Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
Antibacterial Cyclic Lipopeptide Enamidonins with an EnamideLinked Acyl Chain from a Streptomyces Species Sangkeun Son,† Sung-Kyun Ko,†,‡ Seung Min Kim,†,∥ Eun Kim,†,‡ Gil Soo Kim,†,‡ Byeongsan Lee,† In-Ja Ryoo,† Won-Gon Kim,‡,§ Jung-Sook Lee,‡,⊥ Young-Soo Hong,†,‡ Jae-Hyuk Jang,*,†,‡ and Jong Seog Ahn*,†,‡ †
Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Korea Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon 34141, Korea § Superbacteria Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea ⊥ Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup 56212, Korea
J. Nat. Prod. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/19/18. For personal use only.
‡
S Supporting Information *
ABSTRACT: Three cyclic lipopeptides, including one known (1) and two new (2 and 3) compounds, that possess the rare enamide linkage group were discovered from Streptomyces sp. KCB14A132, an actinobacterium isolated from a soil sample collected from Jeung Island, Korea. The NMR and MSbased characterization showed that they differed in the amino acid residues in the peptide backbone. Application of Marfey’s analysis, GITC derivatization, and modified Mosher’s method, as well as ECD measurements provided the absolute configurations of enamidonin (1) and those of new compounds enamidonins B and C (2 and 3). The two new enamidonin analogues were shown to exhibit antibacterial activity against Gram-positive bacteria including methicillin-resistant and quinolone-resistant Staphylococcus aureus. Furthermore, evaluation of the extraction conditions and a close inspection of the LC-MS chromatograms revealed that the N,N-acetonide unit of the enamidonin family was formed during the acetone extraction process. The chemically prepared deacetonide derivatives of enamidonins were found to lack antibacterial activity, demonstrating that the dimethylimidazolidinone residue is necessary for antibacterial activity.
C
foam cell formation. They are distinguished from enamidonin by the lack of a double bond at C-12′ in the fatty acyl chain.6 Although the enamidonin family possesses structurally unique moieties, further detailed chemical analysis for stereochemical determination has not been performed. As part of our program to access the chemical diversity of actinomycetes inhabiting the Korean Peninsula,7 soil samples were collected from Jeung Island of Korea and subjected to microbial studies. In the course of LC-MS profiling of culture extracts obtained from subsequent bacterial isolates, the actinobacterium Streptomyces sp. KCB14A132 was chosen for comprehensive chemical analysis because three abundant metabolites were detected in the acetone extract that displayed molecular weights and UV absorptions reminiscent of enamidonin (1). The scale-up culture of this strain followed by solvent extraction and fractionation led to the isolation of enamidonin (1) and two new analogues, enamidonins B and C (2 and 3). These compounds showed activity against Grampositive pathogenic bacteria whose sensitivity and specificity
yclic lipopeptides biosynthesized by bacteria have attracted considerable interest as important leads for the development of therapeutic agents for treating bacterial infections.1 Members of this class of compounds possess variations in the length of the fatty acids and amino acid substitutions, which results in highly diverse structural analogues with different bioactivities. Their proven antibacterial properties make them a distinguished and promising class of natural products, as exemplified by the approved drugs daptomycin and polymyxin B.2 Actinobacteria, renowned as a prime source of antibiotics, have been studied for their ability to biosynthesize structurally diverse cyclic lipopeptides. Following the characterization of the first lipopeptide amphomycin from Actinoplane f riuliensis,3 different classes of cyclic lipopeptides, such as the calcium-dependent antibiotic, tsushimycin, and laspartomycin, have been discovered from actinobacteria belonging to the genus Streptomyces.4 Among them, enamidonin (1), which was isolated from a soil Streptomyces sp. in 1995, is a rare class of cyclic lipopeptides featuring dimethylimidazolidinone and conjugate acrylamideenamide units.5 The structurally related derivatives of enamidonin, K97-0239A and -B, are inhibitors of macrophage © XXXX American Chemical Society and American Society of Pharmacognosy
Received: June 21, 2018
A
DOI: 10.1021/acs.jnatprod.8b00497 J. Nat. Prod. XXXX, XXX, XXX−XXX
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The absolute configurations of the chiral centers present at C-2, C-5, C-16, and C-16′ were determined mainly through chemical derivatizations. The absolute configuration for the secondary hydroxy group at C-16′ was determined by Mosher ester analysis.9 Compound 1 was treated with (R)- and (S)-αmethoxy-α-trifluoromethylphenylacetyl chloride (MTPA-Cl) to afford (S)- and (R)-MTPA esters (1a and 1b), respectively. The 1H NMR chemical shift differences between 1a and 1b (ΔδS−R) around C-16′ determined the absolute configuration to be R (Figure 1).
varied depending on the amino acid substitutions in the enamidonins. We herein report their structures including absolute configurations as defined by chemical and spectroscopic methods and their antimicrobial activities against a panel of pathogenic bacteria. Furthermore, it is herein proposed that a dimethylimidazolidinone residue in the enamidonin family is formed via N,N-acetonide protection of diamine during the acetone extraction process.
Figure 1. ΔδS−R values for the Mosher esters of enamidonin (1).
In the previous report, the stereochemical study of enamidonin had been performed only for Phe by HPLC analysis of the 1-(9-fluorenyl)ethyl chloroformate derivative, assigning the L-configuration.5 In this study, to determine the absolute configurations of the α-carbons in Phe and the two Dpr residues by Marfey’s analysis, compound 1 was subjected to acid hydrolysis with a short reaction time (6 N HCl, 100 °C, 1 h) and derivatization using Marfey’s reagent (1-fluoro-2,4dinitrophenyl-5-L/D-leucinamide, L/D-FDLA).10 Comparison of the FDLA derivatives by LC-MS showed that Phe was in the L-configuration, which was in agreement with the previous report. Meanwhile, LC-MS analysis of FDLA derivatives revealed that two peaks corresponding to both FDLA derivatives of L- and D-Dpr were present in the reaction mixture, indicating the presence of both L- and D-Dpr residues in enamidonin (Figure S2). In many cases, assignment of the regiochemistry of enantiomeric amino acids in peptide compounds is carried out using Marfey’s analysis of a diagnostic peptide fragment.11 The absolute configuration of each Dpr residue was determined by two separate experiments, namely, chemical derivatization, followed by Mosher amide analysis, and repeated Marfey’s analysis. To determine the stereochemistry at C-16, the N,N-acetonide group was first removed using 60% acetic acid at 70 °C. The resulting primary amine at C-16 was then derivatized with (R)- and (S)-MTPACl to produce bis(S)- and (R)-MTPA derivatives (1d and 1e), respectively (Figure 2). Consequently, the 1H NMR chemical shift differences (ΔδS−R) revealed the S-configuration of C-16. This experiment again confirmed the R-configuration of C-16′. The ROESY results of 1 were also consistent with the configurations determined at C-16 (Figure S3). The diagnostic ROESY correlations of H-5 and 2-NH with one of the acetonide methyl groups H3-19 (δH 1.26), but not with H3-20 (δH 1.18), established that they are all on the same face of the molecule. Furthermore, a strong ROESY cross-peak was observed for H-16 with H3-20, but not with H3-19, indicating a cisoidal relation between H-16 and H 3 -20. These observations suggested the S configuration at C-16, which is consistent with the result of Mosher amide analysis. The absolute configuration of the remaining chiral center at C-2 was determined by a repeated Marfey’s analysis. Attempts to purify the Phe-Dpr dipeptide from the partial acid
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RESULTS AND DISCUSSION The molecular formula of 1 was established as C37H51N7O7 according to HRESIMS and NMR data. Detailed analysis of 1D and 2D NMR spectroscopic data including HSQC, COSY, and HMBC in DMSO-d6 revealed the presence of an unsaturated fatty acid moiety and the amino acids phenylalanine (Phe), glycine (Gly), and 2,3-diaminopropionic acid (Dpr) (Table S1 and Figure S1). Subsequent analysis of the HMBC spectra allowed assignment of 2,2-dimethylimidazolidin-4-one, the distinctive structural feature of the compound, and establishment of the cyclic peptide sequence as Dpr-PheGly-Dpr. Thirteen carbons of the C14 unsaturated fatty acyl chain were connected by COSY correlations, and the connection to an amide carbonyl was established by HMBC correlations. 1H−1H coupling constants (3JH‑2′,H‑3′ = 14.0 Hz and 3JH‑5′,H‑6′ = 15.1 Hz) and ROESY correlations (H-7′/H2-9′ and H-8′/H-6′) defined double-bond geometries as 2′E, 5′E, and 7′E. The 12′E geometry was determined on the basis of the 13C chemical shifts of the adjacent methylenes C-11′ (δC 31.5) and C-14′ (δC 28.5).8 The peptide backbone was determined to be connected to the fatty acid moiety via an enamide bridge by HMBC correlations, establishing the planar structure of 1, which is identical to the previously reported compound enamidonin.5 Their similarity was based on the virtually identical 1H and 13C NMR data and the signs (both positive) of optical rotation. B
DOI: 10.1021/acs.jnatprod.8b00497 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 3. Key 2D NMR correlations of 2 and 3. The HMBC correlations used for determining a dimethylimidazolidinone residue are indicated by blue arrows.
Figure 2. ΔδS−R values for the bis-MTPA derivatives of enamidonin (1).
hydrolysate of 1 by reversed-phase HPLC were unsuccessful because the desired peptide was almost completely obscured by coeluting single amino acids. This was resolved by D-FDLA derivatization of the partial hydrolysate, which yielded the FDLA-derivatized dipeptide (bis-D-FDLA-Phe-Dpr, m/z 840 [M + H]+) that was well resolved from other HPLC peaks. A similar approach using 1-fluoro-2,4-dinitrophenyl-5-D-alaninamide (D-FDAA) was previously used in a case study on desotamide A.12 The desired peak corresponding to bis-DFDLA-Phe-Dpr was successfully purified by semipreparative reversed-phase HPLC and subsequently hydrolyzed and derivatized with D-FDLA. Retention time comparison of the D-FDLA derivative with that of authentic L- and D-Dpr confirmed the dipeptide contains D-Dpr (Figure S4). Thus, two separate experiments determined the regiochemistry of the L- and D-Dpr residues in 1, allowing assignment of the tetrapeptide backbone as D-Dpr-L-Phe-Gly-L-Dpr. The molecular formula of 2 was determined to be C36H57N7O8 through HRESIMS and NMR analysis. The 13C NMR data of 2 showed a peak pattern similar to those of 1: six amide carbons at δC 173.6−163.8, eight olefinic carbons at δC 144.2−104.0, and α-carbons at 69.1−51.6, indicating a structural resemblance to 1. The major differences in the NMR data were the absence of aromatic signals, indicative of variations in the peptide moiety. Other noticeable differences observed in the HSQC spectrum were the presence of an additional secondary alcohol (δC/H 60.3/4.84) and two methyls with doublet (δC/H 15.3/0.76) and triplet (δC/H 10.5/0.85) signals. Four amino acids, including isoleucine (Ile), threonine (Thr), and two Dpr residues, as well as a 2,2dimethylimidazolidin-4-one moiety bridge between Thr and Dpr, were established by COSY (5-NH to H3-8 and H-11 to H3-13) and HMBC (H-11/C-17 and NH-15/C-17) correlations (Figure 3). The full planar structure of 2 was then confirmed to be a new compound similar to enamidonin (1), but with Ile and Thr replacing Phe and Gly, respectively. The Marfey’s analysis of the acid hydrolysate of 2 revealed Sconfigurations of α-carbons in both Ile and Thr. To determine the absolute configurations of the β-carbons of Ile and Thr, 2,3,4,6-tetra-O-acetyl-β- D -glucopyranosyl isothiocyanate (GITC) derivatization of the hydrolysate and HPLC comparison with standard samples were performed,13 which established that 2 possesses L-Ile and L-Thr residues.
Compound 3 had the molecular formula C34H53N7O7, as indicated by HRESIMS and NMR data. The 13C NMR spectrum of 3 showed close similarity to that of compound 1, except for the absence of signals corresponding to aromatic carbons. The 1H and HSQC spectral data clearly revealed that 3 has two extra doublet methyl signals (δC/H 22.5/0.88 and 22.3/0.87), suggesting the replacement of Phe in 1 by the Leu residue. This observation was confirmed by interpretation of COSY extending from 5-NH through H3-8/H3-9 and diagnostic HMBC correlations of 5-NH to carbonyls of Dpr (C-4) and Gly (C-10). Thus, the structure of 3 was assigned as depicted (Figure 3). The absolute configuration of the Leu residue was determined to be L by Marfey’s analysis. The Marfey’s analysis also showed that compounds 2 and 3 contain both L- and D-Dpr residues in common with 1 (Figure S2). On the basis of their chemical structures, it could be assumed that three compounds are synthesized by the same biosynthetic gene cluster, and their structural diversity is expected to be generated by adenylation domain promiscuity of a nonribosomal peptide synthetase. Therefore, the absolute configurations of two Dpr residues in compounds 2 and 3 were proposed to be the same as 1. This deduction was further corroborated by electronic circular dichroism (ECD) curves of 2 and 3 that were closely comparable to that of 1 (Figure 4). The absolute configurations of C-16′ in 2 and 3 were determined to be R on the basis of their excellent NMR
Figure 4. ECD spectra of 1−3 recorded in MeOH. C
DOI: 10.1021/acs.jnatprod.8b00497 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Table 1.
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C (200 MHz) and 1H (800 MHz) NMR Spectroscopic Data for 2 and 3 in DMSO-d6 2
position 1 2 2-NH 3 3-NH 4 5
δC, type 172.0, C 51.6, CH
37.5, CH2
171.9, C 58.0, CH
5-NH 6 7
34.8, CH 24.2, CH2
8 9 10 11
10.5, CH3 15.3, CH3 170.6, C 69.1, CH
12
60.3, CH
12-OH 13 13-NH 14
20.4, CH3
δH, mult (J in Hz) 4.42, dd (14.3, 7.2) 8.97, m 3.41, m; 3.33, ovla 7.91, m 3.98, dd (10.3, 9.4) 7.81, d (9.1) 1.78, m 1.46, m; 1.05, m 0.85, t (7.5) 0.76, d (6.6)
59.3, CH
15-NH 16
38.4, CH2
δC, type 172.0, C 51.9, CH
37.7, CH2
39.3, CH2 24.3, CH 22.5, CH3 22.3, CH3 44.7, CH2
4.84, td (12.6, 6.2) 5.03, br s 1.09, d (6.2)
59.0, CH 38.7, CH2
3.71, dd (9.5, 2.1) 2.88, d (10.0) 3.56, ovl;a 3.14, m
2
δH, mult (J in Hz)
position
4.37, dd (14.4, 7.4) 8.88, d (6.7) 3.41, m; 3.32, ovla 7.94, t (5.8)
171.8, C 51.5, CH
3.26, d (9.9)
173.6, C
14-NH 15
3
4.29, m 6.98, d (9.2) 1.50, ovla 1.43, ovla 1.49, ovla 0.88, d (6.1) 0.87, d (6.1) 169.0, C 4.18, d (14.4); 3.30, d (14.4) 172.8, C
3.74, br d (8.2) 2.93, d (9.4) 3.53, dd (13.9, 7.5); 3.21, m 7.03, t (6.1)
76.3, C
27.4, CH3
δC, type
16-NH 17 18 19 1′ 2′ 3′
77.6, C 27.7, CH3 26.0, CH3 166.5, C 104.0, CH 134.0, CH
3′-NH 4′ 5′ 6′
163.8, C 121.3, CH 142.6, CH
7′ 8′ 9′
128.6, CH 144.2, CH 31.8, CH2
10′ 11′ 12′ 13′ 14′
28.1, CH2 31.5, CH2 129.3, CH 130.7, CH 28.5, CH2
15′
38.9, CH2
16′ 16′-OH 17′
65.2, CH
3
δH, mult (J in Hz)
δC, type
δH, mult (J in Hz)
7.06, m
23.6, CH3
25.8, CH3
1.18, s
1.19, ovl 1.19, ovl 5.67, d (14.0) 7.70, dd (14.0, 11.1) 10.59, m 5.99, d (15.1) 7.20, dd (15.1, 10.1) 6.24, ovla 6.24, ovla 2.14, dd (13.9, 7.0) 1.45, m 1.95, ovla 5.39, ovla 5.39, ovla 2.02, m; 1.96, ovla 1.38, m; 1.32, m 3.56, ovla 4.33, br s 1.03, d (6.1)
166.4, C 104.0, CH 5.67, d (14.0) 134.0, CH 7.70, dd (14.0, 11.0) 10.61, d (11.1) 163.8, C 121.3, CH 5.99, d (15.1) 142.6, CH 7.20, dd (15.1, 10.1) 128.6, CH 6.25, ovla 144.2, CH 6.23, ovla 31.8, CH2 2.14, dd (13.9, 7.0) ovla ovla ovla ovla m; 1.96, ovla
28.1, CH2 31.5, CH2 129.3, CH 130.7, CH 28.5, CH2
1.44, 1.96, 5.36, 5.39, 2.02,
38.9, CH2
1.38, m; 1.32, m
65.2, CH
3.56, m 4.33, ovla 1.03, d (6.2)
23.6, CH3
1.27, s a
resemblances to those of 1 (δH 1.38 and 1.32 at H2-15′; δH 3.56 at H-16′; δH 1.03 at H3-17′). The antibacterial activities of these three compounds (1−3) were evaluated against a panel of pathogenic bacteria including MRSA (methicillin-resistant Staphylococcus aureus) and QRSA (quinolone-resistant Staphylococcus aureus). Contrary to the effects of enamidonin (1), which was active only against Bacillus subtilis with an MIC of 4 μg/mL, the two new compounds (2 and 3) showed broader spectrum activity with MICs of 8−64 μg/mL against MRSA, QRSA, Enterococcus faecalis, and Bacillus subtilis (Table 2), showing that the antibacterial activities of the enamidonin family are highly dependent on the amino acid composition of the tetrapeptide backbone. The origin of a dimethylimidazolidinone residue in the enamidonin family is of interest for the following reasons: (a) A dimethylimidazolidinone residue in the peptide backbone has not been encountered in any other class of natural products; (b) compounds 1−3 were completely absent in the EtOAc extract of whole culture broth; (c) all five members of the enamidonin family, including enamidonin, enamidonins B and C, and K97-0239A and -B, were isolated from acetone extracts of cells.5,6 From these observation, it was hypothesized that the N,N-acetonide unit of a dimethylimidazolidinone residue could be formed during the acetone extraction process. To investigate this, 7-day-old whole culture broth was extracted using acetonitrile rather than acetone and analyzed
Signals were overlapped with other signals.
Table 2. Antibacterial Activities of 1−3 MIC (μg/mL) strain MRSA CCARM 3167 MRSA CCARM 3506 QRSA CCARM 3505 QRSA CCARM 3519 Staphylococcus aureus RN 4220 Streptococcus pneumoniae KCTC 5412 Enterococcus faecalis KCTC 5191 Bacillus subtilis KCTC 1021 Acinetobacter baumannii KCTC 2508 Escherichia coli CCARM 1356 Pseudomonas aeruginosa KCTC 2004 a
Cipa
1
2
3
>128 >128 >128 >128 >128 >128
64 16 32 32 32 >128
32 8 16 16 >128 >128
4 0.125 32 16 0.125 0.5
>128 4 >128
64 16 >128
8 16 >128
1 0.03125 0.25
>128 >128
>128 >128
>128 >128
64 0.5
Ciprofloxacin was used as a positive control.
by LC-MS data. As a result, inspection of LC-MS data revealed that whereas the predicted precursor compounds of 1−3 lacking N,N-acetonide ([M + H]+ ions at m/z 666, 676, and 632, respectively) were detected, compounds 1−3 were not present in the acetonitrile extract (Figure 5A). These precursor compounds showed identical retention times and tandem MS data to chemically prepared deacetonide derivatives of 1−3 (Figures 5F and S5). Precursor compounds were present in D
DOI: 10.1021/acs.jnatprod.8b00497 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 5. HPLC analysis (scan UV 200−420 nm) of enamidonins and deacetonide derivatives. (A) Acetonitrile extract of the whole culture broth; (B) supernatant of the culture broth; (C) acetonitrile extract of the cell pellet; (D) acetone extract of the cell pellet; (E) acetonitrile extract of the cell pellet incubated with acetone; (F) deacetonide derivatives prepared by treatment of enamidonins with 60% AcOH for 1 h.
both supernatant and the cell pellet extract (Figure 5B and C). In addition to small peaks of precursor compounds, the acetonitrile extract incubated with acetone showed the presence of dominant peaks corresponding to 1−3 (Figure 5E). The resulting chromatogram was almost identical to that of the acetone extract of the cell pellet (Figure 5D). These observations confirmed that the N,N-acetonide unit of a dimethylimidazolidinone residue is readily formed via protection of a diamine group by acetone, suggesting 1−3 were indeed the isolation artifacts deriving from acetonide protection during the extraction process. On the basis of the above data, we propose that enamidonin and structurally related compounds, K97-0239A and -B, are most probably isolation artifacts. We also tried to purify natural precursors of 1−3 by largescale culture, acetonitrile extraction, and chromatographic separation; however, these were unsuccessful because precursors decomposed prior to completion of the vacuum liquid chromatography process. Alternatively, three precursor compounds (1c, 2a, and 3a, respectively) were prepared by acetonide deprotection of 1−3 and HPLC purification and then assayed for antibacterial activity against Bacillus subtilis. Interestingly, in contrast to compounds 1−3, which exhibit clear inhibition zones (diameter of inhibition zones: 16.0, 13.5, 13.5 mm, respectively), none of the three deacetonide derivatives were found to show antibacterial activities against B. subtilis (Figure 6). This result revealed that the N,Nacetonide group is necessary for antibacterial activity of the enamidonins.
Figure 6. Disk diffusion assay. Enamidonins and deacetonide derivatives (10 μg each) were spotted onto disks to monitor antibacterial activity against B. subtilis. (A) Enamidonin; (B) enamidonin B; (C) enamidonin C; (D) deacetonide derivative of enamidonin (1c); (E) deacetonide derivative of enamidonin B (2a); (F) deacetonide derivative of enamidonin C (3a).
Bioactive enamide natural products have been found in a variety of microorganisms, including terpeptin (Aspergillus terreus),14 chondriamides (Chondria atropurpurea),15 kanamienamide (Moorea bouillonii),16 and sansanmycins (Streptomyces sp. SS),17 but enamidonin and its analogues represent the only class of cyclic lipopeptidic enamides. We have also verified that the N,N-acetonide unit is formed via protection of diamine during the acetone extraction process. Compared to enamidonins, precursor compounds were found to be unstable and biologically inactive. There are examples of acetonide E
DOI: 10.1021/acs.jnatprod.8b00497 J. Nat. Prod. XXXX, XXX, XXX−XXX
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(OptimaPak C18, 10 × 250 mm, 10 μm; flow rate 3 mL/min; 38% CH3CN−H2O containing 0.05% formic acid over 45 min) to afford 1 (9.7 mg, tR 21.2 min), 2 (8.0 mg, tR 17.1 min), and 3 (5.6 mg, tR 18.8 min). Enamidonin (1): white powder; [α]23 D +77.9 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 205 (4.2), 230 (3.9), 303 (4.5) nm; ECD (MeOH) (Δε) 233 (+25.7), 307 (+4.1); for NMR data, see Table S1; HRESIMS m/z 728.3743 [M + Na]+ (calcd for C37H51N7O7Na+, 728.3742). Enamidonin B (2): white powder; [α]22 D −12.5 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 205 (4.1), 230 (3.9), 303 (4.5) nm; ECD (MeOH) (Δε) 234 (+15.9), 307 (+1.0); for NMR data, see Table 1; HRESIMS m/z 738.4158 [M + Na]+ (calcd for C36H57N7O8Na+, 738.4161). Enamidonin C (3): white powder; [α]22 D +14.4 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 205 (4.0), 230 (3.8), 303 (4.4) nm; ECD (MeOH) (Δε) 233 (+17.3), 304 (+1.1); for NMR data, see Table 1; HRESIMS m/z 694. 3904 [M + Na]+ (calcd for C34H53N7O7Na+, 694.3899). Preparation of Mono-MTPA Ester of 1. Compound 1 (0.5 mg) was dissolved in anhydrous pyridine (1 mL), and a catalytic amount of dimethylaminopyridine (DMAP) was added. After the mixture was stirred for 5 min, 25 μL of (R)-MTPA-Cl was then added, and stirring continued at room temperature for 16 h prior to quenching with 50 μL of H2O. The mixture was subjected to semipreparative reversedphase HPLC (column as above; flow rate 3 mL/min; 30−50% CH3CN−H2O containing 0.05% formic acid over 30 min) to yield mono-(S)-MTPA ester 1a (0.3 mg, tR 15.5 min). Mono-(R)-MTPA ester 1b (0.3 mg, tR 18.0 min) was also prepared and purified with the same procedure using (S)-MTPA-Cl. Acid Hydrolysis and Advanced Marfey’s Analysis. Compounds 1−3 (0.1 mg each) were separately hydrolyzed with 6 N HCl (0.5 mL) at 100 °C for 1 h. The reaction mixture was dried in vacuo, and the residue was dissolved in 1 N NaHCO3 (100 μL). A Marfey’s solution of 1% L-FDLA (5-fluoro-2,4-dinitrophenyl-5-L-leucine amide) in acetone (100 μL) was then added to the solution. The reaction mixture was vortexed and incubated at 40 °C for 1 h. The reaction was quenched by the addition of 20 μL of 2 N HCl, and then the reaction mixture was diluted 2-fold with CH3CN. The resulting solution was analyzed by LC-MS (Waters HSS T3 column, 2.1 × 150 mm; flow rate 0.3 mL/min; 5−100% CH3CN−H2O containing 0.05% formic over 15 min). Compounds 1−3 were also derivatized with DFDLA and analyzed using the same method as described above. Retention times (tR, min) of the L- and D-FDLA derivatives of the acid hydrolysates from compounds 1−3: 1, L-Phe 13.10, 14.25 m/z 460 [M + H]+; 2, L-Ile 13.05, 14.70 m/z 426 [M + H]+; L-Thr 10.81, 11.84 m/z 414 [M + H]+; 3, L-Leu 13.05, 14.70 m/z 426 [M + H]+. Both L- and D-FDLA derivatives of the acid hydrolysates from compounds 1−3 showed two bis-FDLA-derivatized Dpr peaks (m/z 693 [M + H]+) at 14.49 and 14.57 min. Advanced Marfey’s Analysis of the D-FDLA-Derivatized Phe-Dpr Dipeptide. The acid hydrolysate of compound 1 (1.5 mg) was prepared and derivatized with D-FDLA using the same method as described above. An aliquot of the resulting solution was subjected to LC-MS analysis to identify the peak of the FDLAderivatized Phe-Dpr dipeptide (bis-D-FDLA-Phe-Dpr, m/z 840 [M + H]+, tR 16.31 min). The product (tR 33.9 min) was purified by semipreparative reversed-phase HPLC (column as above; flow rate 3 mL/min; 30−100% CH3CN−H2O containing 0.05% formic acid over 40 min). Upon acid hydrolysis with 6 N HCl for 3 h, the dried hydrolysate was derivatized with D-FDLA using the same method as described above. The reaction mixture was analyzed using LC-MS (Waters HSS T3 column, 2.1 × 150 mm; flow rate 0.3 mL/min; CH3CN−H2O containing 0.05% formic acid; 5−50% at 0−5 min and 50−100% at 5−15 min). Retention times (tR, min) of the D-FDLA derivatives of the acid hydrolysate from bis-D-FDLA-derivatized PheDpr: D-Dpr, 12.83 m/z 693 [M + H]+ (bis derivative). Retention times (tR, min) of the D-FDLA derivatives of authentic D- and L-Dpr: D-Dpr 12.83 and L-Dpr 12.98 (bis derivative).
artifacts that exhibited more pronounced bioactivities than natural precursors, such as in the case of broussonetone C, an inhibitor of tyrosinase and xanthine oxidase.18 Our results provide a further example where artifact formation facilitates purification and leads to generation of bioactive compounds from inactive natural products. On the basis of the structures of precursor compounds, it is expected that the biosynthetic process of enamidonin involves the formation of the amide bond between primary β-amine of L-Dpr and the carbonyl of DDpr, which leads to exposure of α-amine in the middle of the peptide. These phenomena are occasionally encountered during the biosynthesis of β-amino acid-containing nonribosomal peptides,19 such as capreomycin 1A and dapdiamides.20 Further studies aimed at investigation of a biosynthetic pathway and in-depth bioevaluation tests are required to understand this rare class of cyclic lipopeptides and to generate more potent derivatives.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 polarimeter. UV data were recorded using an Optizen 2120 UV spectrophotometer, and ECD spectra were obtained on a JASCO J-715 spectropolarimeter. NMR data were measured on a Bruker Biospin Advance II NMR spectrometer (900, 800, and 700 MHz for 1H, and 225, 200, and 175 MHz for 13C) at the Korea Basic Science Institute (KBSI) in Ochang, Korea. Chemical shifts were referenced to the residual DMSO-d6 signal (δC/H 39.51 and 2.50). HRESIMS data were obtained on a Waters SYNAPT G2 Q-TOF mass spectrometer at the KBSI in Ochang, Korea. The fractionation of the extract was performed using a Cosmosil 75C18 ODS gel. Semipreparative reversed-phase HPLC was performed on a Young Lin YL9100 HPLC system equipped with a Young Lin YL9160 PDA detector. LC-MS data were obtained on a Thermo LTQ XL linear ion trap attached to an ESI source that was connected to a Thermo Scientific Dionex Ultimate 3000 Rapid Separation LC system using a Waters HSS T3 column (Waters, 2.1 × 150 mm, 2.5 μm). Bacterial Materials. The strain Streptomyces sp. KCB14A132 was isolated from a soil sample collected from Jeung Island, Korea. The soil sample was suspended in distilled water, and the diluted suspension was spread onto various agar plates, which were incubated at 28 °C until the bacterial colonies appeared. The strain was identified from humic acid-containing agar medium (1.0 g of humic acid, 0.5 g of Na2HPO4, 1.7 g of KCl, 0.05 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, 1 g of CaCl2, and 12 g of agar per 1 L of distilled water, pH 7.2) supplemented with cycloheximide (100 mg/L). The pure colony obtained by repeated streaking was maintained on SY agar medium (10 g of starch, 1 g of yeast extract, 1 g of tryptone, and 17 g of agar per 1 L of distilled water). Phylogenetic analysis based on 16S rRNA gene sequence (GenBank accession code MH027580) comparisons showed a 99.86% match with Streptomyces indigoferus (1381/1383 bp), identifying the strain as Streptomyces. Fermentation and Extraction. Large-scale fermentation of the strain KCB14A132 was performed by growing cultures in 1 L baffled Erlenmeyer flasks each containing 250 mL of YMG medium (10 g of glucose, 20 g of soluble starch, 5 g of yeast extract, 5 g of malt extract, and 0.5 g of CaCO3 per 1 L of distilled water). Two agar plugs (0.5 cm diameter each) punched from an actively growing colony were transferred into each flask and were cultured at 28 °C for 7 days. The whole culture (7.5 L) was centrifuged, and the cells were extracted with 3 L of 70% acetone by sonication. The organic layer was then filtered and evaporated to afford the dried extract. The residue was resuspended in 1 L of H2O and partitioned three times against an equal volume of EtOAc. The EtOAc-soluble material was evaporated to yield 3.5 g of crude extract. The fractionation of the extract was performed utilizing ODS flash column chromatography eluting with a stepwise gradient of MeOH−H2O [2:8, 4:6, 6:4, 8:2, 10:0 (v/v)] to give 10 fractions. Fraction 8 (340 mg) containing compounds 1−3 was rechromatographed using semipreparative reversed-phase HPLC F
DOI: 10.1021/acs.jnatprod.8b00497 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Acid Hydrolysis of the N,N-Acetonide Unit. Compound 1 (1.5 mg) was stirred in 60% AcOH at 70 °C for 2 h. The formation of the expected product was confirmed by LC-MS analysis (ESIMS m/z 666 [M + H]+), and the reaction mixture was purified by semipreparative reversed-phase HPLC (column as above, 10 μm; flow rate 3 mL/min; 30−50% CH3CN−H2O containing 0.05% formic acid over 30 min) to afford compound 1c (0.6 mg, tR 13.3 min). Deacetonide derivatives of 2 (2a, 0.4 mg, tR 10.8 min) and 3 (3a, 0.2 mg, tR 11.3 min) were prepared in the same manner (see Supporting Information for NMR data). 1H and 13C NMR data for 1c are summarized in Table S2. Preparation of Bis-MTPA Derivatives of 1c. Compound 1c (0.3 mg) dissolved in anhydrous pyridine (1 mL) was added to a catalytic amount of DMAP and stirred for 5 min. The solution was then treated with 25 μL of (R)-MTPA-Cl and stirred at room temperature for 2 h. After the reaction was quenched by the addition of 50 μL of H2O, the solution was purified by semipreparative reversed-phase (column as above; flow rate 3 mL/min; 65−82% CH3CN−H2O containing 0.05% formic acid over 30 min) to afford bis(S)-MTPA derivative 1d (tR 26.4 min). Bis(R)-MTPA derivative 1e (tR 26.7 min) was prepared by using (S)-MTPA-Cl and purified in the same manner as described above. 1d: 1H NMR (800 MHz, DMSO-d6) δH 8.71 (1H, t, J = 6.2 Hz), 8.62 (1H, d, J = 8.1 Hz), 8.49 (1H, m), 7.78 (1H, t, J = 5.8 Hz), 7.52−7.44 (11H, m), 7.43 (1H, m), 7.21 (1H, dd, J = 15.0, 10.5 Hz), 7.19 (1H, m), 6.23 (2H, m), 6.01 (1H, d, J = 15.3 Hz), 5.62 (1H, d, J = 14.0 Hz), 5.30 (2H, m), 5.07 (1H, m), 4.45 (2H, m), 4.21 (1H, m), 3.78 (1H, dd, J = 14.4, 6.5 Hz), 3.56 (1H, dd, J = 14.1, 5.0 Hz), 3.49 (3H, s). 3.46 (3H, s), 3.51 (1H, m), 3.49 (1H, m), 3.33 (1H, m), 3.16 (2H, m), 2.82 (1H, dd, J = 14.2, 8.7 Hz), 2.14 (2H, m), 2.01 (2H, t, J = 7.5 Hz), 1.98 (2H, m), 1.94 (2H, m), 1.87 (1H, m), 1.81 (1H, m), 1.60 (1H, m), 1.57 (1H, m), 1.43 (2H, m), 1.30 (3H, d, J = 6.2 Hz); ESIMS m/z 1098 [M + H]+. 1e: 1H NMR (800 MHz, DMSO-d6) δH 10.65 (1H, d, J = 11.4 Hz), 8.61 (2H, m), 8.55 (1H, m), 7.85 (1H, t, J = 6.2 Hz), 7.72 (1H, m), 7.68 (1H, t, J = 12.6 Hz), 7.50−7.42 (10H, m), 7.35 (1H, m), 7.21 (1H, dd, J = 15.2, 10.4 Hz), 6.25 (1H, m), 6.23 (1H, m), 6.02 (1H, d, J = 14.9 Hz), 5.63 (1H, d, J = 13.9 Hz), 5.40 (1H, m), 5.32 (1H, m), 5.08 (1H, d, J = 7.0 Hz), 4.51 (1H, m), 4.41 (1H, m), 4.24 (1H, d, J = 15.3, 7.8 Hz), 3.66 (1H, m), 3.59 (2H, m), 3.57 (6H, s), 3.47 (1H, m), 3.40 (1H, m), 3.20 (1H, m), 3.13 (1H, m), 2.79 (1H, dd, J = 13.7, 8.0 Hz), 2.15 (2H, m), 2.02 (2H, m), 1.98 (2H, m), 1.69 (1H, m), 1.62 (1H, m), 1.44 (2H, m), 1.22 (3H, m); ESIMS m/z 1098 [M + H]+. Derivatization with GITC and LC-MS Analysis. To the acid hydrolysate of 2 (0.1 mg) dissolved in triethylamine (6% w/v in acetone, 200 μL) was added a GITC solution (1% w/v in acetone, 200 μL). The reaction was allowed to stir at room temperature for 20 min and then quenched with 5% acetic acid (200 μL). The mixture was analyzed by LC-MS (Waters HSS T3 column, 2.1 × 150 mm; flow rate 0.3 mL/min; CH3CN−H2O containing 0.05% formic acid; 5% at 0−5 min, 5−40% at 5−65 min, and 40−100% at 65−70 min). Retention times (tR, min) for the GITC derivatives from the acid hydrolysate of 2: L-Ile 59.26, L-Thr 9.49. Retention times (tR, min) for the GITC derivatives of the standard amino acids: L-Ile 59.23; L-alloIle 59.06; L-Thr 9.49; L-allo-Thr 9.36. Antibacterial Activity Assay. To measure the MIC of the compounds, antimicrobial assays against pathogenic bacteria were performed as described previously.21 Briefly, various bacteria including drug-resistant pathogens were grown to mid log phase in the appropriate growth media and seeded in 96-well microtiter plates. Cells (105/mL) were treated with test compounds and ciprofloxacin (Sigma) prepared in DMSO, and the MICs were determined in triplicate by serial 2-fold dilutions of compounds. For a disk diffusion assay, the colonies of Bacillus subtilis cultured for 24 h at 28 °C were suspended in sterile distilled water and inoculated onto the surface of nutrient agar. A paper disk (i.d. 6 mm) containing a sample was placed on the agar plate. The resulting agar plate was incubated at 28 °C for 24 h.
Article
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00497. Supplementary tables and figures for HRESIMS and NMR spectra (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*(J.-H. Jang) Tel: +82-43-240-6164. Fax: +82-43-240-6169. Email:
[email protected]. *(J. S. Ahn) Tel: +82-43-240-6160. Fax: +82-43-240-6169. Email:
[email protected]. ORCID
Young-Soo Hong: 0000-0001-7768-2239 Jae-Hyuk Jang: 0000-0002-4363-4252 Jong Seog Ahn: 0000-0001-5166-4358 Present Address ∥
Functional Material Team, S&D Co., Ltd., Cheongju, 28156, Korea.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors wish to thank Drs. Yushi Futamura, Shunji Takahashi, and Hiroyuki Osada in RIKEN Center for Sustainable Research Science for helpful discussions about the activity of enamidonin. We also thank Dr. Junnosuke Otaka, Harumi Aono, and Kai Yamamoto in RIKEN for preliminary bioactivity tests. This work was supported by an International Joint Research Project (ASIA-16-011) of the NST (National Research Council of Science & Technology), Young Researcher Program (NRF-2017R1C1B2002602) of the NRF (National Research Foundation of Korea), and KRIBB Research Initiative Program funded by the Ministry of Science ICT (MSIT) of the Republic of Korea. We thank the Korea Basic Science Institute, Ochang, Korea, for providing the NMR and HRESIMS data.
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REFERENCES
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