Molecules 2018, 23, 2735
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and C-8 (
δ
106.8) were used to assign the H-4 (7.40, s) and H-8 (7.10, s) as well as C-40 (
δ
135.8) and
C-50 (δ 115.8) which were assigned to H-40 (7.96, s) and H-50 (7.28, s) by 1H-13C HSQC.
The HMBC spectrum showed key correlation peaks between the proton signal at δH
7.93 (1H, s, H-40) and carbon resonances at δC 163.0 (C-20), 147.7 (C-90), 135.8 (C-30), 115.8
(C-50); and between the proton signal at δH 7.38 (1H, s, H-4) and carbon resonances at
δC 0 163.3 (C-2), 150.3 (C-9), 136.1 (C-3), 118.3 (C-5). Thus, compound
1 was identified as
0
0
0
5,8 -di-(6(6 ),7(7 )-tetrahydroxy-3-sulfoxy-3 -sulfoxycoumarin), a new coumarin for which we propose
the trivial name “dasycladin A”.
Compound
2 was also isolated as a yellowish amorphous powder. The molecular formula
of C9H6O11S2 was determined by HR-ESI–MS (negative mode) with a mass peak at m/z 352.92−8
−
(calcd. for [C9H6O11S2-H] , 352.926). The mass spectrum showed a fragment at m/z 272.8 [M-H-80]
corresponding to the loss of a sulfate moiety. The melting point was measured as 233–238 ◦C. The IR
spectra shows a strong and characteristic S=O stretching vibration at about 1038 cm−1 for the R-OSO3H
groups, 1697 cm−1 for (C=O), 3217 cm−1 (OH). The 1H-NMR spectrum (Table 1) of
2 showed signals
for three aromatic protons at δH 7.94 (1H, s, H-4), 7.64 (1H, s, H-5), 7.05 (1H, s, H-8). In the 13C-NMR
(Table 1) spectrum nine carbon signals, including three aromatic methines and six quaternary carbons
were observed. HSQC and HMBC experiments of Compound
2
were useful to assign all signals in
105.2) were used to assign the
the 1H- and 13C-NMR spectra. C-4 (
δ
133.3), C-5 ( 122.4) and C-8 (δ
δ
protons H-4 (7.94, s), H-5 (7.64, s) and H-8 (7.05, s) by 1H-13C HSQC. The HMBC spectrum showed
correlation peaks between the proton signal at δH 7.94 (1H, s, H-4) and carbon resonances at δC 161.1
(C-2), 151.1 (C-9), 134.2 (C-3), 122.4 (C-5); between the proton signal at δH 7.64 (1H, s, H-5) and carbon
resonances at δC 152.9 (C-7), 151.1 (C-9), 137.7 (C-6), 133.3 (C-4); and between the proton signal at δH
7.04 (1H, s, H-8) and carbon resonances at δC 152.9 (C-7), 151.1 (C-9), 137.7 (C-6), 112.3 (C-10). These
spectroscopic data suggested that compound
signal of C-3 and the up field-shifted of C-6 indicated that the sulfate groups were attached at these
carbons. The structure of was deduced as 7-hydroxycoumarin-3,6-disulfate. Compound represents
2 is 3,6,7-trisubstituted coumarin. The low field-shifted
2
2
a new coumarin for which we propose the trivial name “dasycladin B”.
NMR spectra and HR-ESI–MS data for the novel sulfated coumarins
S1A–E and S2A–E in the Supplementary Material.
1
and 2 are shown in Figures
2.2. HPLC-Method Development
A HPLC method was developed for quantification of the coumarins. Four coumarins were isolated
as described above and used as standards in addition to the two synthetized sulfated phenolic acids
and their educts (see Figure 1). Several different stationary phases were screened for the separation
of the coumarins and phenolic acids in D. vermicularis, such as Zorbax SB-C18 3.5
ODS 3 m, YMC-triart C-18, 3.5 m and Kinetex C-18 2.6 m. However, the best separation was
achieved on the Gemini C 18 110 Å, 3 m (150 mm 4.6 mm). The latter column yielded the best
µm, Hyperclone
µ
µ
µ
µ
×
results concerning separation efficiency and peak shape, resulting in an optimum separation within
less than 25 min (Figure 2).
5,80-Di-(6(60),7(70)-tetrahydroxy-3-sulfoxy-30-sulfoxycoumarin) (
followed by 7-hydroxycoumarin-3,6-disulfate (2;
1
) eluted first (13.12 min),
16.58 min), the sulfated phenolic acids
4-(sulfooxy)benzoic acid and 4-(sulfooxy)phenylacetic acid ( 17.73 min, 18.3 min),
then 6,7-dihydroxycoumarin-3-sulfate ( ; 18.81 min), 3,6,7-trihydroxycoumarin ( ; 20.63 min),
4-(hydroxyl)phenylacetic acid (7; 21.62 min), and finally 4-(hydroxyl)-benzoic acid (8; 22.07 min).
5;
6;
4
3