Probing Lipid Peroxidation
FULL PAPER
based on H(1)–H(5). Indeed, the multiplet at d=6.31 ppm,
appearing in enhanced absorption, can be straightforwardly
assigned to the vinylic H(3) protons of both isomers of con-
jugated derivatives of LA (CLA1, CLA2). Additionally, po-
larizations of the protons H(2) and H(4) of CLA1 of CLA2,
respectively, appear at d=5.82 ppm. Those conjugated acids
are formed by hydrogen abstraction of one of the double al-
lylic hydrogen atoms H(3) of LA by the triplet excited state
of BZP. Reverse hydrogen transfer, that is, hydrogen trans-
Experimental Section
General: BZP, CHD, and MBA were commercially available, whereas
DMBA was synthesized through Birch reduction and subsequent methyl-
ation of 9. The 1H and 13C NMR spectra were recorded at 300 and
75 MHz, respectively, on a Bruker AC-300 spectrometer in CDCl3; chem-
ical shifts are reported in ppm downfield from an internal solvent signal.
GC-MS analysis was performed on an Agilent mass selective detector
coupled to an HP GC system. High-resolution mass spectra (EI, FAB)
were recorded on a VG Auto Spec Fisons spectrometer instrument. All
reactions were monitored by analytical TLC with silica gel 60 F254
(Merck), revealed with cerium ammonium sulfate/ammonium molybdate
reagent. Column chromatogaphy was performed on silica gel 60 (0.063–
0.2 mm) and HPLC was carried out by using a C-18, Kromasil column.
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C
fer from the ketyl radical (H(1) of BZPH ) to LA leads to
the formation of a conjugated diene system. This is in agree-
ment with DFT calculations and EPR spectroscopy experi-
ments.[14] The background CIDNP spectrum indicates strong
polarization of H(1)–H(5) of parent LA, which points to the
reversibility of hydrogen abstraction: H(1) of the ketyl radi-
cal is transferred back to regenerate parent LA as well as its
trans isomer. The signal corresponding to trans-LA is found
at d=5.25 ppm (Figure 7b). The triplet at d=4.51 ppm (J=
9.9 Hz) is attributed to the tertiary proton of LA–BZP, pro-
General irradiation procedure
Procedure A: Solutions of CHD, MBA or DMBA (1.5ꢄ10À3 m) and BZP
(1.0ꢄ10À4 m) in acetonitrile were irradiated in a multilamp photoreactor
at lmax =350 nm (Gaussian distribution) under an argon atmosphere.
They were monitored by UV spectrophotometry, following the disappear-
ance of the absorption band at 254 nm
Procedure B: Deaerated solutions of CHD, MBA or DMBA (1.3ꢄ
10À1 m) and BZP (3.3ꢄ10À2 m) in acetonitrile were irradiated through
Pyrex with a 400 W medium-pressure mercury lamp. Reactions were
monitored by TLC and NMR spectroscopy. After 10 h, the reaction mix-
tures were concentrated under reduced pressure and submitted to chro-
matography. The CHD and BZP reaction mixture was purified by silica
gel column chromatography, using hexane/ethyl acetate (90:10 v/v) as the
eluent. The reaction mixtures obtained by irradiation of MBA or DMBA
and BZP were separated by reverse-phase HPLC chromatography, using
acetonitrile/water/acetic acid (70:29.7:0.3 v/v/v) as the eluent.
C
duced by coupling of primary ketyl and LA radicals. The
mechanism of the photoreaction of LA with BZP is present-
ed in Scheme 3.
LA also undergoes partly reversible hydrogen abstraction
during photoreaction with BZP. Follow-up hydrogen transfer
between primary radicals leads to the formation of conjugat-
ed derivatives of LA. On the other side, reverse hydrogen
transition can direct cis–trans isomerization of the parent
Laser flash photolysis measurements: A pulsed Nd:YAG laser (SL404G-
10 Spectrum Laser Systems) was used for the excitation at 355 nm. The
single pulses were about 10 ns in duration and the energy was 16 mJ per
pulse. The laser flash photolysis apparatus consisted of the pulsed laser, a
Xe lamp, a monochromator, and a photomultiplier made up of a tube,
housing, and power supply. The output signal from the oscilloscope was
transferred to a personal computer for data analysis. All experiments
were carried out at room temperature. The sensitizer (BZP) dissolved in
acetonitrile (3.5 mm) had an absorbance of about 0.3 at 355 nm. Solutions
were deaerated by bubbling nitrogen through the solution. The rate con-
stants of triplet excited-state quenching by CHD, MBA, and DMBA
were determined by the Stern–Volmer equation (1/t=1/t0 +kq-
C
LA. The unpaired electron spin population in LA is pre-
dominantly located on protons H(1)–H(5).
Conclusion
The photoreaction between BZP and model systems CHD,
MBA, and DMBA proceeds by hydrogen abstraction of the
allylic hydrogen by the triplet excited state of BZP. In neat
acetonitrile, all three model compounds show similar reac-
AHCTUNGTRENNUNG
3
tivity towards BZP*, the lifetimes of which in the presence
1
Photo-CIDNP experiments: H NMR and CIDNP spectra were recorded
on a 200 MHz Bruker AVANCE DPX spectrometer. Irradiation was car-
ried out by using a frequency-tripled Spectra Physics Nd:YAG INDI
laser (355 nm, ca. 40 mJ per pulse, ca. 10 ns) and a Hamamatsu (Japan)
Hg–Xe lamp (SP4, L8252 lamp, 150 W, 300 ms). The following pulse se-
quence was used: presaturation laser/lamp flash–308–RF–detection pulse
(2.2 ms)–free induction decay. The concentrations of BZP, CHD, MBA,
DMBA, and LA were 0.01m. The hyperfine coupling constants (hfc) of
the free radicals were calculated by using the Gaussian 03 package.[15] All
calculations (geometry optimizations and single-point calculations) were
conducted at the B3LYP[16] level of theory with the basis set TZVP.[17]
of dienes were found to be in good agreement with previous
studies on CHD in benzene.[4a]
Partly reversible hydrogen abstraction of the allylic hydro-
gen atoms of CHD, MBA, and DMBA was also detected by
photo-CIDNP on different timescales. CIDNP polarizations
of the diamagnetic products were in a full agreement with
the results derived by LFP.
Similar processes have been found for LA, yet LA-related
radicals were only reported when generated by hydrogen
transfer from highly substituted model compounds, allowing
their observation by steady-state EPR spectroscopy.[14]
Herein, we have experimentally established the formation
Compound 1: 1H NMR (300 MHz; CDCl3, TMS): d=7.6–7.1 (m, 10H),
5.9 (m, 2H), 5.5 (m, 2H), 4.1 (m, 1H), 2.7 ppm (m, 2H); 13C NMR
(75 MHz; CDCl3, TMS): d=146.1, 133.0–125.0, 79.5, 44.4, 27.2 ppm;
HRMS: m/z calcd for C19H19O: 263.14359 [M H]+; found: 263.14379.
Compound 2: 1H NMR (300 MHz; CDCl3, TMS): d=7.6–7.1 (m, 10H),
5.8 (m, 1H), 5.6 (m, 1H), 5.3 (m, 1H), 4.1 (m, 1H), 3.6 (m, 1H), 1.6 ppm
(s, 3H); 13C NMR (75 MHz; CDCl3, TMS): d=175.2, 144.7, 144.3, 127.3,
127.3, 127.0, 125.7, 125.6, 124.6, 124.5, 121.5, 78.5, 47.7, 44.4, 21.2 ppm;
HRMS: m/z calcd for C21H18O2: 302.13068 [MÀ18]+; found: 302.12991.
C
C
of LA and shown that LA converts into two dominating
conjugated isomers on the ms timescale. Such processes are
the basis of alterations of membrane structures caused by
oxidative stress.
1
Compound 3: H NMR (300 MHz; CDCl3): d=7.6–7.1 (m, 10H), 5.9 (m,
1H), 5.7 (m, 1H), 5.5 (m, 1H), 4.1 (m, 1H), 3.6 (m, 1H), 1.7 ppm (s,
3H); 13C NMR (75 MHz; CDCl3): d=177.4, 145.4, 145.3, 133.1, 128.4,
Chem. Eur. J. 2011, 17, 10089 – 10096
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10095