Introduction

Antibiotics are one of the pillars of modern medicine. The misuse of these drugs for prophylactic purposes or for enhancing the growth of livestock is known to exert a selection pressure on microbial communities, which ultimately favors the spread of resistant organisms. Due to the irrational application of antibiotics, the number of pathogens that do not respond to first-line therapeutics steadily increases. This, in turn, urges the need for the discovery of novel drugs, especially for the treatment of nosocomial infections.1

Due to their structural diversity and high affinity to biological targets, natural products are ideal lead structures for drug development. Predatory bacteria can be considered as a promising and largely untapped resource to identify new antibiotics.2 Recent evidence suggests that secondary metabolites of predatory bacteria contribute to their feeding strategy by killing or paralyzing prey organisms.3, 4 Furthermore, it is conceivable that compounds, which mediate cell lysis, can also be used to facilitate the digestion and consumption of prey. Myxobacteria are particularly noteworthy in the context of drug discovery, as single strains possess the genomic potential to produce a huge diversity of natural products.5, 6 Many of the encoded compounds feature unique structural scaffolds and exhibit rare modes of action.7

Here, we report the isolation and taxonomic identification of the predatory, myxobacterial strain HKI 722 as well as the bioactivity-guided isolation of five antimicrobial compounds. Two of the metabolites (1, 2) were identified as new myxothiazol derivatives (Figure 1), whereas the remaining compounds represent previously described members of this natural product family, including myxothiazol A (3), myxothiazol Z (4) and desmethylmyxothiazol (5).

Figure 1
figure 1

Structures of isolated myxothiazols 15 from strain HKI 722.

Results

Isolation and taxonomy of the producing strain

Strain HKI 722 was isolated in the course of sampling predatory bacteria from the shores of the river Saale, Germany, using a baiting technique. The isolate grew well in the complex medium MD1, but was not able to grow in chemically defined minimal media with glucose as sole carbon source. The growth optimum was observed in the temperature range between 30 °C and 35 °C. On solid media, the bacterium formed red colored swarms. Microscopic analysis revealed non-flagellated vegetative cells, which appeared as slender rods with tapering ends. They exhibited a diameter of 0.5 μm and a length from 3.0 to 10.0 μm. The formation of fruiting bodies, which is a phenotypic hallmark of many myxobacteria,8 was not observed. Sequencing of the 16S rDNA gene of strain HKI 722 supported a phylogenetic classification as Myxococcus fulvus (100% identity with the 16S rDNA gene sequence of M. fulvus strain btx2; accession number: KC862605).

Fermentation and isolation procedure for myxothiazols

For the isolation of biologically active compounds, strain HKI 722 was cultured in shaking Erlenmeyer flasks on a 90-l scale. At the end of cultivation, the cells were separated from the fermentation broth by centrifugation. Metabolites that had been secreted into the culture broth during fermentation were recovered with the adsorber resin XAD-2. Following the removal of the supernatant by filtration, the adsorbed compounds were exhaustively eluted from the resin with a 1:1 mixture of methanol and acetone. The resulting extract was concentrated to dryness, resuspended in water and extracted with dichloromethane. The previously observed antimicrobial activity was tracked to the organic phase, which was hence subjected to reversed-phase HPLC, yielding 5.4 mg of myxothiazol S1 (1) and 7.8 mg of myxothiazol S2 (2). The production of the previously described myxothiazols was considerably larger. Overall, 28.8 mg of 3, 22.2 mg of 4 and 93.6 mg of 5 were isolated.

Structure determination

The molecular formula of 1 was assigned to be C17H21N3O4S2 by high-resolution ESI-MS, which corresponds to nine degrees of unsaturation. An inspection of the 13C NMR spectrum confirmed the deduced number of carbon atoms (Table 1). Furthermore, a total of 10 signals could be ascribed to sp2-hybridized carbons based on their chemical shifts, including a ketone function resonating at δC 207.8 (C-3). The 1H NMR spectrum of 1 showed 18 non-exchangeable protons. First-order multiplet analysis and homonuclear COSY revealed two independent spin systems, namely a 1-hydroxyethyl residue and a –CH(CH3)–CH(OR)–CH=CH– partial structure (Figure 2). The latter covered the carbon atoms C-4 to C-7 and C-17. HMBC data allowed the linkage of C-4 with a 2-carbamoyl-acetyl moiety. H-4, H-5 and H3-17 showed long-range correlations to C-3. The same carbon atom was also observed by the methylene protons of C-2, which exhibited another specific HMBC to C-1 (δC 169.7). An HMBC from H3-16 to C-5 established the presence of a methoxy group at C-5. The two remaining proton singlets at δH 7.17 and 7.99 belonged to sp2-hybridized methine groups and were distinguished by large 1JCH values (187 Hz in case of CH-9 and 192 Hz in case of CH-12), which places them next to either a nitrogen or a sulfur atom.9 Both singlets were individually correlated with two quaternary aromatic carbons in the HMBC spectrum. The elemental composition of 1 as well as the observed chemical shifts was consistent with the presence of two thiazole rings.10, 11, 12, 13 HMBCs from H-6 and H-7 to C-8 as well as from H3-15 to C-13 eventually led to two major fragments that could only be connected via C-10 and C-11, thus establishing the planar structure of 1. To determine the configuration of the carbinol group in 1, the (R)- and (S)-Mosher esters were prepared. In case of the methyl protons in position 15, an ΔδSR value of +0.007 p.p.m. was measured indicating the (R) configuration.14 Since the esterification of the secondary alcohol in 1 did not affect the chemical shifts of further protons and the observed chemical shift difference was also rather small, this stereochemical assignment must be considered with care. The relative orientation of H-4 and H-5 was examined by using molecular modeling, 3JHH coupling constant analysis and synthetically prepared reference compounds. A combination of molecular mechanics and quantum mechanics calculation was performed on all four remaining stereoisomers of 1 with 4R,5S,14R, 4S,5R,14R, 4R,5R,14R and 4S,5S,14R configurations. In case of the 4R,5R,14R and 4S,5S,14R configuration, all structures within 5 kJ mol−1 of the lowest-energy conformer showed with an H4-C4-C5-H5 torsion angle of 180° solely an anti conformation (III in Figure 3). In contrast, the anti (H4-H5180°, III in Figure 3) and one gauche (H4-H555°) H-H arrangement (I and V in Figure 3) have almost the same energy in the 4R,5S,14R and 4S,5R,14R configurations and both arrangements are present in the set of lowest-energy conformations (5 kJ mol−1 window from the lowest-energy conformation generated). The dihedral angles acquired from these models were translated into coupling constants, using quantum mechanics calculations on the one hand and by application of the advanced Karplus-type equation proposed by Smith and Barfield15 on the other hand (Table 2). The experimental 3JH4-H5 value (5.3 Hz) almost corresponded to the gauche conformations of the 4R,5S and 4S,5R stereoisomers (Table 2) and consequently ruled out all anti conformations, whose J-values were nearly double the size and ranged from 10 to 11 Hz. The results from the theoretical experiments were verified by analysis of synthetically prepared reference compounds, namely the syn (6) and the anti diastereomers (8) of (E)-5-hydroxy-4-methyloct-6-en-3-one (Figure 4). Consistent with the computational calculations, we determined for 6 a coupling constant of 4.1 Hz and for 8 a coupling constant of 8.7 Hz. It has to be noted that, according to the literature data, the coupling constants of β-hydroxy-α-methyl ketones and β-methoxy-α-methyl ketones are in good accordance.16, 17, 18, 19, 20, 21 While the α- and β-protons of anti diastereomers consistently exhibit J-values between 8 and 10 Hz, the corresponding coupling constants in syn diastereomers are typically observed between 4 and 6 Hz.16, 17, 18, 19, 20, 21 The 3JH4-H5 value of 1 would therefore suggest a syn orientation which, again, supports either a 4R,5S or a 4S,5R configuration. This result is in agreement with the configuration of compound 3, whose stereochemistry was determined as 4R,5S by degradation experiments.22 Since 1 and 3 likely derive from the same biosynthetic pathway,23 it is reasonable to assume that both compounds possess the same absolute configuration in positions 4 and 5. Together with the results from the Mosher analysis, we hence propose a 4R,5S,14R configuration for 1.

Table 1 NMR spectroscopic data of 1 in chloroform-d1
Figure 2
figure 2

1H,1H COSY (bold lines) and selected 1H,13C long-range correlations (arrows) in myxothiazol S1 (1) and myxothiazol S2 (2).

Figure 3
figure 3

Newman projection of myxothiazol S1 (1) along the axis of the C-4/C-5 bond.

Table 2 Conformational analysis of 1
Figure 4
figure 4

Structures of compounds 6 and 8.

For compound 2, high-resolution ESI-MS gave an exact mass at m/z 252.1059 for the [M+H]+ ion, which is consistent with a molecular formula of C13H17NO2S and corresponds to six degrees of unsaturation. The 13C NMR spectrum exhibited only 12 discrete resonances, but one of the signals showed a very high intensity, suggesting the presence of a magnetically equivalent carbon atom. Except for a singlet resonating at 8.14 p.p.m., all proton NMR signals of 2 were split into multiplets (Table 3). Interpretation of the coupling patterns together with COSY data led to the elucidation of a 1,6-dimethyl-hepta-2E,4E-dienyl moiety. The two conjugated double bonds are oriented s-trans, as evidenced by the vicinal coupling between H-7 and H-8 (J 10.3 Hz) and comparison with literature values.10 It is hence obvious that no steric effects occur that prevent the acyclic diene from achieving a near planar conformation. The remaining resonances were assigned to a thiazole-4-carboxylate unit based upon an analysis of HMBC data and comparison of the observed chemical shifts with literature values.24 According to HMBCs the thiazole ring was clearly attached to the former fragment (Figure 2), concluding the structure determination. The absolute configuration of compound 2 was not determined experimentally. For biosynthetic reasoning, the same stereochemistry as in compound 3 is proposed. The isolated compounds 35 were identified as myxothiazol A, myxothiazol Z and desmethylmyxothiazol, respectively, after comparison of their spectroscopic data with literature values.10, 13, 25, 26

Table 3 NMR spectroscopic data of 2 in chloroform-d1

Antimicrobial activities

The purified compounds 15 were profiled in the agar diffusion assay against bacteria and fungi that are likely to be encountered in the natural environment of the soil inhabiting M. fulvus (Table 4). Except for 3, all myxothiazols were active against Bacillus subtilis. The compounds 1 and 2 also inhibited the growth of Pseudomonas aeruginosa, but lacked activity against the actinomycete Mycobacterium vaccae. The opposite antibacterial profile was observed for the known metabolites 35. Furthermore, 3 and 4 possessed strong antifungal properties against Sporobolomyces salmonicolor and Penicillium notatum. The observed zones of inhibition were comparable to that of the reference compound amphotericin B. In contrast, the compounds 1, 2 and 5 exhibited negligible or no antifungal activity.

Table 4 Antimicrobial activities of the isolated myxothiazols 1–5

Discussion

The myxothiazols are thiazole-containing natural products, which are produced by different species within the myxobacterial family Cystobacteraceae.10, 11, 12 In this study, we confirmed that the soil-derived M. fulvus strain HKI 722 is capable to produce a small series of myxothiazol derivatives with complementing antimicrobial activities. Two of the isolated compounds, myxothiazol A (3) and Z (4), had previously been demonstrated to possess significant antifungal properties due to an inhibition of the cytochrome bc1 complex of the respiratory chain. This activity could be linked to their β-methoxyacrylate residues.27, 28 As expected, no comparable potency was observed for the new analogs myxothiazol S1 (1), myxothiazol S2 (2) or for the known desmethylmyxothiazol (5), because these metabolites lack the aforementioned pharmacophore. Instead 1 and 2 were active against P. aeruginosa, whose growth was not affected by any of the other myxothiazols. The arsenal of compounds, which is secreted by strain HKI 722, enables the bacterium to attack a variety of microorganisms that are present in its natural habitat. Whether the isolated myxothiazols are important for predation is currently under investigation.

Materials and methods

General experimental procedures

UV spectra were recorded on a Varian UV–visible Cary spectrophotometer. IR spectra were recorded on an IFS-55 spectrometer (Bruker, Karlsruhe, Germany). Optical rotation was measured using a 0.5-dm cuvette with a P-1020 polarimeter (JASCO, Tokyo, Japan) at 25 °C. High-resolution mass determination was carried out using an Exactive Mass Spectrometer (Thermo Scientific, Bremen, Germany). NMR spectra were recorded at 300 K on Avance III spectrometers (Bruker) with chloroform-d1 as solvent and internal standard. The solvent signal was referenced to δH 7.26 and δC 77.0, respectively. Preparative HPLC was conducted on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) consisting of a LC-20AT pump and a SPD-M20A photodiode array detector.

Isolation of predatory bacteria

The isolation of strain HKI 722 and other predatory bacteria was achieved by a baiting technique. Briefly, 25–50 mg of soil samples was placed on pure water agar (WAT agar: CaCl2 × 2H2O 0.1% (w/v), agar 1.5% (w/v), 3-(N-morpholino)propanesulfonic acid 20 mM, 50 μg ml−1 cycloheximide, pH 7.2), which had been spotted with suspensions of living Escherichia coli DH5α cells. The plates were then incubated at 30 °C for 5 days. Lysis of the E. coli spots during the incubation period indicated the presence of predators. To obtain pure cultures, smears of the lytic zones were transferred to fresh WAT-E. coli or VY/2 agar plates (Baker’s yeast 0.5% (w/v), CaCl2 × 2 H2O 0.1% (w/v), vitamin B12 0.000005% (w/v), agar 1.5% (w/v)), respectively. Subsequent cultivations were conducted at 30 °C in Erlenmeyer flasks filled with MD1 broth. MD1 medium consisted of casitone 0.3% (w/v), CaCl2 × 2H2O 0.07% (w/v), MgSO4 × 7H2O 0.2% (w/v), vitamin B12 0.00005% (w/v), 1 ml trace elements solution SL-4 (EDTA 0.05% (w/v), FeSO4 × 7H2O 0.02% (w/v), ZnSO4 × 7H2O 0.001% (w/v), MnCl2 × 4H2O 0.0003% (w/v), H3BO3 0.003% (w/v), CoCl2 × 6H2O 0.020% (w/v), CuCl2 × 2H2O 0.0001% (w/v), NiCl2 × 6H2O 0.0002% (w/v), Na2MoO4 × 2H2O 0.0003% (w/v)), pH 7.2.

Isolation of genomic DNA and 16S rDNA analysis

Genomic DNA was isolated from a 3-ml aliquot of a growing MD1 culture using the DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA, USA). The 16S rDNA gene was amplified from the genomic DNA using the primer pair p1 (5′-GGCGTAAAGCGCGTGTAGGC-3′) and p2 (5′-CAWRSAGTCGAGTTGCAGA CTB-3′). The reaction (50 μl total volume) included 2 mM MgSO4, 0.2 mM each dNTP, 5% dimethyl sulfoxide, 50 pmol of each primer and 1.25 U Pfu DNA polymerase. Thermal cycling conditions were as follows: initial denaturation, 5 min, 95 °C; amplification, 30 cycles (95 °C for 1 min, 65 °C for 1 min and 72 °C for 1 min); final extension, 20 min, 72 °C. The PCR product was purified by agarose gel electrophoresis and subsequently sequenced.

Fermentation and isolation of secondary metabolites

Large-scale cultivation of strain HKI 722 was carried out in 5-l Erlenmeyer flasks. The flasks were shaken (120 r.p.m.) at 30 °C for 7 days. After cultivation, the cells were separated from the fermentation broth by centrifugation at 11 000 r.p.m. for 10 min. Metabolites that had been secreted into the culture broth during cultivation were recovered by adsorption onto 15 g l−1 Amberlite XAD-2 resin (Supelco, Bellefonte, PA, USA) overnight. The resin was separated from the supernatant by filtration, washed with 1 l deionized water and extracted three times with 500 ml of a 1:1 mixture of methanol and acetone. The organic extracts were combined and evaporated to dryness under reduced pressure. The resulting residue was resuspended in 100 ml deionized water and extracted three times with 100 ml dichloromethane. The dichloromethane layers were combined, stirred with 5 g water-free Na2SO4, filtrated and dried under reduced pressure. This extract was then chromatographed with a Shimadzu UFLC liquid chromatography system (Shimadzu) equipped with a photodiode array detector and a Nucleodur PFP RP column (250 × 10 mm, 5 μm; Macherey-Nagel, Düren, Germany) and a DAD. The myxothiazols were eluted using a linear gradient of methanol in water+0.1% trifluoroacetic acid with wavelength monitoring at 220 and 310 nm. Final purification of each compound was achieved on a Nucleodur C18 HTec column (250 × 10 mm, 5 μm, Macherey-Nagel).

Myxothiazol S1 (1)

IR (film): 3337, 2927, 1667, 1455, 1365, 1298, 1175, 1106, 1073, 1020, 802 cm−1. UV/Vis λmax (MeOH) nm (log ɛ): 218 (4.30), 248 (4.30), 313 (4.04). [α]25D +83.2 (c 0.95, MeOH). HR-ESIMS: m/z 396.1051 [M+H]+, calcd 396.1046 for C17H22N3O4S2.

Myxothiazol S2 (2)

IR (film): 3028, 2924, 1688, 1651, 1454, 1370, 1295, 1173, 1103, 1069, 1017, 799 cm−1. UV/Vis λmax (MeOH) nm (log ɛ): 234 (4.51). [α]25D +102.3 (c 0.87, MeOH). HR-ESIMS: m/z 252.1059 [M+H]+, calcd 252.1053 for C13H18NO2S.

Synthesis of the syn diastereomers of (E)-5-hydroxy-4-methyloct-6-en-3-one (6)

A mixture of 2 ml (20 mmol) of borane dimethyl sulfide was dissolved in 12 ml of dry diethyl ether and heated under an argon atmosphere in a water bath to 35 °C. To this solution, 7.4 ml (46 mmol) of (+)-α-pinene was added dropwise and the solution was stirred for 30 min. Following the precipitation of the (+)-α-pinene borane in an ice-water bath, the solvent was evaporated using a dry argon stream. The pinene borane complex was redissolved in 10 ml of dry n-hexane under an argon atmosphere and cooled in an ice bath. To this solution, 1.6 ml (17.6 mmol) of trifluoromethanesulfonic acid was added. The mixture was stirred for 30 min at room temperature. Upon completion of the reaction, the upper phase was removed, mixed with 10 ml of dry dichloromethane and cooled in an acetone dry ice bath (−78 °C). Afterwards, 1 ml (6 mmol) of Hünig’s base and 320 μl (3 mmol) of diethylketone were consecutively added under stirring before the mixture was supplemented with 430 μl (6 mmol) of trans-crotonaldehyde. The solution was then stirred for 40 min, during which it turned into a viscous white slime. The latter was extracted three times with 20 ml dichloromethane. The organic layers were combined and dried under reduced pressure to give a colorless dull oil, which was suspended in a mixture of 10 ml of methanol and 3 ml of water. To an ice-cooled solution, 4 ml of 30% hydrogen peroxide solution was added and the solution was stirred for 2 h at room temperature. Afterwards, the solution was poured into 30 ml of water, which was extracted three times with dichloromethane. The dichloromethane extracts were combined and concentrated to 20 ml under reduced pressure. This solution was washed with 20 ml of saturated aqueous NaHCO3 solution and with 20 ml of saturated aqueous NaCl solution. The extract was finally dried with sodium sulfate and dried under reduced pressure giving a colorless oil containing the syn diastereomers of (E)-5-hydroxy-4-methyloct-6-en-3-one (6), which was finally purified by reversed-phase HPLC to yield 101 mg (0.65 mmol, 21.7% yield) of 6.

syn-(E)-5-hydroxy-4-methyloct-6-en-3-one (6)

1H-NMR (600 MHz, chloroform-d1) δH [p.p.m.] (J [Hz]) 1.04 (3 H, t, J 7.3, H-1), 1.13 (3 H, d, J 7.3, H-9), 1.70 (3 H, dd, J 6.5, 1.6, H-8), 2.51 (2 H, q, J 7.3, H-2), 2.66 (1 H, dq, J 7.3, 4.1, H-4), 4.33 (1 H, m, H-5), 5.45 (1 H, ddq, J 15.3, 6.6, 1.6, H-6), 5.71 (1 H, ddq, J 15.3, 6.5, 1.2, H-7). 13C-NMR (150 MHz, chloroform-d1) δC [p.p.m.] 7.5 (C-1), 11.0 (C-9), 17.7 (C-8), 35.5 (C-2), 50.6 (C-4), 72.8 (C-5), 128.0 (C-7), 130.6 (C-6), 215.8 (C-3).

Synthesis of the anti diastereomers of (E)-5-hydroxy-4-methyloct-6-en-3-one (8)

A solution of 20 mg (0.14 mmol) of 6 in 20 ml of dry diethyl ether was mixed with 93.5 mg (0.56 mmol) of p-nitrobenzoic acid and 146.8 mg (0.56 mmol) of triphenylphosphine under an argon atmosphere. The solution was then cooled in a water-ice bath (10 °C) and 110 μl (0.56 mmol) of diisopropyl azodicarboxylate was added dropwise. After extended stirring (14 h at room temperature and 3 h at 40 °C), the solution was extracted with a saturated NaHCO3 solution. The aqueous phases were pooled and extracted twice with dimethyl ether. Subsequently, the ether solutions were combined and evaporated to give a yellow oil. The purification of the anti diastereomers of (E)-5-methyl-6-oxooct-2-en-4-yl 4-nitrobenzoate (7) via reversed-phase HPLC yielded 24.4 mg (0.08 mmol, 57.1% yield) of product. To cleave the p-nitrobenzoic ester, 3.2 mg (0.01 mmol) of 7 was hydrolyzed with 4 mg (0.061 mmol) of sodium azide in 2 ml of dry methanol.29 The anti diastereomers of (E)-5-hydroxy-4-methyloct-6-en-3-one (8) were obtained in a yield of 0.71 mg (0.0068 mmol, 68.0% yield) following purification by reversed-phase HPLC.

anti-(E)-5-methyl-6-oxooct-2-en-4-yl 4-nitrobenzoate (7)

1H-NMR (300 MHz, chloroform-d1) δH [p.p.m.] (J [Hz]) 1.02 (3 H, t, J 7.3, H-8), 1.12 (3 H, d, J 7.2, H-9), 1.74 (3 H, dd, J 6.6, 1.6, H−1), 2.54 (2 H, q, J 7.3, H-7), 3.02 (1 H, dq, J 8.9, 7.2, H-5), 5.42 (1 H, ddq, J 15.3, 8.2, 1.6, H-3), 5.64 (1 H, dd, J 8.9, 8.2, H-4), 5.96 (1 H, dq, J 15.3, 6.6, H-2), 8.13 (2 H, d, J 8.9, H-3′), 8.27 (2 H, d, J 8.9, H-4′). 13C-NMR (75 MHz, chloroform-d1) δC [p.p.m.] 7.6 (C-8), 13.3 (C-9), 17.9 (C-1), 35.4 (C-7), 49.7 (C-5), 77.9 (C-4), 123.5 (C-4′), 126.2 (C-7), 130.6 (C-3′), 133.3 (C-6), 135.7 (C-2′), 150.5 (C-5′), 163.4 (C-1′), 212.1 (C-6).

anti-(E)-5-hydroxy-4-methyloct-6-en-3-one (8)

1H-NMR (500 MHz, chloroform-d1) δH [p.p.m.] (J [Hz]) 1.06 (3 H, t, J 7.3, H-1), 1.13 (3 H, d, J 7.3, H-9), 1.71 (3 H, dd, J 6.5, 1.6, H-8), 2.55 (2 H, q, J 7.3, H-2), 2.66 (1 H, dq, J 8.7, 7.3, H-4), 4.41 (1 H, m, H-5), 5.46 (1 H, ddq, J 15.3, 6.6, 1.6, H-6), 5.69 (1 H, ddq, J 15.3, 6.5, 1.2, H-7). 13C-NMR (125 MHz, chloroform-d1) δC [p.p.m.] 7.5 (C-1), 11.1 (C-9), 17.7 (C-8), 35.4 (C-2), 50.6 (C-4), 72.8 (C-5), 128.0 (C-7), 130.6 (C-6), 215.7 (C-3).

Preparation of Mosher esters of 1

To a solution of 1 mg (2.5 μmol) of 1 in 100 μl of dry deuterated chloroform, 1 μl of dry pyridine was added under an argon atmosphere. To this solution, 1 μl of (R)- or (S)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl; Sigma-Aldrich, Steinheim, Germany) was added and the solution was shaken at room temperature for 1 h. After incubation, the solutions were directly analyzed by 1H NMR spectroscopy.

Molecular modeling and Karplus calculations

Structures of myxothiazol S1 (1) in the 4R,5R,14R, 4S,5S,14R, 4R,5S,14R, as well as 4S,5R,14R configuration were manually generated using the Molecular Operating Environment (MOE) 2012.10 developed by Chemical Computing Group.30 The conformational space was then thoroughly explored within the same program using the Merck Molecular Force Field 94 (MMFF94)31 and the stochastic search method with standard settings. Sets between 500 and 600 low-energy structures were produced, which were then further optimized using density functional theory within the Gaussian 09 program package.32 The B3LYP33 hybrid functional was combined with the 6–31 g(d,p) basis set.34, 35, 36, 37, 38, 39, 40, 41 Effects of the solvent chloroform were approximated with an implicit solvent model (IEF-PCM)42, 43, 44 based on the self-consistent reaction field. The conformations were ranked according to the sum of electronic and thermal free energy based on normal-mode analysis. All structures within an energy window of 5 kJ mol−1 from the lowest-energy conformation were chosen for the calculation of chemical shieldings and coupling constants. The valence triple-ζ basis set 6–311+g(d,p)45, 46, 47, 48 was used in these calculations. After additional energy optimization with the larger basis set, the NMR parameters were calculated with the GIAO (gauge invariant/including atomic orbitals) formalism49, 50, 51 and the two-step approach for the coupling constants developed by Deng et al.52

Empirical calculation of the coupling constants 3JH4-H5 from the corresponding dihedral angles was performed with MestRe-J,53 using the Smith-Barfield equation15 parameterized with the corresponding substituents.

Agar diffusion assay

Antimicrobial activities of 15 were determined in a primary screen against B. subtilis ATCC 6633, P. aeruginosa K799/61, M. vaccae IMET 10670, S. salmonicolor SBUG 549 and P. notatum JP 36. To this end, holes with 7 mm diameter were aseptically punched in the respective agar medium. Subsequently, the agar plates were inoculated with the test organisms. In all, 1 mg of every test compound was dissolved in 1 ml of methanol, and 50 μl of this solution was transferred to a single hole. Ciprofloxacin, amphotericin B and methanol served as positive and negative controls, respectively. After evaporation of the solvent, the agar plates were incubated depending on the growth conditions of the test organisms. A noticeable antimicrobial activity resulted in an inhibition zone of >10 mm.