Phenoxy radical detection using P NMR spin trapping

ion and subsequent deprotonation gives rise to phenoxy radicals, which play a pivotal role in lignin chemistry. Overall spin trapping systems could be powerful tools to understand the radical intermediates involved in lignin chemistry. In Table 4 our data on spin trap experiments with DIPPMPO and different phenols are shown. There are different oxidation systems for the generation of phenoxy radicals. Initially, the phenoxy radicals were produced by hexacyanoferrate(III) in a biphasic system of benzene–water. This oxidation system is simple and well known: during this reaction the O—H bond is homolytically cleaved by the donation of an electron to a powerful one-electron acceptor as a transition-metal ion in a high-valence state, to generate the phenoxy radicals. This system was applied to the oxidation of 2,4,6-trichlorophenol and 2,4,6-tri-tert-butylphenol in the presence of DIPPMPO. At the onset of our investigation, we used such phenols because the positions of the radical delocalization were blocked by the groups in the 2, 4, and 6 positions and the formation of oligomeric products, formed via coupling of oxygento carbon-centered radicals, was a minor reaction. In the absence of substrate, no reaction occurred between the spin trap DIPPMPO and K3Fe(CN)6. As such the P NMR showed only a peak at 22.2 ppm related to the native spin trap. However, in the presence of substrates, the P NMR showed the formation of one radical adduct with a chemical shift at 25.1 ppm for the 2,4,6trichlorophenol and 25.2 ppm for the 2,4,6-tri-tert-butylphenol. This signal was thus assigned to the adducts of the phenoxy radical with DIPPMPO. Similar results were obtained when we used HRP with H2O2 as oxidation system in a buffered solution (pH 4.5) with the addition of a small amount of dimethylformamide (DMF) to overcome the problem of substrate insolubility. In the absence of substrate, no reaction occurred between the spin trap DIPPMPO and HRP, and the P NMR showed only a peak at 22.2 ppm related to the native spin trap. The P NMR spectra showed a single peak at 25.1 ppm for the 2,4,6-trichlorophenol and 25.2 ppm for the 2,4,6-tri-tert-butylphenol, as previously obtained in the presence of K3Fe(CN)6. In the case of 2,4,6-tri-tert-butylphenol oxidation and in the presence of only HRP and H2O2, the substrate was recovered unchanged (99%) and no signal in the P NMR spectrum was observed. The bulky butyl groups in positions 2 and 6 hindered the approach of the enzyme to the phenolic group. However, in the presence of an oxidation mediator of small size, such as 1-hydroxybenzotriazole (HBT), finally the signal of the phenoxy radical adduct was observed. The >N—O species generated from the interaction of HBT by HRP, and in view of its small size and matching value of bond dissociation energy (BDE) (85 kcal/ mol for HBT vs. 84–87 kcal/mol for phenols), allowed the abstraction of a H-atom from the O—H bond of 2,4,6-tri-tertbutylphenol and afforded the corresponding phenoxy radical. It is important to note that during the oxidation of HBT with HRP and H2O2 in the absence of the substrate, we were able to detect two adducts at 23.5 (doublet) and 17.9 ppm and traces of OH and HOO adducts at 25.3 and 16.9–17.0 ppm. We interpreted these results as a consequence of adduct formation of the HBT radical with DIPPMO, and the structure of these radical adducts is actually under investigation (Scheme 2). Alternatively, in the presence of 2,4,6-tri-tert-butylphenol, we were able to detect only a signal at 25.2 ppm related to the phenoxy radical, with traces of residual HOO adducts. It is known Table 3. P NMR signals for DIPPMPO reaction adducts of carbon-centered radicals Species Generating system Chemical shift (ppm) DIPPMPO — 22.2 DIPPMPO/ CH3 DMSOþ 3% H2O2þUV light 23.1 DIPPMPO/ CH2OH Methanolþ 3% H2O2þUV light 22.6 DIPPMPO/ CH(OH)CH3 Ethanolþ 3% H2O2þUV light 27.3 DIPPMPO/ C(O)CH3 AcetoneþUV light 30.2 Table 4. P NMR signals for DIPPMPO reaction adducts of different phenols Substrates Generating system Chemical shift (ppm) — — 22.2 — K3Fe(CN)6 22.2 — HRP-H2O2 22.2 — HRP-HBT-H2O2 17.9/23.5 2,4,6-trichlorophenol K3Fe(CN)6 25.1 2,4,6-trichlorophenol HRP-H2O2 25.1 2,4,6-tributylphenol K3Fe(CN)6 25.2 2,4,6-tributylphenol HRP-H2O2 22.2 2,4,6-tributylphenol HRP-HBT-H2O2 25.2 2,4-dimethylphenol HRP-H2O2 25.2/27.0 Isoeugenol HRP-H2O2 17.5/25.2/27.0 Scheme 2. Mechanism of trapping HBT radical generated by HRP/H2O2 with DIPPMPO J. Phys. Org. Chem. (2009) Copyright 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/poc PHENOXY RADICAL DETECTION