93
1988a,b; Hamberg and Fahlstadius, 1990). Plants possess the allene
GC–MS analyses
from ␣-linolenic acid 13(S)-hydroperoxide) to cyclopentenone
plays some regulatory roles in plants (Böttcher and Pollmann,
and Zimmerman, 1983; Wasternack and Kombrink, 2010).
lylating reagents leads to TMS-peroxy derivatives (Turnipseed
et al., 1993). These TMS derivatives can serve as a useful tool
for biomimetic studies of peroxide decompositions. Previously
we studied the thermal conversions of fatty acid TMS-peroxides
accompanying their GC–MS analyses (Grechkin et al., 2005).
mimic the metabolism of hydroperoxides occurring in differ-
ent living organisms. For instance, the hemiacetals are the true
primary products of CYP74B and CYP74C hydroperoxide lyases
(Grechkin and Hamberg, 2004; Grechkin et al., 2006). Reinves-
tigation of these thermal reactions occurring during the GC–MS
analyses enabled us to detect the cyclopentenones. Moreover,
the thermal treatments of hydroperoxides (Me esters), sealed
in ampoules, repeatedly afforded the cyclopentenones 10-oxo-
11-phytoenoic, 10-oxo-9(13)-phytoenoic, 12-oxo-10-phytoenoic
and 12-oxo-9(13)-phytoenoic acid methyl esters. These conver-
sions mimicking the AOS pathway are described in the present
paper.
methyl esters or methyl esters/TMS derivatives by GC–MS as
described before (Grechkin et al., 2005). GC–MS analyses were per-
formed using a Shimadzu QP5050A mass spectrometer connected
to Shimadzu GC-17A gas chromatograph equipped with an MDN-
5S (5% phenyl; 95% methylpolysiloxane) fused capillary column
(length, 30 m; ID 0.25 mm; film thickness, 0.25 m). Helium at a
flow rate of 30 cm/s was used as the carrier gas. Injections were
made in the split mode using an initial column temperature of
120 ◦C, injector temperature 230 ◦C. The column temperature was
raised at 10 ◦C/min until 240 ◦C. The electron impact ionization
(70 eV) has been used. The high resolution GC–MS analyses were
performed using a DFS mass spectrometer (Thermo Scientific) con-
nected to the Trace GC Ultra gas chromatograph (Thermo Scientific)
equipped with an DB-5MS (5% phenyl; 95% methylpolysiloxane)
fused capillary column (length, 30 m; ID 0.25 mm; film thickness,
0.1 mm). Helium at a flow rate of 30 cm/s was used as the carrier
gas. Injections were made in the split mode. The temperature con-
ditions were the same as described above for Shimadzu GC–MS
system. The electron impact ionization (70 eV) has been used. The
accurate m/z values were measured using the perfluorokerosene as
a reference for mass calibration.
2.4. Thermal treatment of fatty acid hydroperoxides sealed in
ampoules
The methyl esters of 13-HPOD or 9-HPOD (1 mg each) sealed
in ampoules under argon were subjected to thermal treatment
at 230 ◦C for 15 or 30 min. The products of thermal treatment
were separated by reversed phase HPLC (RP-HPLC) on Macherey-
Nagel Nucleosil 5 ODS column (250 mm × 4.6 mm) eluted with
methanol–water (linear gradient from 76:24 to 96:4, by vol.) at
a flow rate of 0.4 mL/min. The diode array detection (190–350 nm)
with SPD-M20A instrument (Shimadzu) has been used. The prod-
ucts were collected, dissolved in hexane and analyzed by GC–MS.
2. Experimental procedures
2.1. Materials
incubation of linoleic acid with tomato fruit at 0 ◦C, pH 6.0, under
continuous oxygen bubbling as described before (Grechkin et al.,
2008). 13-HPOD was obtained by incubation of linoleic acid with
soybean lipoxygenase type V as described before (Chechetkin et al.,
2008). All hydroperoxides were purified by normal phase HPLC
(NP-HPLC).
reference standards
10-Oxo-PEA was obtained as described before (Grechkin et al.,
2008) by incubation of 9(S)-HPOD with the recombinant tomato
AOS, LeAOS3 (CYP74C3). Solution of (9S)-HPOD (100 g) in ethanol
(10 L) was added to LeAOS3 suspension (5 g of purified pro-
tein) in 100 mM phosphate buffer (1 mL), pH 7.0, and the reaction
was allowed to proceed for 15 min at 23 ◦C. Then the incu-
bation mixture was acidified with acetic acid to pH 5–6 and
extracted with hexane/ethyl acetate (1:1, v/v). The extract was
concentrated in vacuo about 2-fold and treated with ethereal
diazomethane at −20 ◦C for 1 min. The products (Me esters)
were separated by RP-HPLC on Macherey-Nagel Nucleosil 5 ODS
column (250 mm × 4.6 mm) eluted with methanol–water (lin-
ear gradient from 76:24 to 96:4, by vol.) at a flow rate of
0.4 mL/min. Fractions of cis- and trans-10-oxo-PEA (Me esters) were
collected and re-chromatographed by NP-HPLC on two serially con-
nected Separon SIX columns (150 mm × 3.2 mm; 5 m) eluted with
hexane–isopropanol 99.2:0.8 (by volume), flow rate 0.4 mL/min.
Alternatively, the LeAOS3 products were separated as the free
fatty acids. The same HPLC conditions have been used with one
exception: all solvents were acidified with 0.1% of acetic acid. Pure
samples of trans- and cis-10-oxo-PEA were trimethylsilylated as
described above to obtain their TMS esters.
2.2. Derivatization of fatty acid hydroperoxides and other
oxylipins
Samples of 13-HPOD or 9-HPOD (1 mol each) were taken
to dryness under
a stream of nitrogen and treated with a
mixture of pyridine/hexamethyldisilazane/trimethylchlorosilane
2:1:2 (by volume) for 10 min at ambient temperature. Sily-
lation reagents were evaporated in vacuo and the remaining
material was extracted with 1 mL of hexane. The solvent was
evaporated under a stream of argon and the oily residue of
TMS/TMS derivatives was dissolved in 0.1 mL of hexane for GC–MS
analyses. Alternatively, 13-HPOD and 9-HPOD were trimethylsi-
lylated as methyl esters after the preliminary methylation with
ethereal diazomethane. The resulting Me/TMS derivatives were
either analyzed by GC–MS, or heated in ampoules as described
below.
Mukhtarova, Lucia S.
