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dissolved in 1.5 mL of 50 mM KH2PO4 (pH ) 5.95) and incubated
with 0.35 ηkat ꢀ-glucuronidase (Type IX-A; from E. coli; Sigma) at
37 °C for 2 h. The reaction mixture was extracted with n-BuOH (4 ×
0.5 mL). The aqueous layer was evaporated to dryness and purified by
HPLC on a DIOL phase (Knauer LiChrosorb DIOL 10 µm; 300 × 7
mm; 0.8 mL/min; UV detection λ ) 210 nm). MeCN as well as H2O
containing 0.05% TFA (v/v) were used as eluents I and II, respectively.
Gradient: 3% II for 10 min, 11-20% II within 25 min. D-Glucuronic
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D
obtained from Sigma.
Antibacterial Bioassays. Compounds 1 and 2 in serial dilutions
[starting test concentration 128 µg/mL, dilution factor 1/2n, final test
concentration 1 µg/mL; three replicates for each concentration] were
tested in microdilution assays in Mueller-Hinton medium according to
established protocols.24 The bacterial strains Escherichia coli
BW25113,25 Pseudomonas pudida KT2440 (ATTC 47054), and
Enterobacter cloacae subsp. dissolVens (ATTC 23373) served as test
organisms. Each strain was grown on a separate 96-well plate along
with negative and sterile controls as well as the antibiotics gentamycin
and ampicillin as positive controls.
Antiproliferation/Cytotoxic Assay. The human pancreatic carci-
noma cell line was a gift of the Bosch Institut fu¨r Klinische
Pharmakologie (IKP Stuttgart, Germany) and the human neuroblastoma
cell line from our own stock. Ham’s F12 medium, fetal calf serum
(FCS), streptomycine, and penicillin were from Gibco.
The cells were seeded in low density (SH-SY5Y: 50.000 and Patu:
20.000 cells/3.5 cm plastic wells) and cultured in Ham’s F12, completed
with 10% FCS and 1% penicillin/streptomycine in the absence or
presence of 10 or 50 µM of 1 for 1-4 days. For determination of cell
densities, cell cultures dishes were washed two times with fresh medium
in order to remove detached dead cells, fixed in 4% paraformaldehyde
(PFA) in phosphate buffer (PB) for 10 min, permeabilized in 0.5%
TRITON X-100/PB for 5 min, and stained with DAPI/PB (1:10 000)
for 15 min. From each cell culture dish, digital images of 26 different
areas of 1.54 mm2 were made, and the densities of stained nuclei
determined by means of the software Image J, revealing for each dish
a total number of cells/40 mm2. Data are given as means ( SD from
six cell culture dishes (three experiments). In order to detect apoptotic
cells, viable cell cultures were stained with Hoechst 33342 (1:5000)
for 10 min. Apoptotic cells were identified by their strong stain of
fragmented DNA clusters.
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Acknowledgment. We thank Dr. Robert Amann, Institut fu¨r Chemie,
Universita¨t Hohenheim, for fruitful discussions and careful consideration
of the manuscript.
Supporting Information Available: 1H NMR and selective TOC-
SYs and MS/MS spectrum of 1. This material is available free of charge
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