Fermentation and isolation of wychimicins A, B, C and D

A slant culture of MI503-A4 was inoculated into a 500 ml baffled Erlenmeyer flask containing 110 ml of medium consisting of galactose, dextrin, Bacto Soytone, corn steep liquor, glycerol, (NH4)2SO4, and CaCO3. The culture (5 l) was incubated on a rotary shaker (200 rpm) at 30 °C for 7 days. Active compounds were isolated from the extracts of mycelium cakes with MeOH and ethyl acetate followed by fermentation broth with ethyl acetate. The organic layer was purified by silica gel column chromatography and eluted with ethyl acetate:methanol:formic acid (100:0:0, 40:1:0.1, 36:4:0.1, and 40:10:0.125) via reversed-phase octadecylsilyl silica gel column chromatography using a Sephadex LH20 column. The active fractions were further purified via reversed-phase octadecylsilyl silica gel HPLC to obtain four novel compounds, which were termed wychimicins A (82.8 mg), B (47.0 mg), C (62.8 mg), and D (35.5 mg).

Structure elucidation of wychimicins A–D

The physicochemical properties of these compounds are summarized in the supplementary information (S-1). Wychimicins were brown solid compounds. The UV spectra of wychimicins revealed absorption maxima at 281–283 nm in acidic methanol and bathochromic shifts to 317–327 nm in alkaline conditions, as presented in Table S1. The molecular formulae of wychimicins A (1), B (2), C (3), and D (4) were C47H60ClNO11, C47H61ClNO11, C46H58ClNO11, and C46H59NO11, respectively, as determined by HRESI-MS and NMR spectra. 1H and 13C NMR data are summarized in Tables S2–5.

Structure determination of 1

1 has 47 carbons consisting of 14 fully substituted carbons, 20 methine groups, 5 methylene groups, and 8 methyl groups as confirmed by the 1H, 13C, DEPT135, and HMQC spectra (Figs. S1–6), and six exchangeable protons were indicated by these data and the molecular formula. NMR analyses of 1 were summarized in Fig. 2. Spin coupling systems from 5-H (δH 3.57, t 9.8 Hz) to the olefinic proton 11-H (δH 5.63), from 6-H (δH 1.85) to 28-H (δH 0.65), and from 5-H to 10-H (δH 2.23) were established by 1H-1H COSY, as presented in Fig. S4. According to the 13C NMR chemical shifts, oxygen atoms were substituted at C-8 (δC 69.9) and C-9 (δC 75.2) in the substructure. The long-range couplings from 5-H to C-3 (δC 208.7), C-4 (δC 53.6), and C-27 (δC 16.7); from 11-H to C-13 (δC 57.2) and C-29 (δC 27.5); from 27-H (δH 1.20) to the carbonyl carbon C-3, C-4, C-5 (δC 37.9), and C-13; and the from ethyl group of 29-H (δH 1.80, 1.97), which was connected with 30-H (δH 0.95) to C-11 (δC 118.8), C-12 (δC 138.7), and C-13, were confirmed to reflect an 11, 12-dehydro-4, 6, 8, 9, 12, 13-substituted trans-decalin moiety.

Fig. 2

NMR studies of Wychimicins

The COSY spectrum revealed the sequence from 13-H of the decalin ring to the oxymethine proton 17-H (δH 3.83) through the olefinic proton 14-H (δH 5.51), 15-H (δH 5.05), and methylene (δH 2.27 and 2.33). Other spin coupling systems from the methyl proton 32-H (δH 1.08) to the olefinic proton 19-H (δH 4.84) via the methine proton 23-H (δH 2.81), olefinic proton 22-H (δH 5.68), 21-H (δH 5.45), and the methine proton 20-H (δH 3.65) and from 23-H to the methylene proton 24-H (δH 1.85, 1.91) were observed. Long-range couplings from the methyl proton 31-H (δH 1.73) to the oxymethine carbon C-17 (δC 84.5), olefinic carbon C-18 (δC 142.0), and C-19 (δC 122.6); from the methylene proton 24-H to the quaternary carbon C-25 (δC 84.5), methine carbon C-20 (δC 41.9), and C-26 (δC 199.9); and from 20-H to C-25 and C-26 established a C6 chain containing a 3-methylcyclohex-1-ene ring from C-13 of decalin. As another substructure, the sequence from the overlapping methine proton 1′-H (δH 4.84) to 6′-H (δH 1.24) through the methylene 2′-H (δH 1.89, 1.95), the methine 3′-H (δH 4.26), 4′-H (δH 4.04), and 5′-H (δH 3.74) and from 4′-H to the NH proton (δH 6.30) suggested a 2′, 4′, 6′-trideoxy-4′-animohexose moiety. The long-range correlations from 1′-H to C-5′ (δC 69.3) and C-17 and from 5′-H to C-1′ (δC 95.9) established the sugar moiety attached to the C6 chain from decalin. The remaining protons were the two singlet methyl protons 8″-H (δH 2.19) and 9″-H (δH 2.46) and the aromatic singlet proton 3″-H (δH 7.20). Long-range couplings were observed from 3″-H to C-1″ (δC 155.2), C-2″ (δC 125.9), C-4″ (δC 129.5), and C-5″ (δC 125.3); from the methyl proton 8″-H (δH 2.19) to C-1″, C-2″, and C-3″ (δC 133.6); from the methyl proton 9″-H to C-4″, C-5″, and C-6″ (δC 119.8); and from the sugar moiety NH proton to C-6″ and C-7″ (δC 169.3), thereby establishing 5-saturated 2-hydroxy-3,6-dimethylbenzoic acid connected with the sugar moiety at the 4′ position via an amide bond. The MS spectrum of 1 revealed the fragment m/z 312.1000 (C15H19NO4Cl), establishing that C-4″ should be Cl. The bathochromic shift under acidic condition in the UV spectra of 1, and the carbon signals of C-20 (δC 41.9), C-24 (δC 38.7), C-25 (δC 84.5), C-26 (δC 199.9), and three characteristic sp2 carbons C-1 (δC 166.8), C-2 (δC 105.6), and C-3 (δC 208.7) were related to those of tetrocarcin [18] and kijanimicin [19], respectively, suggested that 1 contains a spirotetronate moiety. Finally, 1 possesses a 13-membered macrocyclic ring with a spirotetronate containing trans-decalin and sugar moieties connected to C-17 by an O-glycosidic linkage.

Structure determination of 2

2 possesses 47 carbons consisting of 13 fully substituted carbons, 21 methine groups, 5 methylene groups, and 8 methyl groups as confirmed by the 1H, 13C, DEPT135, and HMQC spectra (Figs. S7–12). NMR analyses of 2 were summarized in Fig. 2. The molecular formula indicated that 2 lost a Cl atom compared with 1. The NMR spectra of 2 were similar to those of 1 excluding the phenyl moiety. The newly appeared protons 3″-H (δH 7.08) and 4″-H (δH 6.61) coupled with each other. Long-range coupling from the methyl proton 8″-H (δH 2.21) to C-1″ (δC 158.2), C-2″ (δC 124.6), and C-3″ (δC 133.2); from the methyl proton 9″-H (δH 2.48) to C-4″ (δC 122.0), C-5″ (δC 132.2), and C-6″ (δC 116.8); from 3″-H to C-1″ and C-5″; from 4″-H to C-2″ and C-6″; and from NH to C-6″ and C-7″ (δC 170.4) were established. The MS spectrum of 2 revealed the fragment m/z 278.1389 (C15H20NO4). From these data, 2 was determined to be a 4″-dechloro derivative of 1.

Structure determination of 3

3 possesses 46 carbons consisting of 13 fully substituted carbons, 21 methine groups, 5 methylene groups, and 7 methyl groups confirmed as by the 1H, 13C, DEPT135, and HMQC spectra (Figs. S13–18). NMR analyses of 3 were summarized in Fig. 2. The molecular formula indicated that 3 lost a CH3 moiety compared with 1. The NMR spectra of 3 were similar to those of 1 excluding the phenyl moiety. The newly appeared protons 2″-H (δH 6.77) and 3″-H (δH 7.29) coupled with each other. Long-range couplings from the methyl proton 8″-H (δH 2.48) to C-4″ (δC 126.1), C-5″ (δC 132.8), and C-6″ (δC 121.0); from 2″-H to C-4″ and C-6″; from 3″-H to C-1″ (δC 156.5) and C-5″ (δC 132.8); and from NH (δH 6.32) to C-6″ and C-7″ (δC 168.7) were detected. The MS spectrum of 3 revealed the fragment m/z 298.0843 (C14H17NO4Cl). From these data, 3 was determined to be a 2″-demethyl derivative of 1.

Structure determination of 4

4 possesses 46 carbons consisting of 13 fully substituted carbons, 22 methine groups, 5 methylene groups, and 7 methyl groups as confirmed by the 1H, 13C, DEPT135, and HMQC spectra (Figs. S19–24). NMR analyses of 4 were summarized in Fig. 2. The molecular formula indicated that 4 lost a Cl atom compared with 1. The NMR spectra of 4 were similar to those of 1 except excluding the phenyl moiety. The protons 2″-H (δH 6.83), 3″-H (δH 7.20), and 4″-H (δH 6.70) were observed in the COSY spectrum. Long-range couplings from the methyl 8″-H proton (δH 2.51) to C-4″ (δC 122.7), C-5″ (δC 135.2), and C-6″ (δC 117.7); from 2″-H to C-4″ and C-6″; from 4″-H to C-2″ (δC 115.8) and C-6″; and from NH (δH 6.48) to C-6″ and C-7″ (δC 169.9) were observed. The MS spectrum of 4 revealed the fragment m/z 264.1235 (C14H18NO4). From these data, 4 was determined to be a 2″-demethyl-4″-dechloro derivative of 1.

Absolute structure of wychimicins

Because the complete structure of wychimicins including their stereochemistry could not be determined by NMR, X-ray crystallographic analysis was attempted. Crystallization of wychimicins A–D was performed in various solvents. Only 4 was able to obtain a single crystal in ethyl acetate/methanol, which was used to perform a single-crystal X-ray diffraction study. The Flack parameter of 0.10 (11) permitted definition of the configuration as 4S, 5R, 6S, 8S, 9R, 10S, 13R, 17R, 20S, 23S, 25R, 1’R, 3’S, 4’S, 5’R (Fig. 3a, b).

Fig. 3

X-ray results of 4. a ORTEP of 4. b Absolute configuration of 4

According to physicochemical analyses, including CD spectra (Figs. S29–33), and NMR studies, wychimicins have the same aglycone and sugar moiety. Only the phenyl moieties differ in structure. These results allowed the absolute configuration of wychimicins A–D to be determined in Fig. 1.

Wychimicins belong to a family of spirotetronates. This family of compounds has various biological activities, as exemplified by the antitumor agents tetrocarcin A1 [20] and kijanimicin [21]; the antiviral drugs JK-1, JK-2 [22], MM46115 [23], and quartromicins [24]; the antibacterial compounds chlorothricin [25], abyssomicin [26], and lobophorins [27]; and the cholecystokinin B inhibitor tetronothiodin [28]. The spiro moiety of wychimicins had the R configuration, similarly as abyssomicin.

Biological activities

Table 1 presents the antimicrobial activities of wychimicins against gram-positive and gram-negative bacteria. The minimum inhibitory concentrations of wychimicins were determined by the agar dilution method. All compounds exhibited excellent antimicrobial activities against gram-positive bacteria such as S. aureus (including MRSA) and Enterococcus faecalis/faecium (including VRE). However, only 1 and 3 exhibited strong antibacterial activity against S. aureus and E. faecalis/faecium, and lower activity were noted for 2 and 4. This result indicated that the presence of Cl at the 4″ position in the benzoic acid moiety enhances antibacterial activity.

Table 1 Antimicrobial activities of Wychimicins