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228 Shai Rahimipour et al.
impurity present in the commercial DMPO was removed by treat-
ment with neutral decolorizing charcoal (28). Reversed-phase high-
performance liquid chromatography (HPLC) was performed on a
Spectra-Physics SP-8800 liquid chromatography system equipped
with an Applied Biosystems 757 variable wavelength absorbance
detector. HPLC prepacked columns used were: Lichrocart, contain-
6.77 (s, 1H, Trp), 6.91 (d, 1H, Emo), 7.0 (d, 1H, Emo), 7.10 (s, 1H,
His), 7.17 (d, 2H, Arg), 7.18 (s, 1H, Trp), 7.35 (s, 1H, Trp), 7.51
(s, 1H, Trp), 7.56 (s, 1H, Trp), 7.91 (br, 1H, Tyr), 7.96 (d, 1H, Trp),
8.20 (d, 1H, Ser), 8.34 (s, 1H, His), 8.39 (d, 1H, Leu), 8.49 (d, 1H,
Arg), 8.53 (d, 1H, His), 8.64 (s, 1H, Gly), 8.36 (d, 1H, Lys). Mass
spectrometry: found m/z [M
C74H91N18O19 [M 1536.62. Amino acid analysis after hy-
H]ϩ
drolysis with 6 M HCl at 110 C for 22 h: Glu 1.00, His 1.00, Ser
ϩ ϭ 1536.17; calcd for
H]ϩ
ing Lichrosorb RP-18 (250
purification and Lichrospher 100 RP-18 for analytical purposes (250
4 mm; 5 m), Merck (Darmstadt, Germany). HPLC purification
ϫ
10 mm; 7
m) for semipreparative
ϩ
ϭ
Њ
ϫ
0.87, Tyr 0.98, Lys 1, Leu 0.98, Arg 1.05, Pro 1.01, Gly 0.98. Trp
could not be detected due to its destruction under the acidic condi-
and analyses were achieved by using 0.1% trifluoroacetic acid (TFA)
in water as buffer A and 0.1% TFA in 75% aqueous acetonitrile as
buffer B. Eluent composition was 10% B for the first 10 min and
increased linearly to 100% B 50 min after the injection time. Nuclear
tions of hydrolysis. UV–vis (DMSO):
443 (4).
max/nm (log ⑀), 285 (4.28),
magnetic resonance (NMR) spectra were recorded at 5ЊC on Brucker
Electrochemistry
spectrometers (270 and 500 MHz). Mass spectrometry was per-
formed on a Micromass Platform LCZ 4000 (Manchester, UK) uti-
lizing electron spray ionization method. For amino acid composition
analysis peptides were hydrolyzed in 6 N HCl at 100ЊC for 24 h
under vacuum, and the hydrolyzates were analyzed with a Dionex
Automatic Amino Acid Analyzer.
Cyclic voltammetry was measured on a Metrohm Polarecord E 506
and a VA scanner E 612 with a VA stand 663 (Metrohm AG, Her-
isau, Switzerland). All measurements were carried out in DMF (kept
over molecular sieves) as a solvent containing 0.1 M tetra-n-butyl-
ammonium perchlorate (n-Bu4NClO4, Fluka, Buchs, Switzerland,
dried overnight at 100ЊC before using) as a supporting electrolyte at
room temperature. The working electrode was a hanging mercury
drop, whereas glassy carbon was used as a counter electrode and an
Ag wire served as the reference electrode. All the reported potentials
were cited against a saturated calomel electrode (SCE). Benzophe-
1,6,8-Trihydroxy-3-carboxylic acid-anthraquinone (emodic
acid)
Emodin 1 (4 g, 14.8 mmol) was acetylated by acetic anhydride (40
mL) and a catalytic amount of H2SO4 (0.8 mL) at 60
ЊC for 40 min.
none was used as an internal standard (E ϭ Ϫ1.80 V vs SCE) where
½
The resulting yellow precipitate (5 g, 85% yield) was filtered,
washed with H2O and dried. A solution of crude triacetylemodin 2
(3 g, 7.57 mmol) in acetic acid (125 mL) and acetic anhydride (125
mL) was then oxidized with a solution of CrO3 (2 g, 20 mmol) and
E is the half reduction potential (31).
½
EPR and ENDOR studies of radicals generated by the
reduction of Emo and its [D
-Lys6]GnRH conjugate
dissolved in water (5 mL) and acetic acid (60 mL) at 70ЊC for 3.5
Chemical and electrochemical reduction. For EPR and ENDOR
spectroscopy Emo and its GnRH conjugate were reduced to the cor-
responding semiquinone with Zn in DMF or with Zn in DMSO-d6/
D2O (10:1; vol/vol). Reduction was also carried out electrochemi-
h. Next, the green solution thus obtained was concentrated (to 250
mL), hot water (1200 mL) was then added, and the solution was
refrigerated overnight. The residue was filtered, washed with cold
water and dried to yield triacetylemodic acid 3 with an 84% yield.
Finally, the acetyl groups were hydrolyzed with 2 M NaOH (100
cally at 0ЊC on a helical gold cathode using platinum wire as the
counter electrode. Reductions were performed either in DMF con-
taining 0.1 M tetra-n-butylammonium perchlorate as a supporting
electrolyte or in phosphate buffered saline (PBS, pH 7.4). Electron
spin resonance measurements were performed by a Varian-E9 in-
strument, and a Bruker-ESP-300 (Bruker, Karlsruhe, Germany) sys-
tem was used for all ENDOR and TRIPLE-resonance studies. Si-
multaneous recording of the electronic absorption bands was per-
formed on a J&M TIDAS-16 instrument (J&M, Aalen, Germany),
which was attached directly to the optical cavity of the EPR spec-
trometer (32).
mL) at 80
ЊC for 2 h. After acidification and filtration Emo 4 was
obtained with an 85% yield and used directly for conjugation with
1
[D
-Lys6]GnRH. HPLC retention time (tR): 43 min. H NMR (270
MHz; methanol-d4): ␦ ϭ 6.62 (d, 1H), 7.26 (d, 1H), 7.84 (d, 1H),
8.3 (d, 1H). Mass spectrometry: found m/z [M
H]ϩ ϭ 298.7; calcd
for C15H8O7 [M 299. UV–vis (dimethyl sulfoxide
H]ϩ
[DMSO]): max/nm (log ), 290 (4.19), 445 (3.98).
Ϫ
Ϫ
ϭ
⑀
[
([
D
-Lys6(6-oxy-1,3,8-trihydroxy anthraquinone)]GnRH
D
-Lys6(Emo)]GnRH)
Enzymatic reduction. The activity of the reductase was deter-
mined spectrophotometrically from the kinetic measurements of re-
duced cytochrome C production at 550 nm (33). The formation of
superoxide or hydroxyl radicals by enzymatic reduction was fol-
lowed by the spin trapping technique. The reaction mixture (final
concentration) consisted of NADPH-cytochrome P450 reductase
(0.1–0.2 unit/mL), NADPH (1 mM), FeCl3 (0.1 mM), ethylenedi-
amine-tetraacetic acid (0.2 mM), DMPO (50–100 mM), Emo or its
[D
-Lys6]GnRH was synthesized on an automatic multiple peptide
synthesizer (AMS-422, Abimed Analysen-Technik GmbH, Langen-
feld, Germany) with Rink amide resin as a polymeric support fol-
lowing the company’s protocol for Fmoc strategy as described
(29,30). To a dimethylformamide (DMF) solution (1 mL) of the
dried peptide (31 mg, 25
containing 4-methylmorpholine (NMM) (8.2
solution (0.5 mL) of benzotriazole-1-yl-oxy-tris-pyrrolidino-phos-
phonium hexafluorophosphate (PyBOP) (13 mg, 27.5 mol) was
added. The mixture was stirred for 2 h at room temperature. The
mol) and Emo (8.25 mg, 27.5
mol)
L, 75 mol), a DMF
corresponding [
ence or absence of 10% DMSO. Measurements were made in a 200
L EPR flat cell at 22 C.
Photoreduction of Emo and [D
-Lys6(Emo)]GnRH. All irradiations
D
-Lys6]GnRH analog (0.1 mM) in PBS, in the pres-
Њ
progress of the reaction was followed by the disappearance of [
D
-
Lys6]GnRH as revealed by analytical HPLC. Upon completion of
the reaction the crude peptide was precipitated with ice-cold tert-
butyl methyl ether (10 mL), collected by centrifugation and dried.
It was then purified to homogeneity by semipreparative HPLC to
were carried out with a KL 1500 electronic projector lamp (Schott,
Mainz, Germany). Unless otherwise stated, all irradiations were car-
ried out with an appropriate filter with a bandpass of 320–510 nm
and
ϭ 400 nm. Samples were irradiated directly inside the EPR
max
yield 23 mg (15
mol; 60%). HPLC (tR): 39.1 min (tR
ϭ
31.6 min
cavity with an optical fiber while the EPR spectra was recorded.
Emo semiquinone was generated under anaerobic conditions by the
irradiation of Emo (2 mM) in DMF containing 5% triethylamine
(vol/vol). For spin trapping studies a mixture of DMPO (0.1 M),
for [D
-Lys6]GnRH in the same conditions). 1H NMR (500 MHz;
H2O:D2O, 9:1; pH 3.1, the partial assignment was based on 2D Nu-
clear overhauser effect, nuclear overhauser enhancement spectros-
copy and Homonuclear Hartman Hann Spectroscopy): ␦ ϭ 0.85 (d,
3H, Leu), 0.90 (d, 3H, Leu), 1.54–1.57 (m, 6H, Leu, Arg, Pro, pGlu),
1.64 (m, 4H, Leu, Arg, Pro, Lys), 1.74 (m, 2H, Arg, Lys), 1.82 (m,
1H, Lys), 2.43 (m, 1H, Tyr), 2.59 (m, 1H, Tyr), 2.81 (d, 1H, Tyr),
2.9 (m, 3H, Trp, His), 3.42 (m, 1H, Pro), 3.53 (m, 2H, Ser, Pro),
3.59 (m, 1H, Ser), 3.82 (d, 1H, Gly), 3.85 (d, 1H, Gly), 4.15 (m,
1H, pGlu), 4.23 (m, 2H, Ser, Tyr), 4.34 (m, 3H, Leu, Pro, Lys),
4.48 (m, 2H, Arg, Trp), 4.55 (br, 1H, His), 6.31 (s, 1H, Tyr), 6.38
(d, 1H, Emo), 6.49 (br, 1H), 6.62 (s, 1H, Tyr), 6.69 (d, 1H, Emo),
Emo (1, 0.1 mM) or the corresponding [
mM) in the specified solvent and pH was irradiated in a 200
D
-Lys6]GnRH analog (0.1
L
EPR flat cell. To generate anaerobic conditions Argon was bubbled
through the corresponding sample 10 min before measurements. All
spin trapping studies were performed with a Bruker Electron Spin
Resonance ER200D-SRC spectrometer at 25ЊC.
Simulation of EPR spectra. For computer simulations of the EPR
spectra the public domain program WINSIM (NIEHS, NIH, Re-
search Triangle Park, NC) was used (34).
Rahimipour, Shai
