1
Supplementary
Structural eElucidation
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 13C-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 C-10 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 DFA. 1H-NMR spectroscopy showed the absence of the methylene group at δH 3.32ppm and the presence of a methyl group at δH 1.13ppm. 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 MFA-1. 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 DδC 3.0 ppm at C-2 compared to MFA-2.
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)
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
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)
Supplementary Fig. 6 1H-1H-COSY (lines) observed in fomannoxin amide
Supplementary Fig. 7 HMBC correlations (blue lines) observed in fomannoxin amide
Supplementary Table 1 Physico-chemical properties of DFA, MFA-1, MFA-2 and fomannoxin amide
Appearance / white powder / white powder / white powder / white powder
Molecular weight
HR-ESI-MS (m/z)
measured (M+H)+
calculated (M+H)+ / 238.2
239.0917
239.0920
Δ = 0.31 ppm / 222.3
223.0969
223.0970
Δ = 0.44 ppm / 220.2
221.0815
221.0814
Δ = 0.64 ppm / 203.3
204.3
n.d.
n.d.
Molecular formula / C12H14O5 / C12H14O4 / C12H12O4 / C12H13NO2
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 amideNo. / 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) / 7.70 (1H, d, 8.4) / 130.4 (d) / 7.70 (1H, d, 8.4) / 132.2 (d) / 7.86 (1H, d, 8.4) / 128.4 (d) / 7.68 (1H, dd, 8.4,1.6 )
4 / 108.4 (d) / 6.79 (1H, d, 8.4) / 108.3 (d) / 6.78 (1H, d, 8.4) / 109.5 (d) / 6.95 (1H, d, 8.4) / 108.0 (d) / 6.80 (1H, d, 8.4)
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, 1.6)
8 / 28.7 (t) / 3.25 (1H, dd, 16.2, 8.1)
3.12 (1H, dd, 16.3, 9.8) / 29.3 (t) / 3.16 (2H, dd, 9.4, 8.2) / 127.7 (d) / 5.06 (1H, d, 3.9) / 33.3 (t) / 3.40 (1H, dd, 15.8, 9.6)
3.01 (1H, dd, 15.8, 7.7)
9 / 86.5 (d) / 4.82 (1H, dd, 9.7, 8.2) / 90.1 (d) / 4.63 (1H, dd, 9.2, 8.4) / 94.0 (d) / 4.86 (1H, d, 4.0) / 85.7 (d) / 5.30 (1H, dd, 8.4, 8.4)
10 / 72.5 (s) / - / 70.2 (s) / - / 141.8 (s) / - / 143.7 (s) / -
11 / 66.7 (t) / 3.32 (2H, s) / 24.8 (q) / 1.13 (3H, s) / 112.0 (t) / 5.02 (1H, s)
4.90 (1H, s) / 111.8 (t) / 5.05 (1H, s)
4.90 (1H, s)
12 / 20.0 (q) / 1.09 (3H, s) / 25.9 (q) / 1.12 (3H, s) / 17.2 (q) / 1.69 (3H, s) / 16.9 (q) / 1.71 (3H, s)