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3.29 (dd, J = 5.2 Hz, J = 14.1 Hz, 1H, H-15b), 3.52 (ddd, 1H,
J = 3.4 Hz, J = 6.2 Hz, J = 11.8 Hz, H-3), 5.12 (m, 1H, H-14), 6.95–
7.18 (m, H-17–H-21), 8.57 (d, J = 5.3 Hz, 1H, H-13), 8.63 (1H, s,
H-6), 13.02 (s, 1H). 13C NMR (101 MHz, CDCl3) d 20.7 (C-11), 32.3
(C-4), 37.3 (C-15), 54.5 (C-14), 75.9 (C-3), 110.1 (C-9), 120.2
(C-7), 123.2 (C-5), 127.3 (C-19), 128.7 (C-17, C-21), 129.3 (C-18,
C-20), 135.8 (C-16), 139.0 (C-6), 141.0 (C-10), 159.1 (C-8), 163.2
402.07382 (calculated mass for [C20H17O6NCl]ꢀ: m/z 402.07499),
MS/MS (HCD@90.00): m/z (%) 211.02 (100), 358.08 (74), 166.99
(51), 314.06 (28), 254.02 (14), 239.01 (6).
2.7. Cultivation of the Hep G2 cell line
Human hepatocellular carcinoma cells (Hep G2, ACC 180, DSMZ,
Braunschweig, Germany) were cultivated in DMEM medium
enriched with 10% fetal calf serum using standardized culture con-
ditions (37 °C, 8.5% CO2). After the cultivation of cells for 48 h in
96-well (cytotoxicity assay) or 24-well plates (caspase-3 assay, lac-
tate dehydrogenase release assay) the cell medium was changed to
serum-free medium when cells reached a microscopic confluence
of approximately 80%. The cytotoxicity assays were performed un-
der serum-free conditions to exclude any binding of the tested
compound to serum proteins. After 24 h of serum-free cultivation
1, 2, 3 or 4 (stock solution: 1 mM in methanol) were added in con-
(C-12), 169.8 (C-1), 174.8 (C-22). ½a D23
ꢂ
= 68 (c 0.41 mg/mL, metha-
nol), ESI-MS (negative mode): HRMS m/z 402.07422 (calculated
mass for [C20H17O6NCl]ꢀ: m/z 402.07499), MS/MS (HCD@90.00):
m/z (%) 211.02 (100), 358.08 (74), 166.99 (51), 314.06 (28),
254.02 (14), 239.01 (7).
2.5.2. 3S14S-Ochratoxin A (2)
1H NMR (400 MHz,) d 0.78 (d, J = 6.3 Hz, 3H, H-11), 1.75 (dd,
J = 17.3 Hz, 11.9 Hz, 1H, H-4a), 2.34 (dd, J = 17.3 Hz, 3.2 Hz, 1H,
H-4b), 3.10 (dd, J = 13.8 Hz, 7.3 Hz, 1H, H-15a), 3.28 (dd,
J = 13.9 Hz, 4.6 Hz, 1H, H-15b), 3.64 (ddd, J = 11.7 Hz, 6.0 Hz,
3.3 Hz, 1H, H-3), 5.16 (m, 1H), 6.95–7.18 (m, H-17–H-21), 8.57
(d, J = 6.4 Hz, 1H, H-13), 8.62 (s, 1H, H-6), 13.01 (s, 1H), 13C NMR
(101 MHz, CDCl3) d 20.9 (C-11), 32.5 (C-4), 37.5 (C-15), 54.6 (C-
14), 76.2 (C-3), 110.3 (C-9), 120.4 (C-7), 123.4 (C-5), 127.5 (C-19),
128.9 (C-17, C-21), 129.5 (C-18, C-20), 135.9 (C-16), 139.2 (C-6),
141.3 (C-10), 159.3 (C-8), 163.5 (C-12), 170.0 (C-1), 174.8 (C-22).
centrations ranging from 10 nM to 50 lM and incubated for 24 h
(caspase-3) and 48 h (CCK-8 and LDH), respectively. The cells of
the control group were incubated with according solvent concen-
trations. Studies with all stereoisomers were performed in tripli-
cate with cells from three independent passages (n = 9). Each
experiment was carried out simultaneously with all four com-
pounds allowing a better comparability of the effects.
½
a 2D3
= 101 (c 0.33 mg/mL, methanol). ESI-MS (negative mode):
ꢂ
HRMS m/z 402.07420 (calculated mass for [C20H17O6NCl]ꢀ: m/z
402.07499), MS/MS (HCD@90.00): m/z (%) 211.02 (100), 358.08
(76), 166.99 (51), 314.06 (28), 254.02 (14), 239.01 (7).
2.8. Cytotoxicity assay
The cytotoxicity of ochratoxin A derivatives was evaluated col-
orimetrically using the Cell Counting Kit-8 (CCK-8) (Dojindo Labo-
ratories, Tokyo, Japan) according to the literature and the
manufacturer’s instructions.15,16 Briefly, cells were seeded on 96-
well microplates (4 ꢁ 103 cells/well). After toxin exposure the
dye solution (WST-8) was added to the cells, followed by the incu-
bation for 1 h at 37 °C. The reduction of WST-8 dye by cellular
dehydrogenases of viable cells increases the absorbance at
k = 450 nm and was measured with a microplate reader. The re-
sults for toxin-treated cells were normalized to the values of the
untreated negative control.
2.6. Synthesis of 3R14R-ochratoxin A (3) and 3S14R-ochratoxin
A (4)
Synthesis and purification were carried out as described under
4.4 but 9 was coupled with D-phenylalanine methyl ester hydro-
chloride (11). The reaction product 3R14R-ochratoxin A (3) eluted
at a retention time of 24.0 min, 3S14R-ochratoxin A (4) at a reten-
tion time of 26.2 min.
2.6.1. 3R14R-Ochratoxin A (3)
1H NMR (400 MHz, C6D6) d 0.77 (d, J = 6.3 Hz, 3H, H-11), 1.75
(dd, J = 12.0 Hz, J = 17.3 Hz, 1H, H-4a), 2.33 (dd, J = 3.3 Hz,
J = 17.3 Hz, 1H, H-4b), 3.09 (dd, J = 7.3 Hz, J = 13.9 Hz, 1H, H-15a),
3.27 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H, H-15b), 3.58 (m, 1H, H-3),
5.15 (m, 1H, H-14), 6.95–7.18 (m, H-17–H-21), 8.56 (d, J = 5.7 Hz,
1H, H-13) 8.62 (s, 1H, H-6), 13.01 (1H, s). 13C NMR (101 MHz,
CDCl3) d 20.7 (C-11), 32.3 (C-4), 37.3 (C-15), 54.5 (C-14), 75.9
(C-3), 110.1 (C-9), 120.2 (C-7), 123.2 (C-5), 127.3 (C-19), 128.7
(C-17, C-21), 129.3 (C-18, C-20), 135.8 (C-16), 139.0 (C-6), 141.0
(C-10), 159.1 (C-8), 163.2 (C-12), 169.8 (C-1), 174.8 (C-22).
2.9. Caspase-3 activity assay
The assay was carried out according to the literature.17,16 Cells
were seeded in 24-well plates (2 ꢁ 104 cells/well). After toxin incu-
bation, cells were washed with cold PBS buffer and incubated with
100 lL of cell lysis buffer (10 mM TRIS, 100 mM NaCl, 1 mM EDTA,
0.1% Triton X-100, pH 7.5) for 15 min on ice. The cell lysates were
centrifuged at 7000g for 10 min at 4 °C. Fifty microliters of the
supernatant were incubated with 50
PIPES, 10 mM EDTA, 0.5% CHAPS, 10 mM DTT) containing 8
fluorogenic caspase-3 substrate (Ac-Asp-Glu-Val-Asp-7-Amino-4-
trifluoromethylcoumarin, DEVD-AFC) at 37 °C for 1 h. The fluores-
cence of 7-amino-4-trifluoromethylcoumarin (AFC), released by
proteolytic cleavage, was measured with a microplate reader (exci-
tation: k = 400 nm; emission: k = 505 nm). Released AFC concen-
trations were quantified using an AFC standard for the
calibration and were normalized to the cellular protein content
in each sample. Protein concentrations were determined with the
bicinchoninic acid assay using bovine serum albumin (BSA) as
standard for the calibration.
lL reaction buffer (50 mM
lM
½
a 2D3
ꢂ
= ꢀ104 (c 0.44 mg/mL, methanol). ESI-MS (negative mode):
HRMS m/z 402.07428 (calculated mass for [C20H17O6NCl]ꢀ: m/z
402.07499), MS/MS (HCD@90.00): m/z (%) 211.02 (100), 358.08
(76), 166.99 (51), 314.06 (28), 254.02 (13), 239.01 (6).
2.6.2. 3S14R-Ochratoxin A (4)
1H NMR (400 MHz, C6D6) d 0.77 (d, J = 6.3 Hz, 3H, H-11), 1.76
(dd, J = 17.3 Hz, 11.5 Hz, 1H, H-4a), 2.32 (dd, J = 17.3 Hz, 3.4 Hz,
1H, H-4b), 3.08 (dd, J = 14.0 Hz, 7.3 Hz, 1H, H-15a), 3.28 (dd,
J = 14.2 Hz, 5.0 Hz, 1H, H-15b), 3.50 (m, 1H, H-3), 5.21 (m, 1H, H-
14), 6.96–7.20 (m, H-17–H-21), 8.56 (d, J = 6.6 Hz, 1H, H-13), 8.63
(s, 1H, H-6), 12.99 (s, br, 1H, C-22 OH). 13C NMR (101 MHz, CDCl3)
d 20.9 (C-11), 32.5 (C-4), 37.5 (C-15), 54.6 (C-14), 76.2 (C-3), 110.3
(C-9), 120.4 (C-7), 123.5 (C-5), 127.5 (C-19), 128.9 (C-17, C-21),
129.5 (C-18, C-20), 135.9 (C-16), 139.2 (C-6), 141.2 (C-10), 159.3
2.10. Lactate dehydrogenase release assay
The assay was carried out according to the literature.18 Cells
were seeded in 24-well plates (2 ꢁ 104 cells/well). After toxin incu-
bation, the cells were washed with cold PBS buffer and incubated
(C-8), 163.5 (C-12), 169.9 (C-1), 174.8 (C-22).
0.44 mg/mL, methanol). ESI-MS (negative mode): HRMS m/z
½
a 2D3
ꢂ
= ꢀ66 (c
with 100 lL of cell lysis buffer (10 mM TRIS, 100 mM NaCl, 1 mM