Formation of methyl radicals derived from cumene hydroperoxide in reconstructed human epidermis: an EPR spin trapping confirmation by using 13C-substitution

Article abstract of DOI:10.1080/10715762.2019.1624741

Dermal exposure to cumene hydroperoxide (CumOOH) during manufacturing processes is a toxicological issue for the industry. Its genotoxicity, mutagenic action, ability to promote skin tumour, capacity to induce epidermal hyperplasia, and aptitude to induce allergic and irritant skin contact dermatitis are well known. These toxic effects appear to be mediated through the activation to free radical species such as hydroxyl, alkoxyl, and alkyl radicals characterised basically by electron paramagnetic resonance (EPR) and spin-trapping (ST) techniques. To be a skin sensitiser CumOOH needs to covalently bind to skin proteins in the epidermis to form the antigenic entity triggering the immunotoxic reaction. Cleavage of the O–O bond allows formation of unstable CumO^?/CumOO^? radicals rearranging to longer half-life specific carbon-centred radicals R^? proposed to be at the origin of the antigen formation. Nevertheless, it is not still clear which R^? is precisely formed in the epidermis and thus involved in the sensitisation process. The aim of this work was to elucidate in conditions closer to real-life sensitisation which specific R^? are formed in a 3D reconstructed human epidermis (RHE) model by using ¹³C-substituted CumOOH at carbon positions precursors of potentially reactive radicals and EPR-ST. We demonstrated that most probably methyl radicals derived from β-scission of CumO^? radicals occur in RHE through a one-electron reductive pathway suggesting that these could be involved in the antigen formation inducing skin sensitisation. We also describe a coupling between nitroxide radicals and β position ¹³C atoms that could be of an added value to the very few examples existing for the coupling of radicals with ¹³C atoms.

Full text of DOI:10.1080/10715762.2019.1624741

Free Radical Research
ISSN: 1071-5762 (Print) 1029-2470 (Online) Journal homepage: https://www.tandfonline.com/loi/ifra20
Formation of methyl radicals derived from
cumene hydroperoxide in reconstructed human
epidermis: an EPR spin trapping confirmation by
using 13C-substitution
Fatma Sahli, Amélie Godard, Bertrand Vileno, Jean-Pierre Lepoittevin &
Elena Giménez-Arnau
To cite this article: Fatma Sahli, Amélie Godard, Bertrand Vileno, Jean-Pierre Lepoittevin &
Elena Giménez-Arnau (2019): Formation of methyl radicals derived from cumene hydroperoxide in
reconstructed human epidermis: an EPR spin trapping confirmation by using 13C-substitution, Free
Radical Research, DOI: 10.1080/10715762.2019.1624741
To link to this article: https://doi.org/10.1080/10715762.2019.1624741
Accepted author version posted online: 27
May 2019.
Submit your article to this journal
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ifra20

Formation of methyl radicals derived from cumene hydroperoxide in reconstructed
human epidermis: an EPR spin trapping confirmation by using 13C-substitution
Fatma Sahli,a Amélie Godard,a Bertrand Vileno,b Jean-Pierre Lepoittevin,a Elena
Giménez-Arnaua,*
aDermatochemistry Laboratory, University of Strasbourg-CNRS UMR 7177, Institut le Bel, 4 Rue Blaise
Pascal, 67081 Strasbourg, France
bPOMAM Laboratory, University of Strasbourg-CNRS UMR 7177, Institut le Bel, 4 Rue Blaise
Pascal, 67081 Strasbourg, France, and French EPR Federation of Research (REseau NAtional de Rpe
interDisciplinaire (RENARD, Fédération IR-RPE CNRS #3443)
Conflicts of interest:
none
Corresponding author:
Elena Giménez-Arnau, Dermatochemistry Laboratory, University of
Strasbourg-CNRS UMR 7177, Institut le Bel, 4 Rue Blaise Pascal, 67081 Strasbourg, France. Phone: +33
3 68 85 15 25, E-mail: egimenez@unistra.fr
Co-authors e-mail addresses:
f.sahli@unistra.fr; amelie.godard@etu.unistra.fr; vileno@unistra.fr;
jplepoit@unistra.fr
ORCIDs:
Bertrand Vileno: 0000-0001-7527-2068

Elena Giménez-Arnau: 0000-0001-8275-5447

Abstract
Dermal exposure to cumene hydroperoxide (CumOOH) during manufacturing processes is a toxicological
issue for the industry. Its genotoxicity, mutagenic action, ability to promote skin tumor, capacity to induce
epidermal hyperplasia and aptitude to induce allergic and irritant skin contact dermatitis are well known.
These toxic effects appear to be mediated through the activation to free radical species such as hydroxyl,
alkoxyl and alkyl radicals characterized basically by electron paramagnetic resonance (EPR) and spin-
trapping (ST) techniques. To be a skin sensitizer CumOOH needs to covalently bind to skin proteins in
the epidermis to form the antigenic entity triggering the immunotoxic reaction. Cleavage of the O-O bond
allows formation of unstable CumO•/CumOO• radicals rearranging to longer half-life specific carbon-
centered radicals R• proposed to be at the origin of the antigen formation. Nevertheless, it is not still clear
which R• are precisely formed in the epidermis and thus involved in the sensitization process. The aim
of this work was to elucidate in conditions closer to real-life sensitization which specific R• are formed
13
in a 3D reconstructed human epidermis (RHE) model by using C-substituted CumOOH at carbon
positions precursors of potentially reactive radicals and EPR-ST. We demonstrated that most probably
methyl radicals derived from -scission of CumO• radicals occur in RHE through a one-electron reductive
pathway suggesting that these could be involved in the antigen formation inducing skin sensitization. We
also describe a coupling between nitroxide radicals and  position 13C atoms that could be of an added
value to the very few examples existing for the coupling of radicals with 13C atoms.
13
Keywords:
cumene hydroperoxide, skin sensitizers, C-substitution, EPR spin-trapping spectroscopy,
reconstructed human epidermis

Introduction
Cumene hydroperoxide (CumOOH) is produced mainly through the oxidation of cumene in the so-
called “cumene process”, an industrial procedure for synthesizing phenol and acetone from benzene
and propylene based on the acid-catalyzed Hock rearrangement [1]. CumOOH is used as catalyst,
curing agent, and initiator in the manufacture of polymers and fiber-reinforced plastics. Plus, it is a
strong oxidizing agent making it an important intermediate in the fabrication of epoxy and polyester
resin coatings. However, dermal exposure to CumOOH during manufacturing processes is an important
toxicological issue for the industry, currently lacking of regulatory guidelines for occupational exposure
limits [2]. CumOOH genotoxicity, mutagenic action and ability to promote skin tumor and induce
epidermal hyperplasia have been described [3-5]. CumOOH is also known to cause allergic contact
dermatitis (ACD) and has been reported as a significant sensitizer in guinea pigs, with positive specific
responses reported in allergy cross-reactivity studies with allergenic cyclohexene hydroperoxides [6].
These toxic effects are believed to be mediated through the activation of the hydroperoxide to radical
species such as hydroxyl (HO•), alkoxyl (CumO•) and alkyl (R•) radicals.
Electron paramagnetic resonance (EPR) together with spin-trapping (ST) is a powerful combination
for the characterization of transient radicals in chemical and biological systems [7]. Basically, the formed
radical (either too short-lived or of too low concentration to be directly detected) reacts with a diamagnetic
reagent (spin-trap) to form a more persistent radical (spin-adduct), whose EPR signature can help the
characterization of the trapped transient reactive species. EPR-ST has been used for dermatological
purposes mainly for the detection of free radicals in UV-irradiated skin, melanoma investigation and in
vivo EPR imaging directly in skin of human volunteers [8-10]. EPR-ST has also been used to characterize
radicals in isolated mouse keratinocytes and in murine skin treated with CumOOH [11-13]. In addition,
EPR-ST proved the formation of in vivo lipid-derived free radicals triggered by CumOOH when mouse
skin was exposed causing severe oxidative stress [14,15]. To understand ACD mechanisms for risk
assessment purposes, we have proved that EPR-ST is also a powerful technique for the detection and
identification of radical species derived from sensitizing hydroperoxides (ROOHs) present in consumer
products [16-18]. To be skin sensitizers, ROOHs need to covalently bind via radical processes to skin
proteins after having penetrated the epidermis, to form this way the antigenic entity trigger of the

immunotoxic process. As for the CumOOH tumor promoting action, initial cleavage of the O-O bond
forms unstable RO•/ROO• radicals that further rearrange to longer half-life carbon centered radicals R•
proposed to be at the origin of the antigen formation [15,19,20]. Still, it is not still clear which R• are
precisely formed in the epidermis and thus involved in the sensitization processes.
We have recently reported an EPR-ST methodology allowing to investigate the formation of free
radicals issued from skin allergens in a reconstructed human epidermis (RHE) 3D model, thus much
closer to what may happen in vivo, by using CumOOH as proof of concept [21]. RHE are multi-layered
keratinocyte cultures, major cell type in the epidermis playing a key role in skin inflammatory reactions.
We disclosed that in the EpiSkinTM RHE model carbon radicals are mainly formed, both by incubating
the assay medium and by topical application with CumOOH. Previous in vitro EPR-ST studies reported
in keratinocytes cultures suggested these carbon radicals as being methyl radicals issued from -scission
fragmentation of tertiary alkoxyl radicals (CumO•) formed through cleavage of the O-O bond (Scheme 1)
[10]. Unlike keratinocytes cultures isolated from skin biopsies, EpiSkinTM RHE is an in vitro reconstructed
human epidermis histologically similar to in vivo human epidermis. It is a complex 3D structure including
a stratum corneum that allows assessing skin penetration concerns with phospholipids and ceramides
content influencing the RHE barrier properties [22]. The aim of the present study was to elucidate, in
the skin allergy context and in conditions closer to real-life sensitization, if methyl radicals are also
13
13
formed in EpiSkinTM RHE by using C-substituted CumOOH derivatives ( C-MeCumOOH) and
1
2
((13C-Me)2CumOOH) (Figure 1), and thus be able to suggest their participation in the formation of the
immunogenic complex inducing ACD.
Materials and methods
Synthesis of 13C-substituted CumOOHs
13
13
2
13
1
2
C-MeCumOOH and ( C-Me) CumOOH were synthesized C-substituted at the methyl positions
following the synthetic pathway shown in Scheme 2.
Chemicals and instrumentation

Starting materials were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France) and used as
received. 13C-Labeled reagents and deuterated solvents were purchased from Euriso-Top (Saint Aubin,
France). Air-/moisture-sensitive reactions were conducted in flame-dried glassware under an atmosphere
of dry argon. Solvents were dried on alumina, under argon, using a Glass Technology GTS 100 device.
Reactions were followed by thin layer chromatography (TLC) on 0.25 mm silica gel plates (60F254;
Merck, Darmstadt, Germany). After migration, the plates were inspected under UV light (254 nm) or
sprayed with a solution containing phosphomolybdic acid (5 g), cerium (IV) sulfate (2 g) and sulfuric
acid (12 mL) in water (188 mL), followed by heating. Column chromatography purifications were
performed on silica gel 60 (Merck, Geduran®, 40–63 m). Neutralized silica was prepared by adding to
a homogenous water solution of silica gel 60 a saturated solution of NaHCO3 until pH about 8. After
decantation the silica precipitate was washed with water to reach a pH of 7, filtered then dried in a oven
for at least 24 h. 1H and 13C NMR spectra were recorded on a Bruker Avance 500 spectrometer at 500
MHz and 125 MHz, respectively. Chemical shifts () are reported in ppm and are indirectly referenced
1
13
to TMS via the solvent signal (CDCl3:  H  7.26,  C  77.16). Spectra multiplicities are denoted as
d (doublet), dd (doublet of doublets), t (triplet) and m (multiplet).
13
4
Synthesis of 1-( C)acetophenone ( )
To a suspension of aluminum chloride (2.07 g, 15.5 mmol, 1.4 eq) in anhydrous benzene (6 mL) at 0°C, 2-
(13C)acetyl chloride was added drop wise (0.8 mL, 11.07 mmol, 1 eq) under argon atmosphere. Once the
addition completed, the mixture was stirred for 17 h at room temperature. After that time, the mixture was
placed into an ice bath and distilled water (6 mL) was added drop wise until it became white, followed by
addition of HCl 2 M (12 mL). The two phases were separated, and the aqueous phase was extracted with
diethyl ether (4 × 40 mL). The organic phases were combined, washed with brine, dried over MgSO4,
13
4
filtered and concentrated to obtain 1-( C)acetophenone ( )as a yellow oil (1.04 g, 8.58 mmol, 77 %
yield). CAS Registry Number: [71777-36-1]. 1H NMR (CDCl3) δ 2.61 (d, 3H, 1J H1-C1= 127.6 Hz, H1),
7.44-7.50 (m, 2H, H5 and H7), 7.54-7.60 (m, 1H, H6), 7.95-7.99 (m, 2H, H4 and H8); 13C NMR (CDCl3)
δ 26.8 (C1), 128.4 (2C, C5 and C7), 128.7 (2C, C4 and C8), 133.2 (C6), 137.1 (d, 2JC3-C1 = 13.5 Hz, C3),
198.4 (d, 1J C2-C1= 42.5 Hz, C2).

13
5
Synthesis of 1-( C)2-phenylpropan-2-ol ( )
13
4
In a two-necked flask under argon 1-( C)acetophenone ( )(450 mg, 3.71 mmol, 1 eq) was dissolved in
6 mL of anhydrous tetrahydrofuran at -78 °C. Methyllithium (2.8 mL, 4.46 mmol, 1.6 M in Et2O, 1.2 eq)
was added drop by drop and the mixture was stirred at -78 °C for 21 h then at room temperature for 5 h.
Ammonium chloride was added (7 mL) and the solution was extracted with diethyl ether (4 × 20 mL).
Organic phases were combined, dried over MgSO4, filtered and concentrated under reduce pressure to
give an orange oil. The crude product was purified by chromatography on silica gel (pentane/EtOAc 9/1
13
1
5
then 5/5) to afford 1-( C)2-phenylpropan-2-ol ( ) as a yellow oil (269.4 mg, 1.96 mmol, 53 % yield). H
NMR (CDCl3) δ 1.59 (d, 3H, 1JH1-C1 = 126.5 Hz, H1),1.59 (d, 3H, 3JH3-C1 = 4.1 Hz, H3), 7.24-7.27 (m,
1H, H7), 7.34-7.37 (m, 2H, H6 and H8), 7.49-7.51 (m, 2H, H5 and H9);13C NMR (CDCl3) δ 31.9 (2C,
C1 and C3), 72.7 (d, 1JC2-C1= 39.3 Hz, C2), 124.5 (2C, C5 and C9), 126.8 (C7), 128.4 (2C, C6 and C8),
149.2 (C-4).
13
1
Synthesis of 1-( C)cumene hydroperoxide ( )
To an aqueous solution of hydrogen peroxide (30 %, 12.7 mL) at 0°C, one drop of concentrated sulfuric
13
5
acid was added. After 30 min stirring, 1-( C)2-phenylpropan-2-ol ( ) (268.1 mg, 1.95 mmol, 1 eq) was
added and the reaction mixture was vigorously stirred at 0°C during 4 h, followed by extraction with ethyl
acetate (3 × 10 mL). The organic layers were combined, dried over MgSO4,filtered, and concentrated
under reduce pressure. The crude product was purified by flash chromatography on neutralized silica gel
13
1
(pentane/EtOAc 85/15 then 50/50) to obtain 1-( C)cumene hydroperoxide ( )as an orange oil (136.0 mg,
0.88 mmol, 32 % yield).1H NMR (CDCl3) δ 1.62 (d,3H, 3JH3-C1= 4.4 Hz, H3), 1.62 (d, 3H, 1JH1-C1= 127.7
Hz, H1),7.29-7.34 (m, 2 × 1H, H6 and H8),7.38-7.41 (m, 1H, H7), 7.47-7.49 (m, 2 × 1H, H5 and H9);
13C NMR (CDCl3) δ 26.2 (2C, C1 and C3), 84.1 (d, 1JC2-C1= 39.9 Hz, C2), 125.5 (2C, C5 and C9), 127.6
(C7), 128.7 (2C, C6 and C8), 144.7 (C4).
13
6
Synthesis of 1,3-( C)2-phenylpropan-2-ol ( )
Previous synthesis of (13C)methylmagnesium iodide: in a 50 mL two-necked flask under argon,
magnesium (305 mg, 12.53 mmol, 1.2 eq) was flame-dried before to add a spatula tip of iodine and
anhydrous diethyl ether (10 mL). (13C)Methyl iodide (0.65 mL, 10.44 mmol, 1 eq) was then added to the

reaction mixture heated slightly with a hairdryer to initiate the reaction. Diethyl ether was added (10 mL)
and the reaction heated again slightly. The reaction mixture was stirred at room temperature during 24 h.
A solution of (13C)methylmagnesium iodide 0.5 M in diethyl ether was in this way obtained.
In a two-necked flask under argon, zinc chloride (79.1 mg, 0.58 mmol, 0.2 eq) was placed and dried
under reduced pressure. Lithium chloride (295.0 mg, 6.94 mmol, 2.4 eq) was added and the flask was
again dried under reduced pressure. To the mixture, (trimethylsilyl)methylmagnesium chloride (2.30 mL,
1.2 mmol, 0.4 eq) was added drop wise at room temperature and the reaction was stirred for 15 min.
(13C)Methylmagnesium iodide (10 mL, 4.9 mmol, 0.5 M in diethyl ether, 1.7 eq) was then added drop
13
4
wise and the reaction stirred for 45 min and then cooled to 0°C. 1-( C)Acetophenone ( )(350 mg, 2.89
mmol, 1 eq) was added during 1 h with the use of a syringe pump and the reaction was stirred for 3 h at
0°C. After this time, the reaction mixture was quenched with ammonium chloride (10 mL), extracted with
ethyl acetate (3 × 10 mL) and washed with brine (10 mL). The organic phases were combined and dried
over MgSO4, filtered and concentrated under reduced pressure to obtain an orange oil. The crude product
was purified by chromatography on silica gel (pentane/EtOAc 92/8) and 1,3-(13C)2-phenylpropan-2-ol
1
3
6
( ) was obtained as a yellow oil (303.7 mg, 2.19 mmol, 76% yield). H NMR (CDCl ) δ 1.59 (dd, 3H,
3 H1/3-C1/3
1JH1/3-C1/3 = 121.3 Hz, J
1 H1/3-C1/3
= 1.7 Hz, H1 or H3), 1.59 (d, 3H, J
= 130.7 Hz, H-1 or H-3),
7.25-7.28 (m, 1H, H7), 7.34-7.37 (m, 2H, H6 and H8), 7.50-7.51 (m, 2H, H5 and H9); 13C NMR (CDCl3)
δ 31.8 (2C, C1 and C3), 74.0 (t, 1JC2-C1/3= 38.9 Hz, C2), 124.5 (2C, C5 and C9), 126.8 (C7), 128.3 (2C,
C6 and C8), 149.2 (C4).
13
2
Synthesis of 1,3-( C)cumene hydroperoxide ( )
To an aqueous solution of hydrogen peroxide (30 %, 7.5 mL) was added at 0°C one drop of concentrated
13
6
sulfuric acid. After 30 min, 1,3-( C)2-phenylpropan-2-ol ( )(155.9 mg, 1.13 mmol, 1 eq) was added and
the reaction mixture was vigorously stirred at 0°C during 4 h, followed by extraction with ethyl acetate (3
× 10 mL). The organic layers were combined, dried over MgSO4, filtered, and concentrated under reduce
pressure. The crude product was purified by flash chromatography on neutralized silica gel (pentane/
13
2
EtOAc 5/1 and 1/1) to obtain 1,3-( C)cumene hydroperoxide ( )as an orange oil (36.7 mg, 0.24 mmol,
21 % yield).1H NMR (CDCl3) δ 1.62 (d, 3H, 1JH1/3-C1/3 = 123.8 Hz, H1 or H3), 1.62 (d, 3H, 1JH1/3-C1/3
=
132.1 Hz, H1 or H3), 7.29-7.32 (m, 1H, H7), 7.37-7.41 (m, 2H, H6 and H8), 7.47-7.49 (m, 2H, H5 and

H9); 13C NMR (CDCl3) δ 26.2 (2C, C1 and C3), 84.1 (t, 1JC2-C1/3= 39,9 Hz, C2), 125.5 (2C, C5 and C9),
127.6 (C7), 128.7 (2C, C6 and C8), 144.7 (C4).
EPR studies in solution
CumOOH was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and used as
received. DEPMPO was synthesized as reported in the literature [23].HEPES (≥ 99.5 %), ferrous
sulfate heptahydrate FeSO4.7H2O and acetonitrile CH3CN (99.8%) were acquired from Sigma-
Aldrich (Saint-Quentin Fallavier, France). Aqueous solutions were prepared with deionized
water.HEPES buffer solution (10 mM, pH 6.8) was prepared by dissolving 1.19 g HEPES in
400 mL deionized water, with additional 4 g NaCl and 0.1 g KCl. To attain pH 6.8 NaOH pellets
were added. If the pH went too high, it was lowered back by carefully adding HCl (2 M) until
pH remained stable to 6.8. Deionized water was added for a final volume of 500 mL.Stock
solutions were prepared for CumOOH, 13C-MeCumOOH and (13C-Me)2CumOOH (10 mM,
HEPES/CH3CN 9/1), DEPMPO (100 mM in HEPES) and FeSO4.7H2O (10 mM in HEPES). 12.5
4
2


L of DEPMPO solution were mixed with FeSO .7H O (0.5 L for a final 0.1 mM concentration

in the reaction mixture), 5 L of the hydroperoxide solution added and final volume completed

to 50 L. This way, final concentrations in the reaction mixture were 25 mM spin-trap and 0.54
mM hydroperoxide. The reaction mixture was subjected to stirring and further introduced into
the EPR quartz capillary tube.
EPR studies in reconstructed human epidermis
EpiSkinTM (Lyon, France; 0.38 cm2) is a RHE from normal human keratinocytes cultured for 13 days on
a collagen matrix at the air-liquid interface. Immediately after arrival in the laboratory, the RHE were
removed from the agarose-nutrient solution in the shipping multiwell plate under a sterile airflow.They
were placed in a plate in which each well was previously filled with 2 mL EpiSkinTM maintenance or
growth medium at room temperature.Samples were placed in the incubator at 37 °C, 5% CO2 and saturated
humidity, at least 24 h before incubation. EpiSkinTM (Lyon, France) furnished the assay medium used
for incubations. RHE were topically treated first with DEPMPO (20 L, 250 mM in HEPES) and post
incubated (37 °C, 5% CO2) during 15 min. After the incubation time, the RHE were placed in the EPR
tissue cell and the hydroperoxide (20 L, 50 mM in acetone) was applied to the epidermis taking care to
ensure that the solution was only applied to it. EPR spectra were then recorded.
EPR instrumentation

EPR spectra were recorded on an EPR X-band spectrometer (ESP300E, Bruker Biospin GmbH,
Germany), equipped with a high sensitivity resonator (4119HS-W1, Bruker Biospin GmbH,
Germany). The g calibration was performed using Bruker standard (strong pitch) with known
isotropic g factor of 2.0028. The principal experimental parameters values were: microwave
power 5 mW, modulation amplitude 0.5-1 G, and conversion time of ca. 160 ms resulting
sweep time of ca. 3 min s for a single scan. Spectra were recorded at room temperature (295K
±1K).Regarding the investigations in solution,samples were introduced into glass capillaries

(Hirschmann, 25 L), sealed at both ends and transferred into the EPR cavity for measurement
as fast as possible after reagents mixing. RHE were placed in an EPR tissue cell equipped
with a silica window (Willmad, #ER162TC-Q) and resulting EPR spectra recorded in situ right
afterwards. Up to 10 scans were accumulated to improve the signal-to-noise ratio (S/N).
EPR simulations
All experimental spectra were analyzed by means of computer simulation using labmade scripts
based on Easyspin toolbox under Matlab (Mathworks) environment [24]. Relevant 14N, 1H, 31P,
13C hyperfine coupling constants (hfccs) of detected spin-adducts are listed in Table 1. Coupling
constants in RHE were calculated following the best simulation attempts for such S/N.
Results
EPR studies in solution
Analyses in solution were initially carried out to evaluate radicals formed from 13C-substitued CumOOHs
in comparison with previous studies we achieved with non-substituted CumOOH [21]. DEPMPO was
chosen as the spin-trap since we observed previously that it allowed a broad view of all sort of radicals
deriving from CumOOH [21]. Radical initiation was triggered by a catalytic amount of Fe(II) (0.1 mM)
inducing a Fenton-like reaction. DEPMPO was employed in an excess concentration (25 mM) with
respect to 13C-substitued hydroperoxides (0.54 mM). First studies were completed with mono-substituted
13
1
C-MeCumOOH . Figure 2(c) shows the experimental EPR spectrum obtained. Computer simulation of
the signal displayed a mixture of different spin-adducts formed by addition of transient short-lived radicals
1
derived from to the -carbon of DEPMPO nitronyl group. In agreement with our previous investigations
on CumOOH [21], spin-adducts were identified through their hyperfine coupling constants (hfccs) [25,

26] as originating from HO• (Figure 2(e)), carbon centred radicals, probably methyl (Figure 2(f)), methoxy
and/or possible peroxyl radicals (Figure 2(h)). But here, when compared to the non-substituted sample,
an additional hyperfine splittingappeared (Figure 2(g)), attributed to the presence of the paramagnetic
13
probe of a C nucleus with nuclear spin I = ½. Analysis of the spectrum leaded to hfccs values of aN
(G) = 15.5, aH (G) = 22.6, aP (G) = 48.3 and a new value a13C of 6.3 G (Table 1). As only half of methyl
13
radicals were C-substituted, it was possible to observe two spin-adducts corresponding to two types
of carbon radicals trapped with similar aN, aH and aPhfccs, being either non-substituted (Figure 2(f))
13
13
or C-substituted (Figure 2(g)). There are only few examples in the literature describing hfccs for C
atoms, for example the description of p-benzyne radicals [27], -substituted ethyl radicals [28] or some
vinyl and allyl radicals [29]. However, no reference was found on hfccs values for the coupling with 13C
atoms on  position of a nitroxide radical. Thus, to corroborate that 13C-methyl radicals had been trapped
13
2
13
2
by DEPMPO, studies were conducted with di-substituted ( C-Me) CumOOH having C-substituted
methyl groups exclusively. Figure 3(c) shows the experimental EPR spectrum obtained. Here, computer
simulation displayed a mixture of different spin-adducts formed again by trapping of HO• (Figure 3(e)),
carbon centred (Figure 3(f)), methoxy and/or possible peroxyl radicals (Figure 3(g)). Yet, previously
assigned DEPMPO-13CH3 spin-adduct was again detected in a relative higher proportion when compared
13
N
1
to the studies performed with C-MeCumOOH and having very comparable hfccs values of a (G)
= 15.4, aH (G) = 22.6, aP (G) = 48.3 and a13C (G) = 6.4. Thus, the relative intensity of the DEPMPO-
carbon radical spin-adduct derived from CumOOH (Figure 4(a), star) was significantly reduced while
introducing a single 13C-methyl group in the molecule of CumOOH (Figure 4(b)) and was suppressed in
the case of doubly 13C substitution (Figure 4(c)), to the benefit of the spin-adduct corresponding to the
13C-methyl trapped (Figures 4(b)(c), black circle). These results supported that CumOOH derived carbon
centred radicals were, in these experimental conditions, methyl radicals.
EPR studies in reconstructed human epidermis
In order to carry out the experiments, the RHE tissue was loaded beforehand with the spin-trap. As
described previously in studies with tumour promoting peroxides [12], radicals formed from CumOOH
in the skin model being both short lived and potentially generated at low concentrations, it was desirable
for the spin-trap to be as close as possible to the site of radical generation prior to the topical application

of the hydroperoxides. If similar EPR fingerprints to those of the experiments in solution were obtained, it
was noticeable that other features did not fully overlay with any of the solution studies. Such differences
can be explained by the much more complex system formed by the RHE when compared to simplified in
solution investigations. Figure 4 shows the spectra obtained in solution along and in comparison with the
spectra obtained in RHE for the three hydroperoxides tested. If the appearance of the spin-adduct resulting
13
from trapping the radical C-substituted in RHE was barely visible when using13C-MeCumOOH
1
(Figure 4(f)), it became more evident with (13C-Me)2CumOOH (Figure 4(g)). Major peaks could
be assigned to DEPMPO-OH, DEPMPO-CH3 and DEPMPO-13CH3 spin-adducts by comparison with
previous data in solution (Table 1) [21]. Again, the difference observed when comparing the hfccs values
with those obtained in the EPR-ST solution studies could be explained by the distinct environment
of the spin-adducts in solution compared to that of a complex heterogeneous tissue such as the RHE.
Indeed, magnitudes of the hfccs pointing to the extent of delocalization of the unpaired electron over
the molecule may be affected by the local spin environment and polarity. Moreover, control experiments
were carried out for each compound by simply incubating DEPMPO in the RHE and showed no EPR
signal, corroborating that spin-adducts resulted from trapping radicals issued from the hydroperoxide.
Also, after penetrating the RHE, CumOOH radical initiation has been induced by RHE matrix itself as
no Fe(II) was supplemented, thus better mimicking real life exposure scenarios. These results suggested
methyl radicals being formed in RHE after CumOOH exposure.
2
Discussion
In a recent work, we employed EPR-ST for the first time in the skin allergy field to study the potential
of this methodology to investigate in situ the formation of free radicals from skin allergens in 3D RHE
models [21]. CumOOH was used as proof of concept and we demonstrated the formation of carbon
radicals in RHE. However, no evidence could be obtained concerning their specific nature. In the present
13
work, this was investigated by using C-substitution as a tool to unravel which carbon radicals could
be formed within the epidermis. As the major mechanism described for the formation of carbon radicals
from CumOOH is -scission of cumyl oxyl radicals affording methyl radicals, we developed the synthesis

13
13
2
1
2
of CumOOH containing either one ( C-MeCumOOH ) or both (( C-Me) CumOOH ) methyl groups
13C-substituted and we further studied the formation of free radicals in solution and in RHE.
Preliminary studies in solution allowed setting up the optimal experimental conditions providing
a decent signal-to-noise ratio while keeping low CumOOHs concentrations, i.e. suitable with
physiological conditions. DEPMPO was chosen as an accurate spin-trap based upon (i) the
high persistency of its superoxide and alkylperoxyl spin-adducts when compared to other spin-
traps (e.g. DMPO); (ii) the clear distinction between oxygen- and carbon-derived spin-adducts
[25, 30]. Radical initiation was performed via a Fe(II) Fenton-like reaction. As for CumOOH,
carbon centered, CumO• and HO• radicals were identified for 13C-MeCumOOH 1 and (13C-
Me)2CumOOH 2. A new signal appeared in the case of mono-substituted 1 with hfccsaN (G) =
15.5, aH (G) = 22.6, aP (G) = 48.3 and a13C (G) = 6.3, whose fingerprint significantly increased
in the case of the double-substituted compound 2,fully replacing the signal of carbon centered
radicals trapped from non-substituted CumOOH. Thus, when all methyl groups were 13C-
substituted, only DEPMPO-13CH3 spin-adducts were detected confirming that methyl radicals
were the carbon-centered trapped radicals. This information was of high relevance for further
studies in RHE.
RHE EpiSkinTM shows reasonable similarities to native human tissue in terms if morphology,
stratum corneum presence, lipid composition and biochemical markers [22].It is today a useful
tool for testing irritation and penetration features of chemicals in contact with the skin as
replacement of in vivo testing [31]. For our studies on skin allergy the availability of EpiSkinTM
RHE is a very good alternative to the use of human and animal tissues as the model amends
important legal and ethical issues. Although considerably more permeable than human skin
on average, the model appears to be more consistent in permeability and responsiveness
than human skin which is highly variable. Similar EPR-ST results were observed in RHE when
testing CumOOH, 13C-MeCumOOH 1 and (13C-Me)2CumOOH 2. Though, new features in the
RHE samples did not fully overlay with the reference spectra in solution and were ascribed
to the complexity of the RHE system. Even if the S/N was significantly lower when compared
to solution investigations and thus did not allow quantification of the spin-adducts, we can
still suggest that methyl radicals issued from CumOOH decomposition can be formed in RHE
EpiSkinTM. Indeed, spectrum of the RHE experiment with twice 13C-labeled CumOOH (Figure
4(g)) was rather convincing as an additional spin-adduct assigned to DEPMPO-13CH3 spin-
adduct (black circles) was detected. Moreover, control experiments with DEPMPO, in absence
of 13C-MeCumOOH 1 or (13C-Me)2CumOOH 2,did not exhibit any EPR signal (Figure 4(d)),
•
suggesting that the trapped CH3 radicals originate from the hydroperoxide. Initially formed
•
3

alkoxyl radicals derived from the O-O bond cleavage and giving the CH radicals by -scission
could also react with biomolecules in the RHE before DEPMPO trapping occurred. However,
these potential side-reactions were not the purpose of the present study and will be part of future
investigations. Worthy of note, no Fe(II) was used for radical initiation when probing RHE, to

mimic real life skin allergy situation, whereas we previously showed that Fe(II) was required
to initiate the free radical generation when mixing CumOOH and DEPMPO in solution [21].
As it is shown in Figure 5(d), no EPR signal was observed in the absence of Fe(II), whereas
0.1 mM Fe(II) was enough to initiate the radical reaction with a Fe(II) concentration dependent
answer on the amount of spin-adducts formed. We can then hypothesize that reaction of
organic peroxides in the skin in the presence of one-electron donor agents (i.e. amino and thiol
groups present in amino acids, metal complexes, enzymes) may initiate the electron transfer
processes and further skin proteins haptenation [32-34]. Also, the potential presence in the skin
of Fe(III) heme-containing enzymes could be responsible for the peroxide O-O bond cleavage
and start radical processes. Reactions of model Fe(III) porphyrin complexes with oxidants, such
as peroxyacids and hydroperoxides, have been extensively studied as models. The cleavage
has been confirmed but a clear mechanistic consensus has not evolved on the homolytic or
heterolytic nature of the O-O bond cleavage by these species, both kinds of mechanisms being
possible [35-37].
While skin allergens (haptens) are unable to stimulate an adaptive immune response
after penetration into the epidermis, the immunogenicity is reached by reaction with skin
proteins, to form stable antigenic conjugates recognized and processed for presentation to the
immune system [38]. Therefore, the hapten-protein interaction is the primary key step to induce
sensitization and was shown to occur relatively quickly in RHE [39]. The resulting sequence
of events leading epidermal cells to present the antigen so formed to the immune system and
its activation take 10 to 15 days in humans. The fast trapping by DEPMPO allowed EPR-ST
characterization of the generated methyl radicals but the reaction with epidermal proteins was
not evidenced. Thus, if we cannot testify so far that these radicals are the reacting species for
the antigen formation we can strongly suggest their involvement. Additional tests to decipher
their reactivity towards amino acids in the skin could be isolation from RHE of modified proteins
(reactivity in the absence of spin-trap) by sonication followed by mass spectrometry analysis
and will be addressed in a near future.
Conclusion
This work shows by using 13C-substituted compounds and EPR-ST that the production of methyl radicals
from CumOOH previously reported in isolated keratinocytes and other model systems is also possible in
reconstructed skin tissue through a one-electron reductive pathway, and suggest that these methyl radicals
could be involved in the antigen formation responsible for skin sensitization to CumOOH.
Acknowledgements

This work was part of the DEFCHEMSKALL Franco-German Collaborative International Research
Project financially supported by the ANR (Agence Nationale de la Recherche; project no. ANR-15-
CE15-0023-01) and the DFG (Deutsche Forschungsgemeinschaft;project no. DFG, BL340/6-1). The
authors thank the University of Strasbourg, the CNRS and the REseau NAtional de Rpe interDisciplinaire
(RENARD, Fédération IR-RPE CNRS #3443).

References
[1] Yaremenko IA, Vil’ VA, Demchuk DV, Terent’ev AO. Rearrangements of organic peroxides and
related processes. Beilstein J Org Chem 2016; 12: 1647-1748.
[2] Rider CV, Chan P, Herbert RA, et al. Dermal exposure to cumene hydroperoxide: assessing its toxic
relevance and oxidant potential. Toxicol Pathol 2016; 44: 749-762.
[3] Lai DY, Woo Y, Argus MF, Arcos JC. Carcinogenic potential of organic peroxides: prediction based on
structure-activity relationships (SAR) and mechanism-based short-term tests. J Environ Sci Health
Part C Environ Carcinog Ecotoxicol Rev 1996; 14: 63-80.
[4] Hanausek M, Walaszek Z, Viaje A, et al. Exposure of mouse skin to organic peroxides: subchronic
effects related to carcinogenic potential. Carcinogenesis 2004; 25: 431-437.
[5] Brink A, Richter I, Lutz U, et al. Biological significance of DNA adducts: comparison of increments
over background for various biomarkers of genotoxicity in L5178Y tk(+/-) mouse lymphoma cells
treated with hydrogen peroxide and cumene hydroperoxide. Mutat Res 2009; 678: 123-128.
[6] Bråred Christensson J, Matura M, Bäcktorp C, Börje A, Nilsson JL, Karlberg AT. Hydroperoxides
form specific antigens in contact allergy. Contact Dermatitis 2006; 55: 230-237.
[7] Lauricella R, Tuccio B. Détection et caractérisation de radicaux libres après piégeage de spins. In:
La Spectroscopie de Résonance Paramagnétique Électronique, 2014, Bertrand P Ed. edp Sciences,
Collection Grenoble Sciences, Les Ulis, France, pp. 49-78.
[8] He G, Samouilov A, Kuppusamy P, Zweier JL. In vivo EPR imaging of the distribution and
metabolism of nitroxide radicals in human skin. J Magn Reson 2001; 148: 155-164.
[9] Jurkiewicz BA, Buettner GR. EPR detection of free radicals in UV-irradiated skin: mouse versus
human.Photochem Photobiol 1996; 64: 918-922.
[10] Plonka PM. Electron paramagnetic resonance as a unique tool for skin and hair research. Exp Dermatol
2009; 18: 472-484.
[11] Taffe BG, Takahashi N, Kensler TW, Mason RP. Generation of free radicals from organic
hydroperoxide tumor promoters in isolated mouse keratinocytes. J Biol Chem 1987; 262:
12143-12149.
[12] Timmins GS, Davies MJ. Free radical formation in murine skin treated with tumour promoting organic
peroxides. Carcinogenesis 1993; 14: 1499-1503.
[13] Timmins GS, Davies MJ. Free radical formation in isolated murine keratinocytes treated with organic
peroxides and its modulation by antioxidants. Carcinogenesis 1993; 14: 1615-1620.
[14] Shvedova AA, Kisin ER, Murray AR, et al. Antioxidant balance and free radical generation in vitamin
E-deficient mice after dermal exposure to cumene hydroperoxide. Chem Res Toxicol 2002; 15:
1451-1459.
[15] Shvedova AA, Kisin ER, Murray AR, et al. Pro/antioxidant status in murine skin following topical
exposure to cumene hydroperoxide throughout the ontogeny of skin cancer. Biochemistry (Moscow)
2004; 69: 23-31.
[16] Sahli F, Silva e Sousa M, Vileno B, Lichter J, Lepoittevin JP, Blömeke B, Giménez-Arnau E.
Understanding the skin sensitization capacity of ascaridole: a combined study of chemical reactivity
and activation of the innate immune system (dendritic cells) on the epidermal environment. Arch
Toxicol 2019, DOI: 10.1007/s00204-019-02444-3.

[17] Kao D, Chaintreau A, Lepoittevin JP, Giménez-Arnau E. Synthesis of allylic hydroperoxides and
EPR spin-trapping studies on the formation of radicals in iron systems as potential initiators of the
sensitizing pathway. J Org Chem 2011; 76: 6188-6200.
[18] Kao D, Chaintreau A, Lepoittevin JP, Giménez-Arnau E. Mechanistic studies on the reactivity of
sensitizing allylic hydroperoxides: investigation of the covalent modification of amino acids by
carbon-radical intermediates. Toxicol Res 2014; 3: 278-289.
[19] Bråred-Christensson J, Johansson S, Hagvall L, Jonsson C, Börje A, Karlberg AT. Limonene
hydroperoxide analogues differ in allergenic activity. Contact Dermatitis 2008; 59: 344-352.
[20] Deza G, García-Bravo B, Silvestre JF, Pastor MA, González-Pérez R, Heras-Mendaza F, Mercader P,
Fernández-Redondo V, Niklasson B. Giménez-Arnau A, GEIDAC. Contact sensitization to limonene
and linalool hydroperoxides in Spain: a GEIDAC prospective study. Contact Dermatitis 2017; 76:
74-80.
[21] Kuresepi S, Vileno B, Turek P, Lepoittevin JP, Giménez-Arnau E. Potential of EPR spin-trapping to
investigate in situ free radicals generation from skin allergens in reconstructed human epidermis:
cumene hydroperoxide as proof of concept. Free Radic Res 2018; 52: 171-179.
[22] Netzlaff F, Lehr CM, Wertz PW, Schaefer UF. The human epidermis models EpiSkin®, SkinEthic®
and EpiDerm®: an evaluation of morphology and suitability for testing phototoxicity, irritancy,
corrosivity, and substance transport. Eur J Pharm Biopharm 2005; 60: 167-178.
[23] Barbati S, Clément JL, Olive G, Roubaud V, Tuccio B, Tordo P. InMinisci F. Ed. Free Radicals in
Biology and Environment. Kluwer Academic Publishers, Dordrecht, The Netherlands, 1997, pp.
39-47.
[24] Stoll S, Schweiger A. EasySpin, a comprehensive software package for spectral simulation and
analysis in EPR. J Magn Res 2006; 178: 42-55.
[25] Frejaville C, Karoui H, Tuccio B, et al. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide: A
new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered
radicals. J Med Chem 1995; 38: 258-265.
[26] Karoui H, Chalier F, Finet JP, Tordo P. DEPMPO: an efficient tool for the coupled ESR-spin trapping
of alkylperoxyl radicals in water. Org Biomol Chem 2011; 9: 2473-2480.
13
[27] Usuki T, Mita T, Lear MJ, et al. Spin trapping of C-labeled p-benzynes generated by Masamune-
Bergman cyclization of bicyclic nine-membered enediynes. Angew Chem Int Ed2004: 43;
5249-5253.
[28] Griller D, Ingold KU. On the conformation of β-substituted ethyl radicals. J Am Chem Soc 1974: 96;
6715-6720.
[29] Griller D, Cooper JW, Ingold KU. Kinetic applications of electron paramagnetic resonance
spectroscopy. XVIII. Persistent vinyl, alkyl, and allyl radicals. J Am Chem Soc 1975: 97; 4269-4275.
[30] Stolze K, Udilova N, Nohl H. Spin trapping of lipid radicals with DEPMPO-derived spin traps:
detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase. Free Radic Biol Med
2000; 29: 1005-1014.
[31] OECD testing guideline 439, 2015. In vitro skin irritation: RHE test method. DOI:
10.1787/9789264242845-en.

[32] Moriarty-Craige SE, Jones DP. Extracellular thiols and thiol/disulphide redox in metabolism. Annu
Rev Nutr 2004; 24: 481-509.
[33] Fujimoto A, Masuda T. Chemical interaction between polyphenols and a cysteinyl thiol under radical
oxidation controls. J Agric Food Chem 2012; 60: 5142-5151.
[34] Grossi L. The mechanism of oxidation of aniline by peroxy-compounds: can EPR spectroscopy help
to discriminate between one-electron transfer and a nucleophilic substitution process? Res Chem
Intermed 1993; 19: 697-705.
[35] Traylor TJ, Kim C, Fann WP, Perrin CL. Reactions of hydroperoxides with iron (III) porphyrins:
heterolytic cleavage followed by hydroperoxide oxidation. Tetrahedron 1998; 54: 7977-7986.
[36] Almarsson O, Bruice TC. A Homolytic mechanism of O-O bond scission prevails in the reactions
of alkyl hydroperoxides with an octacationic tetraphenylporphinato-iron(III) complex in aqueous
solution. J Am Chem Soc 1995; 117: 4533-4544.
[37] Labeque R, Marnett LJ. Homolytic and heterolytic scission of organic hydroperoxides by (meso-
tetraphenylporphinato)iron(III) and its relation to olefin epoxidation. J Am Chem Soc 1989; 111:
6621-6627.
[38] Lepoittevin JP. Molecular aspects in allergic and irritant contact dermatitis. In Johansen JD, Frosch
PJ, Lepoittevin JP(Eds.) Contact Dermatitis 5th Ed. Springer-Verlag, Berlin, Heidelberg, 2011, pp.
91-110.
[39] Elbayed K, Berl V, Debeuckelaere C, Moussallieh FM, Piotto M, Namer IJ, Lepoittevin JP. HR-MAS
NMR spectroscopy of reconstructed human epidermis: potential for the in situ investigation of the
chemical interactions between skin allergens and nucleophilic amino acids. Chem Res Toxicol 2013;
26: 136-145.

Table 1
Table 1. Hyperfine coupling constants (hfccs) of spin-adducts identified in solution (sol.) and in RHE*
Cpd.
Coupling constants
aH (G) aP(G) a13C(G)
Assignment
aN (G)
g
CumOOH
(sol.) [21]
1(sol.)
14.2
15.4
11.3
14.2
15.4
15.5
11.3
14.2
15.4
11.6
12.5
14.8
12.9
14.5
14.9
13.1
15.1
13.5 47.4
2.0055
2.0053
2.0055
2.0056
2.0053
2.0053
2.0055
2.0053
2.0051
2.0055
2.0064
2.0058
2.0064
2.0056
2.0056
2.0063
2.0059
DEPMPO-OH
22.6 48.1
13.7 48.5
13.3 47.3
22.6 48.0
22.6 48.3 6.3
13.7 48.2
13.4 47.4
22.6 48.3 6.4
14.4 48.1
13.6 46.0
21.4 48.0
13.7 46.0
21.2 47.3
21.6 47.9 6.2
14.0 46.4
21.4 48.2 6.4
DEPMPO-carbon R
DEPMPO-OCH3 and/or
DEPMPO-OOCum
DEPMPO-OH
2 (sol.)
CumOOH
(RHE)
1 (RHE)
2 (RHE)
DEPMPO-carbon R
DEPMPO-13CH3
DEPMPO-OCH3 and/or
DEPMPO-OOCum
DEPMPO-OH
DEPMPO-13CH3
DEPMPO-OCH3 and/or
DEPMPO-OOCum
DEPMPO-OH
DEPMPO-carbon R
DEPMPO-OH
DEPMPO-carbon R
DEPMPO-13CH3
DEPMPO-OH
DEPMPO-13CH3
* hfccs in RHE were calculated following the best simulation attempts considering the S/N

Scheme 1

Scheme 2

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5
>Converted from a word shape, significant formatting may have been lost:
(Gaa)uss
(b)
(c)
(d)
Solution
+DEPMPO
+Fe(II) 1.0 mM
+CumOOH
Solution
+DEPMPO

Legends
Scheme 1.
Scheme 2.
Radical process leading to the formation of methyl radicals from CumOOH.
13
13
2
13
1
2
4
Synthetic pathway to obtain C-MeCumOOH and ( C-Me) CumOOH . 1-( C)Acetophenone
was the key starting material for both compounds.
13
13
2
Figure 1.
Figure 2.
1
2
Chemical structures of C-MeCumOOH and ( C-Me) CumOOH .
13
1
EPR spectra of C-MeCumOOH /DEPMPO/Fe(II) in solution. (a) Control with single DEPMPO (25
mM); (b) Control with DEPMPO (25 mM) and Fe(II) (1 mM); (c) Experimental spectrum obtained with 13C-
1
MeCumOOH (0.54 mM), DEPMPO (25 mM) and Fe(II) (0.1 mM); (d) Computer simulation of spectrum (c);
(e-h) deconvolution of (d) with: (e) DEPMPO-OH spin-adduct (45%); (f) DEPMPO-carbon R spin-adduct (28%);
(g) DEPMPO-13CH3 spin-adduct (23%); (h) DEPMPO-OCH3 and/or DEPMPO-OOCum spin-adduct (4%).
13
2
Figure 3.
2
EPR spectra of ( C-Me) CumOOH /DEPMPO/Fe(II) in solution. (a) Control with single DEPMPO
(25 mM); (b) Control with DEPMPO (25 mM) and Fe(II) (1 mM); (c) Experimental spectrum obtained with (13C-
2
2
Me) CumOOH (0.54 mM), DEPMPO (25 mM) and Fe(II) (0.1 mM); (d) Computer simulation of spectrum (c);
(e-g) deconvolution of (d) with: (e) DEPMPO-OH spin-adduct (55%); (f) DEPMPO-13CH3 spin-adduct (42%); (g)
DEPMPO-OCH3 and/or DEPMPO-OOCum spin-adduct (3%).
Figure 4.
Comparative experimental EPR spectra in solution (a-c) and in RHE (d-g): (a) CumOOH (0.54 mM)/
13
1
DEPMPO (25 mM)/Fe (II) (0.1 mM) [21]; (b) C-MeCumOOH (0.54 mM)/DEPMPO (25 mM)/Fe (II) (0.1
mM); (c) (13C-Me)2CumOOH (0.54 mM)/DEPMPO (25 mM)/Fe (II) (0.1 mM); (d) DEPMPO (250 mM)/
2
13
1
RHE; (e) CumOOH (10 mM)/DEPMPO (250 mM)/RHE; (f) C-MeCumOOH (50 mM)/DEPMPO (250 mM)/
13
2
2
RHE; (g) ( C-Me) CumOOH (50 mM)/DEPMPO (250 mM)/RHE. Because of the low signal-to-noise ratio,
DEPMPO-OCH3 and/or DEPMPO-OOCum spin-adducts are no indicated for clarification purposes. Spin-adducts
are indicated as follows: DEPMPO-OH white circle, DEPMPO-CH3 star and DEPMPO-13CH3 black circle. Dotted
arrows indicate the formation of signals related to DEPMPO-13CH3 spin-adduct in RHE.
Figure 5.
EPR experimental spectra in solution of CumOOH (0.54 mM)/DEPMPO (25 mM) with varying
concentrations of Fe (II): (a) Fe(II) 1.0 mM; (b) Fe(II) 0.5 mM; (c) Fe(II) 0.1 mM; (d) in the absence of Fe(II).
1