As found before, complete quench occurs in about half an hour. Although the dye may accumulate again in mitochondria, we have not yet looked for co-localization with TMRE. Strikingly different are the effects of inhibitors as shown for antimycin A in Fig. 20. Corresponding experiments employing rotenone or both, rotenone and antimycin A as well as the Fenton reaction gave very similar results (data not shown). Quench is almost instantaneous,

**Figure 19.** Fluorescence decay of **3** (1 µM) at selected times in MCF-7 cells: **A:** 0 min, **B:** 5 min, **C:** 10 min, **D:** 15 min, **E:** 20 min in the absence of inhibitors (control; laser intensity 80 % and exposure 800 ms)

but fluorescence recovered within four to five seconds and then decayed completely over the next 15 min. What could cause fluorescence recovery? The half-life of nitroxides as Tempone or Tempamine in viable systems is in the order of 30 min at most due to the reducing milieu in the cell (Berliner, 1991). Hence, the fluorescence being quenched by the radical could come back. There is however, also the possibility that differences are due to the experimental setup, *i.e.,* a continuous flow device for the medium at the ApoTome to which the inhibitor was added, whereas in these experiments a concentrated solution of antimycin A etc. was manually injected directly into the medium surrounding the adherent cells.

360 Nitroxides – Theory, Experiment and Applications

**A B**

**C D**

**E**

Preliminary spin trapping experiments with **3** were carried out with a regular confocal microscope with excitation at 488 nm. Fig. 19 shows the control in the absence of inhibitors. As found before, complete quench occurs in about half an hour. Although the dye may accumulate again in mitochondria, we have not yet looked for co-localization with TMRE. Strikingly different are the effects of inhibitors as shown for antimycin A in Fig. 20. Corresponding experiments employing rotenone or both, rotenone and antimycin A as well as the Fenton reaction gave very similar results (data not shown). Quench is almost instantaneous,

**Figure 19.** Fluorescence decay of **3** (1 µM) at selected times in MCF-7 cells: **A:** 0 min, **B:** 5 min, **C:** 10 min, **D:** 15 min, **E:** 20 min in the absence of inhibitors (control; laser intensity 80 % and exposure 800 ms)

**Figure 20.** Fluorescence decay of **3** in MCF-7 cells at selected times after addition of 1 µl antimycin A (2 mg/ml) **A:** 0 sec, **B:** 1 sec, **C:** 2 sec, **D:** 4 sec, **E:** 13 min (laser intensity 80 % and exposure 800 ms)
