[i

Figure 3 Attachment of supplementary equipment to the ESR spectrometer as radical detector.

Figure 4 Synthesis scheme for PCA anhydride using DCC in purified methylenechloride.

Minutes

Figure 5 Chromatogram of synthesis product obtained by means of online coupled HPLC-ESR.

Minutes

Figure 5 Chromatogram of synthesis product obtained by means of online coupled HPLC-ESR.

HPLC: Merck-Hitachi-HPLC with DAD (diode array detector), RP-18 (5 |im) column (125/4 mm) and methanol-water solutions (flow rate 1 mL min"1, pH 7, isocratic as well as gradient mode).

ESR: Miniscope MS-100 desk X-band spectrometer (Magnettech GmbH, Germany). GC-MS, IR and NMR-techniques were also used for the identification of unknown products.

In following the synthesis, mixtures of PCA anhydride [II] were investigated by online coupled HPLC-ESR. This anhydride in practice represents a suitable tool for the spin labelling of macro-molecules such as albumin. The purified synthesis product was analysed. In contrast to the available literature, the chromatogram obtained showed four ESR-active compounds after HPLC separation (Figure 5).

These were the PCA anhydride [II] (peak 3 with tR = 4.95 min) and PCA [i] (peak 1 with tR = 1.39 min) as expected as well as two new paramagnetic compounds with typical 14N-hyperfine coupling (peak 2 with tR = 3.34 min and peak 4 with tR = 7.41 min). Further, 1H-NMR, IR and GC-MS experiments showed the compound with tR = 7.71 min (peak 4) to be PCA dicyclohexylurea-amide. No other peaks gave an ESR signal.

Figure 6 represents the ESR spectra of the sample before and after chromatographic separations. Spectra of peak 1, 2 and 4 show a line triplet typical

Figure 6 ESR spectra of the initial substance and four separated paramagnetic compounds.

o ch3

-CH3 + free radicals R generated

• transition metals

• radical leaks

• oxidative stress

• ischaemia/reperfusion

"CH, spin trap PBN

Figure 7 Scheme of formation of stable radicals in biological systems using PBN.

different PBN radical adducts with nearly similar ESR spectra for nitroxyl radicals owing to the interaction between the unpaired electron and the nitrogen nucleus. The uneven number of protons and neutrons results in nuclear spin quantum numbers of —1,0 and + 1, in equal distribution. Those nuclei with a spin different from 0 cause a magnetic field recognized by the unpaired electron in addition to that generated by the magnet of the spectrometer. The result is a hyperfine splitting into three spectral lines as shown in the respective spectra. Peak 3 comprises the biradical PCA anhydride [II] with two nitroxyl groups in close vicinity. The ESR spectrum is determined by additional interactions between these two radical moieties. All the single spectra of the separated peaks add to the composite one seen with the initial unseparated sample.

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