Bacillamidins A–G from a Marine-Derived Bacillus pumilus

Seven long-chain amides, including five previously undescribed bacillamidins A–E (1–5) and two previously reported synthetic analogs, bacillamidins F (6) and G (7), were isolated from extracts of the marine-derived Bacillus pumilus strain RJA1515. The structures of the new compounds were established by extensive analysis of 1D and 2D nuclear magnetic resonance (NMR) data as well as high resolution mass spectrometry (HRMS), and the absolute configurations of the stereogenic carbons of 1–4 were established by comparison of the calculated and the experimental electronic circular dichroism (ECD) spectra. The cytotoxic and antimicrobial activities of 1–7 were evaluated.


Introduction
Marine microorganisms have become a promising source of structurally diverse and bioactive compounds [1,2]. Marine Bacillus species, which are ubiquitous in the marine ecosystem [3], can produce versatile secondary metabolites that exhibit a wide range of biological activities, such as antimicrobial, anticancer, and antialgal activities [4][5][6][7]. The emergence of microbial resistance to currently available antibiotics makes the treatment of infectious diseases more challenging, and the development of new antimicrobial agents is an urgent need [8].
In a previous study by our group, the ethyl acetate (EtOAc) extract of the culture broth of the strain RJA1515, identified as Bacillus pumilus, showed potent antimicrobial activity against Gram-negative bacteria through the inhibition of the enzyme citrate synthase type II [9]. Our ongoing chemical investigation on the same strain led to the isolation of seven long-chain amides including five new compounds, bacillamidins A-E (1-5), and two previously reported synthetic analogs, bacillamidins F (6) and G (7) (Figure 1). This paper describes the isolation, the structural elucidation, and the in vitro cytotoxicity and antibacterial activity assays of the isolated compounds.

Results and Discussion
The strain B. pumilus RJA1515 was cultured in solid agar for 14 days, then the bacterial cultures and the agar were extracted with EtOAc to afford a crude ethyl acetate extract. The extract was fractionated and purified by repeated column chromatography to give bacillamidins A-G (1-7).

Results and Discussion
The strain B. pumilus RJA1515 was cultured in solid agar for 14 days, then the bacterial cultures and the agar were extracted with EtOAc to afford a crude ethyl acetate extract. The extract was fractionated and purified by repeated column chromatography to give bacillamidins A-G (1-7).
Compound 1 was obtained as a white powder. The molecular formula was determined as C 17   The presence of an alkanone moiety was corroborated by hydrogen-hydrogen correlation spectroscopy ( 1 H-1 H COSY) and heteronuclear multiple bond correlation (HMBC) correlations ( Figure 2, Figures S4 and S5). The 1 H-1 H COSY spectrum showed correlations from the methyl doublet at δ H 0.84 (CH 3 -11 and CH 3 -12) to H-10 (δ H 1.48), thus, a gem-dimethyl terminus was established (Table 1). An alkyl chain moiety with eleven carbon atoms was established by HMBC correlations from H-3 (δ H 2.08, t, J = 7.6 Hz) to C-4 (δ C 25.1) and C-5 (δ C 28.4) and from the terminal gem-dimethyl protons (CH 3 -11 and CH 3 -12) to C-9 (δ C 38.4), as well as the presence of a broad singlet, integrated for sixteen methylene protons (H 2 -3 to H 2 -10). The HMBC correlation from H-3 to C-2 (δ C 172.2) suggested the connectivity of an alkyl chain to the C-2 amide carbonyl carbon. An amide group was confirmed by the HMBC correlation from the proton doublet of H-1 at δ H 8.31 to C-2. The HMBC correlations from H-1 to C-1 (δ C 48.4), from H-1 (δ H 4.60) to C-1 (δ C 170.4) and C-2 (δ C 35.6), and from H-2 a (δ H 2.79)/H-2 b (δ H 2.67) to C-1 and C-3 (δ C 171.2) suggested the presence of the aspartic acid moiety [10]. In addition, the HMBC correlations from the two methoxyl groups at δ H 3.61 and δ H 3.60 to C-1 and C-3 , respectively, indicated the locations of two methyl ester groups.
The absolute configuration of C-1' was defined as R by comparison of the experimental optical rotation (  Compound 2 was obtained as a white powder. Its molecular formula was established as C 19  except for the presence of two additional methylene groups (δ H 1.23; δ C 29.9, 29.0), one methyl triplet at δ H 0.83 (3H, t, J = 9.0 Hz), and one methyl doublet δ H 0.78 (3H, d, J = 9.0 Hz). The 1 H-1 H COSY cross-peaks from H-11 (δ H 1.23, m) to H-12 (δ H 1.10, m) and H-14 (δ H 0.78, d, J = 9.0 Hz) and from H-12 to H-13 (δ H 0.83, t, J = 9.0 Hz) and the HMBC correlations ( Figure 2, Figures S12 and S13) from H-13 to C-12 (δ C 29.0) and C-11 (δ C 33.7) and from H-14 to C-10 (δ C 36.0), C-11, and C-12 indicated that 2 had a terminal sec-butyl group in the alkyl chain. All the aforementioned information indicated the presence of a 10-methyldodecanoyl motif in 2, instead of the 9-methyldecanoyl moiety in 1. The absolute configuration of C-1 was established by comparison of the calculated and the experimental ECD spectra ( Figure 4). However, the absolute configuration of C-11 was not determined. Thus, the structure of 2 was determined as (1 R)-10-methyldodecanoyl dimethylaspartate, named bacillamidin B.  S12 and S13) from H-13 to C-12 (δC 29.0) and C-11 (δC 33.7) and from H-14 to C-10 (δC 36.0), C-11, and C-12 indicated that 2 had a terminal sec-butyl group in the alkyl chain. All the aforementioned information indicated the presence of a 10methyldodecanoyl motif in 2, instead of the 9-methyldecanoyl moiety in 1. The absolute configuration of C-1' was established by comparison of the calculated and the experimental ECD spectra ( Figure 4). However, the absolute configuration of C-11 was not determined. Thus, the structure of 2 was determined as (1'R)-10-methyldodecanoyl dimethylaspartate, named bacillamidin B. .53) to C-4' and C-2', in addition to a NH singlet (δH 11.19), suggested the presence of a succimide substructure [11]. The HMBC correlations from NH-1 (δH 8.42, d, 7.6 Hz) to C-1' (δC 49.5) and C-2' indicated the linkage between the amide and succimide portion. The 1 H-1 H COSY and HMBC spectra (Figures 5, S20, and S21) of 3 indicated the same alkyl side chain as a terminal sec-butyl group in 2. The absolute configuration of C-1' was established by comparison of the calculated and the experimental ECD spectra ( Figure 6). The absolute configuration of C-11 was not determined at a current state. Thus, the structure of 3 was determined as (3'R)-N-(2, 5-dioxopyrrolidin-3-yl)-10-methyldodecamide, named bacillamidin C. , suggested the presence of a succimide substructure [11]. The HMBC correlations from NH-1 (δ H 8.42, d, 7.6 Hz) to C-1 (δ C 49.5) and C-2 indicated the linkage between the amide and succimide portion. The 1 H-1 H COSY and HMBC spectra ( Figure 5, Figures S20 and S21) of 3 indicated the same alkyl side chain as a terminal sec-butyl group in 2. The absolute configuration of C-1 was established by comparison of the calculated and the experimental ECD spectra ( Figure 6). The absolute configuration of C-11 was not determined at a current state. Thus, the structure of 3 was determined as (3 R)-N-(2, 5-dioxopyrrolidin-3-yl)-10-methyldodecamide, named bacillamidin C.        Figures S25, S26, S28 and S29) showed that the alkyl side chain of 4 possessed a gem-dimethyl terminus, similar to that of 1. The rest 1 H and 13 C NMR data of 4 was very similar to those of 3 except for the absence of the NH singlet at δ H 11.19 and the presence of a sec-butyl moiety (two methyl doublets at δ H 0.84, J = 6.6 Hz/δ C 20.0, a multiplet at δ H 1.90, /δ C 26.7, and a doublet at δ H 3.19, J = 7.2 Hz/δ C 45.4). Therefore, the structure of 4 was established as shown in Figure 3. The absolute configuration of C-1 was established by comparison of the calculated and the experimental ECD spectra (Figure 7). Therefore, the structure of 4 was determined as (3 R)-N-(1-isobutyl-2, 5-dioxopyrrolidin-3-yl)-9-methyldecanamide, named bacillamidin D.
The negative sign of the Cotton effect for the n-π* transition of the carbonyl group at 260 nm in the ECD spectrum ( Figure S42) was in agreement with the 3 S configuration [13]. Moreover, the J H-3 /H-5 (10.4 Hz) value was indicative of the trans configuration between H-3 and H-5 [13]. In addition, the ECD spectrum of 5 showed a positive Cotton effect at 220 and 242 nm and a negative Cotton effect at 260 and 314 nm, which were in good agreement with those for amicoumacin B and amicoumacin C [15]. Thus, the structure of 5 was determined as (3 S,5 S,8 S,9 S,10 S)-8 ,8 -dihydroxy-9 -oxotetrahydrofuran-10 -yl-13-methyltetradecanamide, named bacillamidin E. Compound 5 was obtained as a white powder and its molecular formula was established as C35H54N2O5 by HRESIMS (m/z 653.3775 [M + Na] + , calcd. 653.3778), implying ten degrees of unsaturation. The UV spectrum of 5 showed absorption maxima at 248 and 300 nm which are characteristic of the amicoumacins [12,13]. The 1 H NMR spectrum (Table 3, Figure S33 .1, 48.3) carbons. These NMR data were similar to those of amicoumacin C [14]. The gross structure of 5 was therefore established based on the HSQC and HMBC correlations (Figure 8), indicating that an amicoumacin unit was linked to an alkanone side chain with a gem-dimethyl terminus.
The negative sign of the Cotton effect for the n-π* transition of the carbonyl group at 260 nm in the ECD spectrum ( Figure S42) was in agreement with the 3'S configuration [13]. Moreover, the JH-3'/H-5'' (10.4 Hz) value was indicative of the trans configuration between H-3' and H-5'' [13]. In addition, the ECD spectrum of 5 showed a positive Cotton effect at 220 and 242 nm and a negative Cotton effect at 260 and 314 nm, which were in good agreement with those for amicoumacin B and amicoumacin C [15]. Thus, the structure of 5 was determined as   Compounds 6 and 7 were obtained as white powders. Their molecular formulas were determined as C 15 H 31 NO and C 17 H 35 NO according to sodium-adduct ion peaks at m/z 264.2301 for 6 and 292.2616 for 7. The structures of 6 and 7 were further established by analyses of their 1 H, 13 C, HSQC and HMBC NMR spectral data (Table S1, Figure 9, Figures S43-S46 and S51-S54) as 13-methyltetradecanamide (bacillamidin F) [16], and 14-methylhexadecanamide (bacillamidin G) [17], respectively. The absolute configuration of C-15 of 7 was not determined at current state. Although 6 and 7 have been already synthesized [16,17], this is the first report of their isolation from a natural source. 108.3, C 10' 140.4, C Compounds 6 and 7 were obtained as white powders. Their molecular formulas were determined as C15H31NO and C17H35NO according to sodium-adduct ion peaks at m/z 264.2301 for 6 and 292.2616 for 7. The structures of 6 and 7 were further established by analyses of their 1 H, 13 C, HSQC and HMBC NMR spectral data (Table S1, Figures 9, S43-S46, and S51-S54), as 13methyltetradecanamide (bacillamidin F) [16], and 14-methylhexadecanamide (bacillamidin G) [17], respectively. The absolute configuration of C-15 of 7 was not determined at current state. Although 6 and 7 have been already synthesized [16,17], this is the first report of their isolation from a natural source.  Table 4). None of the compounds showed significant cytotoxicity in all the cell lines tested. Compounds 1-4 showed antibacterial activity against P. aeruginosa PA-01 and A. baumannii ATCC19606 with minimum inhibitory concentration (MIC) values ranging from 58 to 64 μg/mL. The cytotoxic activity against human hepatoma cell line (HepG2), human lung cancer cell line (A549), human breast cancer cell line (MDA-MB-231), and human gastric cancer cell line (SGC7901) and the antibacterial activity against Pseudomonas aeruginosa PA-01, Acinetobacter baumannii ATCC19606 and Escherichia coli BW25113 of 1-7 were investigated ( Table 4). None of the compounds showed significant cytotoxicity in all the cell lines tested. Compounds 1-4 showed antibacterial activity against P. aeruginosa PA-01 and A. baumannii ATCC19606 with minimum inhibitory concentration (MIC) values ranging from 58 to 64 µg/mL.

Strain and Cultivation
Bacillus pumilus strain RJA1515 was isolated from marine sediments collected in Bamfield, British Columbia at a depth of 84 m by Raymond J. Andersen's group, in January 2010. The strain was cultured on solid agar at room temperature in 160 pans measuring 50 cm × 30 cm, equivalent to 6.8 L volume of the marine medium 1 (MM1) [18]. After 14 days, the bacterial cultures and the agar were extracted twice with EtOAc (2 × 80 L). Then the extracts were re-dissolved in 2.5 L of EtOAc and back extracted three times with 600 mL of water after being combined and dried in vacuo. The organic fraction was then dried in vacuo to give an extract (32.0 g).

Extraction and Isolation
The EtOAc extract (32.