1
Supplementary
Structural Elucidation
The physico-chemical properties of DFA, MFA-1, MFA-2 and fomannoxin amide are
summarized in Supplementary Table 1. 13C- and 1H-NMR data of DFA, MFA-1, MFA-2 and
fomannoxin amide are summarized in Supplementary Table 2. The exact molecular masses
were determined by high-resolution HPLC-ESI-Orbitrap mass spectrometry for DFA as
239.0917 ([M+H]+theor = 239.0920, Δm = 0.31 ppm), for MFA-1 as 223.0969 ([M+H]+theor =
223.0970, Δm = 0.44 ppm), for MFA-2 as 221.0815 ([M+H]+theor = 221.0814, Δm = 0.64
ppm) and for fomannoxin amide as with HPLC-ESI-QQQ mass spectrometry ([M+H]+ =
204.3) corresponding to the molecular formulae C12H14O5 (DFA), C12H14O4 (MFA-1),
C12H12O4 (MFA-2) and C12H13NO2 (fomannoxin amide).
The structure elucidation of DFA, MFA-1, MFA-2 and fomannoxin amide was done on the
basis of extensive 1D and 2D NMR experiments. The 1H-NMR spectrum of DFA showed a
total of 8 signals, with three signals in the aromatic region, five in the region between 4 and 5
ppm and three signals in the aliphatic region (Supplementary Table 2). Integration of the
signals revealed the presence of 11 protons, with the signal at δH 1.09 ppm corresponding to
three protons and the signal at δH 3.32 ppm corresponding to two protons. The
13
C-NMR
spectra as well as distortionless enhancement by polarization transfer (DEPT) spectra further
revealed the presence of five quaternary, three aromatic and one aliphatic methine, two
methylene and one methyl carbon atoms. The correlation of 1H-NMR signals to the
corresponding 13C-carbon atoms was carried out in a heteronuclear single quantum coherence
(HSQC) NMR experiment as shown in Supplementary Table 2, revealing the signals at δH
3.25 ppm and δH 3.12 ppm belonging to a methylene group at δC 28.7 ppm The 1H-1H-COSY
experiment revealed correlations between H-3 and H-4 and between H-8 and H-9
(Supplementary Fig. 2). The HMBC (heteronuclear multiple bond correlation) experiment
allowed assignment of the structure of DFA as follows. The correlation from H-7 to C-1, C-3,
C-5 and C-8, from H-3 to C-1, C-7, C-4 and C-5 and from H-4 to C-2, C-3 and C-6 gave rise
to the benzoic acid substructure of DFA. H-8 showed correlation to C-5, C-6, C-7, C-9 and C10 giving rise to the tetrahydrofuranyl substructure. The HMBC correlation from H-9 to C-11
and C-12 and from H-11 and H-12 to C-9 and C-10 gave rise to the full elucidation of DFA
with the two hydroxyl groups coupled vicinal to C-10 and C-11 (Supplementary Fig. 2).
DFA and MFA-1 showed very similar NMR signals for the benzoic acid and the
tetrahydrofuranyl substructure hence the difference in structure could only be due to changes
at C-10 or C-11. The molecular formula revealed that MFA-1 lacks one oxygen compared to
2
DFA. 1H-NMR spectroscopy showed the absence of the methylene group at δH 3.32 ppm and
the presence of a methyl group at δH 1.13 ppm. Confirmation of the structure by HMBC
correlation from H-11 to C-9, C-10 and C-12 gave rise to the structure of MFA-1
(Supplementary Fig. 3).
The molecular formula of MFA-2 shows the lack of two hydrogen atoms compared to MFA1. Comparison of the NMR signals revealed a shift of δC 127.7 ppm at C-8 and the presence
of a double bond at C-10 (δC 141.8 ppm) and C-11 (δC 112.0 ppm) (Supplementary Fig. 4).
The molecular formula of fomannoxin amide shows the lack of two oxygen and the addition
of one nitrogen and one hydrogen compared to MFA-2. Comparison of the NMR signals
revealed the lack of a hydroxyl group at position C-8 and the presence of a methylene group,
accounting for the lack of one oxygen group. The remaining difference could be explained by
the presence of a carboxylic acid amide substructure at C-2 instead of a carboxylic acid
substructure (Supplementary Fig. 6, Supplementary Fig. 7), which could be elucidated by a
downfield shift of δC 3.0 ppm at C-2 compared to MFA-2.
3
Supplementary Fig. 1 HPLC analysis of a culture filtrate extract from Streptomyces sp. AcH
505 after a cultivation time of 48 h, monitored at 260 nm. Inserts: UV-visible spectra of DFA
(retention time 5.4 min), MFA-1 (retention time 6.7 min), MFA-2 (retention time 7.5 min)
and fomannoxin acid (retention time 9.4 min)
4
Supplementary Fig. 2 1H-1H-COSY (bold lines) and HMBC correlations (arrows) observed
in DFA
Supplementary Fig. 3 1H-1H-COSY (bold lines) and HMBC correlations (arrows) observed
in MFA-1
Supplementary Fig. 4 1H-1H-COSY (bold lines) and HMBC correlations (arrows) observed
in MFA-2
5
Supplementary Fig. 5 HPLC analysis of a culture filtrate extract from Streptomyces sp. GB
4-2 after a cultivation time of 25 h, monitored at 210 nm. Inserts: UV-visible spectra of
fomannoxin amide (retention time 8.9 min) and of fomannoxin alcohol (retention time 9.5
min)
6
Supplementary Fig. 6 1H-1H-COSY (lines) observed in fomannoxin amide
Supplementary Fig. 7 HMBC correlations (blue lines) observed in fomannoxin amide
7
Supplementary Table 1 Physico-chemical properties of DFA, MFA-1, MFA-2 and
fomannoxin amide
DFA
MFA-1
MFA-2
FOMANNOXIN
AMIDE
Appearance
white powder
white powder
white powder
white powder
Molecular weight
238.2
222.3
220.2
203.3
measured (M+H)+
239.0917
223.0969
221.0815
204.3
calculated (M+H)+
239.0920
223.0970
221.0814
n.d.
Δ = 0.31 ppm
Δ = 0.44 ppm
Δ = 0.64 ppm
n.d.
C12H14O5
C12H14O4
C12H12O4
C12H13NO2
HR-ESI-MS (m/z)
Molecular formula
8
Supplementary Table 2 1H- and 13C-NMR Data for DFA, MFA-1, MFA-2 and fomannoxin
amide in DMSO-d6
DFA
MFA-1
MFA-2
FOMANNOXIN AMIDE
No.
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
1
167.2 (s)
-
167.2 (s)
-
167.0 (s)
-
167.5 (s)
-
2
122.6 (s)
-
123.0 (s)
-
123.8 (s)
-
126.8 (s)
-
3
130.4 (d)
7.86 (1H, d,
128.4 (d)
7.68 (1H,
4
108.4 (d)
5
163.6 (s)
-
163.5 (s)
-
163.0 (s)
-
161.7 (s)
-
6
128.4 (s)
-
128.1 (s)
-
129.9 (s)
-
126.7 (s)
-
7
126.4 (d)
7.75 (1H, s)
126.3 (d)
7.74 (1H, s)
127.6 (d)
7.92 (1H, s)
124.7 (d)
7.75 (1H,d,
8
28.7 (t)
7.70 (1H,
d, 8.4)
6.79 (1H,
d, 8.4)
130.4 (d)
108.3 (d)
86.5 (d)
8.4)
6.78 (1H, d,
8.4)
132.2 (d)
109.5 (d)
8.4)
6.95 (1H, d,
dd, 8.4,1.6 )
108.0 (d)
8.4)
6.80 (1H, d,
8.4)
1.6)
3.25 (1H,
3.40 (1H,
dd, 16.2,
dd, 15.8,
8.1)
3.12 (1H,
29.3 (t)
3.16 (2H,
dd, 9.4, 8.2)
127.7 (d)
5.06 (1H, d,
3.9)
33.3 (t)
9.6)
3.01 (1H,
dd, 16.3,
dd, 15.8,
9.8)
7.7)
4.82 (1H,
9
7.70 (1H, d,
dd, 9.7,
90.1 (d)
8.2)
4.63 (1H,
dd, 9.2, 8.4)
94.0 (d)
10
72.5 (s)
-
70.2 (s)
-
141.8 (s)
11
66.7 (t)
3.32 (2H, s)
24.8 (q)
1.13 (3H, s)
112.0 (t)
12
20.0 (q)
1.09 (3H, s)
25.9 (q)
1.12 (3H, s)
17.2 (q)
4.86 (1H, d,
4.0)
5.02 (1H, s)
4.90 (1H, s)
1.69 (3H, s)
85.7 (d)
143.7 (s)
111.8 (t)
16.9 (q)
5.30 (1H,
dd, 8.4, 8.4)
5.05 (1H, s)
4.90 (1H, s)
1.71 (3H, s)