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C. Kurth et al. / Phytochemistry 117 (2015) 417–423
negative ionization mode. The gradient started with 100% A (held
for 0.2 min) and was ramped to 82% A (1.8 min) and to 0% A
(4.5 min) before re-equilibration with 100% A started at 6 min.
Co-injection experiments were performed to confirm the identity
of synthetic and natural products. Therefore, the concentrations
of metabolites in the algal samples were roughly estimated
using an external calibration with the synthetic standards
4.6. Synthesis of 4-(sulfooxy)phenylacetic acid
7.5 g of 4-hydroxyphenylacetic acid (52 mmol, Sigma Aldrich,
Germany) and 8.3 g Pyr⁄SO3 (52 mmol) were dissolved in 100 mL
water free pyridine. The synthesis and work-up followed that
described for SBA synthesis. 1H NMR (400 MHz, DMSO [D6] +
0.8% formic acid) d(ppm): 3.11 (s, 2H), 6.98 (d, 2H), 7.08 (d, 2H),
13C NMR d(ppm): 45.93, 119.89, 129.30, 135.26, 150.82, 174.06.
Elemental analysis as potassium salt found: C 32.24, H 1.87, S
10.83. Calc. for C7H5O6SKa C 35.55, H 2.61, S 11.86. UPLC–MS m/z
[MÀH]À: 231.00.
(10–80 l l
g mLÀ1 in steps of 10 for SBA and 50–400 g mLÀ1 in steps
of 50 for SPA). Then, algal extracts were mixed with the synthetic
standards in similar concentrations. Samples were immediately
measured by UPLC–MS using a modified gradient. This started with
100% A (held for 0.2 min) and was ramped to 91% A (2.5 min) and
to 0% A (5 min) before re-equilibration with 100% A started at
6.5 min. If peak areas were doubled without major changes in peak
symmetry, peaks were considered as co-eluting, confirming the
identity of the tested metabolites.
4.7. Synthesis of other standards
Additionally, the synthetic standards 2-(sulfooxy)benzoic acid,
3-(sulfooxy)benzoic acid, 2,3- and 4-sulfooxy methoxyphenol, as
well as the isomers of mono sulfated 2,3- and 3,4-dihydro-
cycinnamic acids were prepared as described for SBA.
Dihydroxycoumarin sulfate was prepared as described elsewhere
(Welling et al., 2011).
4.4. Screening for sulfated metabolites
Sulfated metabolites show, other than the pseudo molecular ion
[MÀH]À a characteristic mass of [MÀHÀ80]À due to loss of the SO3.
This characteristic property of the mass spectra was used to iden-
tify candidate peaks for further structure elucidation. Since in no
cases could enough material be obtained for a direct NMR structure
elucidation we reverted to the synthesis of standards and co-injection
experiments to prove the identity of the natural products.
4.8. Bioassays on bacterial growth and biofilm formation
Bioassays were conducted with SBA and SPA as previously
described (for full experimental details see Kurth et al. (2015)).
Microtiter dish assay with E. coli and V. natriegens: Briefly, solu-
tions of the test compounds (2.05 mM) were added to bacterial
cultures (ATCC 25404 and ATCC 14048, Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Braunschweig,
Germany) in 96-well plates and incubated at 30 °C. When biofilm
formation started a first plate was removed from the incubator
and from then on hourly samples were taken by collecting addi-
tional plates for biofilm evaluation and UPLC–MS analysis.
4.5. Synthesis of 4-(sulfooxy)benzoic acid
15 g 4-hydroxybenzoic acid (108 mmol, Sigma Aldrich,
Germany) and 17.2 g sulfur trioxide pyridine complex (Pyr⁄SO3)
(108 mmol, Alfa Aesar, Germany) were dissolved in 200 mL water
free pyridine and the reaction mixture was stirred in a round neck
flask at RT for 48 h. Subsequently, pyridine was removed by rotor
evaporation to yield a yellow to brown oil. The oil was dissolved
in as little water as possible and the pH adjusted to ꢀ6–7 with a
25% potassium hydroxide (KOH) solution. The aqueous solution
was then washed three times with the same volume of ethyl acet-
ate. During the first two washing steps a precipitate formed, not
containing product, which was filtered off after phase separation.
The ethyl acetate phase was discarded, the aqueous phase concen-
trated by rotor evaporation to yield a brown to yellow, slightly
damp raw product. The raw product was dissolved in as little water
as possible and pH was adjusted to 10 with 25% KOH. The solution
was stirred for 1 h at 60 °C to cleave the anhydride side products,
providing sulfated product and educt. The solution was neutralized
with diluted H2SO4 and water was removed by rotary evaporation.
The yellowish powder was dissolved in as little water as possible at
40 °C and twice the volume of methanol was added to precipitate
salts. The precipitate was filtered off and washed with a further
single amount of methanol, followed by unification of the
methanolic solutions. The batch was then left standing at RT over
night without stirring and the newly formed crystalline precipitate
was filtered off and discarded. After evaporation of the solvent to
dryness the powder was taken up in 1/10 the amount of methanol
used to precipitate salts and placed into an ultrasonic bath until a
fine suspension of a white powder and yellowish solution was
obtained. The solution was filtered off, evaporated again and the
suspension step repeated with half the amount of methanol. The
white precipitate was collected, washed with acetone and dried
at 70 °C to give 13.5 g of 4-(sulfooxy)benzoic acid (SBA) as a fine
white powder (57%). 1H NMR (400 MHz, DMSO [D6] + 0.8% formic
acid) d(ppm): 7.22 (d, 2H); 7.84 (d, 2H), 13C NMR d(ppm):
119.33, 126.59, 130.45, 156.93, 164.64, Elemental analysis found:
C 38.44, H 1.62, S 14.32. Calc. for C8H7O6SKa C 38.50%, H 2.70%, S
14.70%. UPLC–MS m/z [MÀH]À: 216.99.
Therefore 190
compound and centrifuged (15 min, 13,000 rpm). 100
natant were then transferred into glass vials containing 900
l
L sample were collected in triplicates per assayed
L of super-
L of a
l
l
methanolic solution with 0.1 mg mLÀ1 trans-cinnamic acid as
internal standard and stored at À26 °C until UPLC–MS analysis.
For biofilm evaluation, the remaining liquid contents were poured
quickly into a waste container, and then plates were immediately
washed in phosphate saline buffer and water. After drying,
200
into each biofilm well and stained for 15 min. After washing with
deionized water and drying 200 of 80:20 mixture of
ll of a 0.1% aqueous crystal violet (CV) solution were added
ll
a
ethanol/acetone were filled in each stained well and left for
15 min until CV was dissolved. CV absorption was then measured
in a Mithras plate reader at 570 nm (30 °C), with CV staining of
untreated wells as reference. Biofilm of V. natriegens was not
evaluable due to floc formation under the given experimental
conditions. Growth was monitored until the onset of biofilm
formation by OD measurements.
Biofilm assay on submerged polytetrafluoroethylene (PTFE)
plates with V. natriegens: Briefly, 1.5 cm long PTFE tubes were
each cut twice to 1/3 of the diameter and two PTFE plates with
a diameter of 12 mm were fixed in the cuts. 5–10 of the set-ups
were autoclaved added to Erlenmeyer flasks and incubated with
50 mL V. natriegens (ATCC 14048) cultures with or without test
compounds (2.05 mM). Flasks were sealed and put on a shaker
(150 rpm) at 30 °C. For biofilm sampling, one PTFE set-up from
each flask was removed and washed with water, air dried
and dyed in 0.1% CV solution for 15 min. After washing and
air-drying, tubes of the PTFE set-ups were cut, such that PTFE
plates could be removed and biofilm formation was monitored
by microscopy (for full experimental details see Kurth et al.
(2015)).