I. Makarchuk, A. Nikolaev, A. Thesseling et al.
Electrochimica Acta 381 (2021) 138293
Compound 2: 1-Bromo-3,7,11,15-tetramethylhexadeca-2,6,10,14-
tetraene (geranylgeranyl bromide). To a solution of geranylgeraniol
(1) (5.80 g, 20.0 mmol, 1.0 eq) in Et2O (40 ml) at 0 °C was added
dropwise within 10 min PBr3 (1.80 g, 0.65 ml, 6.60 mmol, 0.33
eq) and the solution was stirred at 0 °C for additional 1 h. After
dilution with H2O, it was extracted with n-hexane (3 × 75 ml)
and the combined organic layers were washed with sat. NaCl solu-
tion, dried (MgSO4), filtered and concentrated. The geranylgeranyl
bromide (6.57 g, 18.6 mmol, 93%) was obtained as a pale-yellow
liquid. 1H NMR (CDCl3): δ (ppm) = 5.54 (t, J = 8.0 Hz, 1 H, =CH),
5.15–5.08 (m, 3 H, 3 × =CH), 4.03 (d, J = 8.0 Hz, 2 H, CH2–Br),
2.15–1.95 (combined m, 12 H, 3 × CH2CH2), 1.74/1.69/1.62/1.61
(4 × s, 15 H, 5 × CH3). 13C NMR (CDCl3): δ (ppm) = 143.62,
135.64, 134.97, 131.26, 124.36, 124.16, 123.38, 120.53, 39.71,
39.65, 39.52, 34.27, 29.68, 26.76, 26.59, 26.10, 25.68, 17.68, 16.04,
16.00.
2.4. Gold colloid solution preparation
The gold nanoparticles were prepared according to the conven-
tional method proposed by Turkevich and Frens [36-38]. Briefly, to
obtain nanoparticles of 15 nm average diameter, 125 mL of a 1 mM
solution of HAuCl4 were boiled before adding 13 mL of a 39 mM
sodium citrate solution. The solution was kept under boiling for
15 min, and was then allowed to cool down to room temperature.
The average size of the nanoparticles was estimated to be 15 nm
from the ratio of the absorbance at 521 and 450 nm [39]. Before
use, the gold colloid was concentrated by centrifugation for 30 min
ꢀ
at 10 000 rpm, and removal of 99% of the supernatant.
2.5. Electrode preparation and electrochemical setup
The surface of the gold rotating disk electrode (RDE) was me-
chanically polished with 0.05 μm alumina paste and activated by
subsequent oxidation at +2.0 V for 5 s, reduction at −0.35 V for
10 s and then cycling 100 times between −0.35 V and 1.5 V at
Compound
4:
Ethyl
2-acetyl-5,9,13,17-tetramethyloctadeca-
4,8,12,16-tetraenoate (Ethyl 2-(geranylgeranyl)acetoacetate). NaH
(14.8 mmol, 1.1 eq) was suspended under argon in anhydrous
THF (10 ml), cooled to 0 °C and ethyl acetoacetate (3) (1.70 ml,
1.75 g, 13.4 mmol, 1.0 eq) was added slowly and stirring was
continued for additional 20 min at 0 °C. Geranylgeranyl bromide
(2) (5.23 g, 14.8 mmol, 1.1 eq) in anhydrous THF (5 ml) was added
and the mixture was stirred at rt for 12 h. To the suspension was
added H2O (20 ml) followed by extraction with Et2O (3 × 20 ml).
The combined organic layers were washed with sat. NaCl so-
lution (2 × 30 ml), dried (MgSO4), filtered and concentrated.
The crude product was purified by flash chromatography (SiO2,
EtOAc/n-hexane 5:95, Rf = 0.18), yield 3.29 g (8.17 mmol, 61%)
of a pale-yellow liquid. 1H NMR (CDCl3): δ (ppm) = 5.12–5.01
(combined m, 4 H, 4 × =CH), 4.19 (q, J = 8.0 Hz, 2 H, O–CH2),
3.44 (t, J = 8.0 Hz, 1 H, α–H), 2.57 (t, J = 8.0 Hz, 2 H, CH2), 2.22
−
1
4 V.s
in 0.1 M H2SO4 solution. A single deposition of 7 μL of
a concentrated gold colloid solution was done on the gold disk,
and the electrode was allowed to dry under air. The modified el−ec-
1
trode was cycled again between −0.35 V and 1.5 V at 0.1 V.s
until a stable voltammogram was obtained. The real surface area
of the electrode was estimated from the integration of the gold-
oxide reduction peak at 1.1 V. The charge associated with the re-
duction of the gold oxide layer was assumed to be 390 μC.cm−2
[40]. The electrode was then immersed for 30 min in a (1:1) mix-
ture of 1 mM 6-mercaptohexan-1-ol and 1-hexanethiol in EtOH
and rinsed with fresh ethanol and dried. Finally, 5 μL of a 10 μM
protein solution in phosphate buffer were deposited on the surface
and left at 4 °C for two hours. Before the electrochemical stud-
ies, the modified electrode was rinsed with fresh buffer to remove
excess protein. All voltammograms were measured in a standard
three-electrode cell using a Princeton Applied Research VERSAS-
TAT 4 potentiostat. An AgCl/Ag 3 M NaCl electrode and a platinum
wire were used as the reference and counter electrode, respec-
tively. However, all the potentials mentioned in the manuscript are
referenced to the standard hydrogen electrode (SHE). All the mea-
surements were carried out at room temperature in a 20 mL pH 7
phosphate buffer solution. Three voltammograms were recorded at
0, 10 and 20 min after the preparation of the electrode to evaluate
the stability of the protein film.
–
(s, 3 H, H3C C = O), 2.10–1.90 (combined m, 12 H, 6 × CH2),
1.69 (broad s, 6 H, 2 × CH3), 1.64/1.63/1.61/1.60/1.50 (5 × s, 9
H, 3 × CH3), 1.27 (t, J = 7.1 Hz, 3 H, CH3). 13C NMR (CDCl3):
δ (ppm) = 203.10, 169.58, 138.46, 135.21, 135.16, 134.92, 131.24,
125.01, 124.98, 124.66, 124.60, 124.37, 124.33, 124.19, 124.10,
123.90, 123.88, 119.71, 119.60, 61.27, 59.82, 59.81, 39.98, 39.95,
39.68, 31.95, 29.10, 29.08, 26.89, 26.75, 26.70, 26.64, 26.52, 26.27,
25.70, 25.67, 23.37, 17.66, 17.61, 16.12, 15.98, 14.08.
Compound AD-4: Geranylgeranyl aurachin D. Ethyl 2-
(geranylgeranyl)acetoacetate (4) (300 mg, 0.74 mmol, 1.0 eq),
aniline (5) (0.08 ml, 0.82 mmol, 1.1 eq) and molecular sieves
˚
3 A (1 g) were heated to reflux in toluene (7 ml) for 16 h. After
cooling, the reaction mixture was filtrated through Celite and
the solvent was removed in vacuum. The crude enamine 6 was
added quickly to heated diphenyl ether (6 ml) and heating to
at least 250 °C was continued for 1 h. After cooling, the prod-
uct was separated from the solvent by column chromatography
(SiO2, EtOAc/n-hexane 5:95 to 1:1), yield 170 mg (4.00 mmol
53%) of a pale-yellow solid. 1H NMR (DMSO–d6): δ (ppm) = 7.98
(d, J = 8.0 Hz, 1 H, Ar–H), 7.78 (d, J = 8.0, 1 H, Ar–H), 7.58
(dd, J = 8.0/8.0 Hz, 1 H, Ar–H), 7.40 (dd, J = 8.0/8.0 Hz, 1 H,
Ar–H), 5.10–4.95 (combined m, 4 H, 4 × =CH), 2.72 (mc, 2 H,
quinolone-CH2), 2.52 (s, 3 H, CH3), 2.20–1.80 (combined m, 12
H, 6 × CH2), 1.60/1.53/1.51/1.50/1.34 (s, 15 H, 5 × CH3). 13C NMR
(DMSO–d6): δ (ppm) = 158.74, 158.73, 154.47, 146.61, 134.60,
134.57, 134.54, 134.47, 134.46, 134.34, 134.27, 130.79, 130.53,
128.49, 127.85, 124.79, 124.76, 124.37, 124.06, 124.02, 123.98,
123.81, 123.67, 120.73, 119.73, 110.37, 110.34, 77.29, 77.21, 39.42,
39.17, 38.83, 31.64, 31.45, 31.39, 31.23, 30.22, 30.14, 26.15, 26.12,
26.02, 25.97, 25.82, 25.78, 25.45, 25.42, 23.35, 23.26, 23.14, 23.10,
23.04, 22.73, 21.66, 21.53, 18.77, 17.46, 17.39, 15.72, 15.64, 15.62,
15.59. HR–MS (Cl) for [M] C30H41NO: 431.3191 (found); 431.3188
(calculated).
2.6. Inhibition studies
20 mM stock solutions of all inhibitors were prepared in DMSO.
A freshly prepared electrode was used for each inhibitor, and the
measurements were repeated with 3 different electrodes in order
to report the mean values and standard deviations. For the sin-
gle measurement at 10 μM, 10 μL of the solution of inhibitor
was added to the 20 mL buffer solution. After an equilibration
time of 10 min, the voltammogram of the immobilized proteins
in presence of the inhibitor was recorded. For the concentration-
response analysis, several aliquots of the inhibitor stock solution
were added to the 20 mL buffer solution. A voltammogram was
recorded 10 min after each addition of inhibitor.
3. Results and discussion
3.1. Preparation of the electrodes
Highly hydrophobic membrane proteins such as cytochrome bd
oxidase are more difficult to handle and study than soluble pro-
teins. Our strategy consists in their immobilization on 3D gold
3