Research Article

1804
mV
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A
S Di 3
S Di 2
S Di 4
S Di 5
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C
S Tri 7
S Di 6
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www.soci.org D Dobberstein, M Bunzel
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700 B
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SDi8 SDi9 S Di 11
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450 D
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S Tri 9
S Tri 10
S Tri 12
S Tri 11
S Tri 13
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Figure 2. Separation of the size exclusion fractions B3 (top; A, B) and B2 (bottom; C, D) by using Sephadex LH-20 chromatography. (A) Elution with
0.5 mmol L −1 aqueous trifluoroacetic acid (TFA)/methanol (MeOH) (50/50, v/v), detection at 280 nm. (B) Elution with 0.5 mmol L −1 aqueous TFA/MeOH
(40/60, v/v), detection at 280 nm. (C) Elution with 0.5 mmol L −1 aqueous TFA/MeOH (50/50, v/v), detection at 325 nm. (D) Elution with 0.5 mmol L −1
aqueous TFA/MeOH (40/60, v/v), detection at 325 nm. Elution with 0.5 mmol L −1 aqueous TFA/MeOH (95/5, v/v) is not shown for both size exclusion
fractions. S Di 2–S Di 11 contained the following dehydrodiferulic acids: S Di 2: 8-8(tetrahydrofuran)-; S Di 3: 8-8(aryltetralin)-, S Di 4: 8-8-, S Di 5: 8-5-, S Di 6: 8-O-4-,
8-5(benzofuran)-, S Di 8andS Di 9: 5-5-, S Di 11: 8-5(decarboxylated)-dehydrodiferulic acid. For S Tri 7toS Tri 13 see text.
chromatogram (Fig. 2), and evaporated. Fractions S tri 7 and
S tri 9–S tri 13 were further separated by using semi-preparative
RP-HPLC.
RP-HPLC was carried out by using a semi-preparative phenylhexyl
column (250×10 mm i.d., 5 µm particle size), ternary gradient
systems made up of aqueous TFA (1 mmol L −1 ), acetonitrile (ACN),
and MeOH, and a flow rate of 2.5 mL min −1 .Injectionvolume
was 60 µL and the separation was performed at either 35 or 45 ◦ C.
Chromatograms were monitored at 280 and 325 nm. The following
gradients were used (eluent A: aqueous TFA (1 mmol L −1 ); eluent
B: ACN/aq. TFA (1 mmol L −1 ) 90/10 (v/v); eluent C: MeOH/aq. TFA
(1 mmol L −1 ) 90/10 (v/v)): Fractionation of S tri 7: initially A 60%, B
25%, C 15%, held for 15 min, linear over 5 min to A 45%, B 35%,
C 20%, held for 5 min, linear over 5 min to A 25%, B 35%, C 40%,
held for 5 min, linear over 5 min to A 0%, B 0%, C 100%, held for
5 min, following an equilibration step. Fractionation of S tri 9and
S tri 10: initially A 75%, B 10%, C 15%, held for 10 min, linear over
5 min to A 60%, B 25%, C 15%, held for 5 min, linear over 5 min
to A 50%, B 25%, C 25%, held for 5 min, linear over 5 min to A
20%, B 25%, C 55%, held for 5 min, following an equilibration step.
Fractionation of S tri 11–S tri 13: initially A 65%, B 20%, C 15%, held
for 15 min, linear over 5 min to A 50%, B 35%, C 15%, held for 5 min,
linear over 5 min to A 40%, B 35%, C 25%, linear over 5 min to A
10%, B 35%, C 55%, held for 5 min, following an equilibration step.
Fractions were collected according to the chromatograms, pooled
and evaporated. Some fractions had to be re-chromatographed to
increase purity for structural characterization.
Characterization of the fractions and isolated compounds
The UV spectra and the molecular weights were determined
by RP-HPLC coupled to both a photodiode array detector
and a mass spectrometer (atmospheric pressure–electrospray
ionization, positive and negative mode). The fragmentor voltage
was either 75 V (positive mode) or 90 V (negative mode), the
scan range m/z 100–1000. Elution on an analytical phenyl-hexyl
column (250 × 4.6 mm i.d.) was carried out by using the following
gradient (eluent A: 0.1% (v/v) formic acid; eluent B: ACN): Initially
A 82%, B 18%, linear over 5 min to A 80%, B 20%, linear over
5 min to A 75%, B 25%, linear over 5 min to A 70%, B 30%, linear
over 10 min to A 65%, B 35%, held for 5 min, linear over 5 min
to A 55%, B 45%, followed by rinsing and equilibration steps.
The column temperature was held at 35 or 45 ◦ C, the injection
volume was 20 µL. Structural identification was performed using
1 H- and HMQC-NMR experiments. Samples were dissolved in
0.7 mL acetone-d6. Chemical shifts (d) were referenced to the
central solvent signals (dH 2.04 ppm; dC 29.8 ppm). J-values were
calculated in hertz.
Screening for ester-bound cyclobutane dimers and monolignol–ferulate
cross-products by GC-MS
An aliquot of the BioBeads fraction B3 (about 0.3 mg) was
silylated in pyridine/BSTFA 1/4 (v/v) for 30 min at 60 ◦ C. The
silylated compounds were separated and detected by GC-MS.
He (1 mL min −1 ) was used as carrier gas. GC conditions were as
follows: HP-5-MS fused-silica capillary column (30 m, 0.32 mm i.d.,
www.interscience.wiley.com/jsfa c○ 2010 Society of Chemical Industry J Sci Food Agric 2010; 90: 1802–1810