Neo-actinomycins A and B, natural actinomycins bearing the 5H-oxazolo[4,5-b]phenoxazine chromophore, from the marine-derived Streptomyces sp. IMB094.

Neo-actinomycins A and B (1 and 2), two new natural actinomycins featuring an unprecedented tetracyclic 5H-oxazolo[4,5-b]phenoxazine chromophore, were isolated from the marine-derived actinomycete Streptomyces sp. IMB094. Their structures were elucidated by spectroscopic analyses. The presence of this ring system was proposed to originate from a condensation between actinomycin D (3) with α-ketoglutarate and pyruvate, respectively. Compound 1 showed potent cytotoxic activities against human cancer HCT116 and A549 cell lines in the nanomolar range (IC50: 38.7 and 65.8 nM, respectively) and moderate antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) strains.

Introduction

<pan class="Chemical">spn>an class="Chemical">Actinomycinspan> are well-known chromopeptides with potent cytotoxic and antibiotic activities, isolated from various Actinomyces strains[1] and consisting of a <sppan>an class="Chemical">tricyclic phenoxazinone chromophore attached to two pentapeptide <span class="Chemical">lactone rings via amide bonds. More than 30 naturally occurring single congeners have been identified so far, and about 40 variants have been obtained by precursor-directed biosynthesis[2-4]. Natural <span class="Chemical">actinomycins differ in amino acid composition of the peptidolactone side chains, whereas the chromophore (2-amino-4,6-dimethylphenoxazine-3-one-1,9-dicarboxylic acid, actinocin) is identical in all reported actinomycins[2]. Recently, S. chrysomallus and S. parvulus were reported to generate new C-demethylactinomycins lacking one or both methyl groups in their phenoxazinone chromophores when cultured with 3-hydroxyanthranilic acid[5].

<pan class="Chemical">spn>an class="Chemical">Actinomycin Dpan> is the most common <sppan>an class="Chemical">actinomycin antibiotic and is used as an anti<span class="Disease">cancer drug, particularly in the treatment of Wilms’ tumor and soft tissue <span class="Disease">sarcomas in children[6, 7]. Actinomycins intercalate DNA and inhibit DNA-primed RNA synthesis[8, 9]. As high toxicity of actinomycins restricts their clinical application, extensive structural redesign studies have been performed to improve their therapeutic index, leading to the synthesis of a number of analogs with structurally modified cyclopeptide rings or chromophore[1, 10].

In our ongoing research for new bioactive metabolites from marine-derived bacteria[11-13], the crude extract of <pan class="Chemical">spn>an class="Species">Streptomyces sp.pan> IMB094 isolated from marine sediment showed potent antibacterial activity towards <sppan>an class="Chemical">methicillin-resistant <span class="Species">Staphylococcus aureus (MRSA) (MIC of <10 μg/mL) and cytotoxicity. Analysis of the exact by LC-UV-MS revealed metabolites with UV absorption similar to <span class="Chemical">actinomycins[14, 15]. In addition to the observed UV absorption maximum at 443 nm which is characteristic for actinomycins[16], the LC-UV-MS profile also showed a UV maximum at 410 nm for two of the metabolites (Supplementary Figure S1), which attracted our interest. Extensive investigation of the secondary metabolite composition of the IMB094 strain resulted in the isolation of a novel actinomycin chromophoric analog, neo-actinomycin A (1, Fig. 1), a new natural product, neo-actinomycin B (2), and two known actinomycins D and X2 (3 and 4). Structurally, the chromophore of neo-actinomycin A (1) contains a fourth oxazole ring fused with the actinocin moiety, forming a tetracyclic 5H-oxazolo[4,5-b]phenoxazine ring, which has never previously been found in naturally occurring actinomycins.

Figure 1

Chemical structures of compounds 1–4.

Results and Discussion

<pan class="Chemical">spn>an class="Chemical">Neo-actinomycin Apan> (1) was isolated as a red powder, with molecular formula of C66H90N12O18 (HRESIMS), indicating 28 degrees of unsaturation. The IR spn>ectrum of 1 suggested the presence of carboxyl (1743 cm−1), <span class="Chemical">amide (1647 cm−1), and aromatic ring (1505 cm−1) functionalities. The 1H NMR n class="Chemical">spectrum of 1 (Table 1) in <span class="Chemical">DMSO-d 6 displayed characteristics of a typical peptide, with five NH <spn>an class="Chemical">protons (δ H 7.2–11.7), 12 amino acid α-protons (δ H 3.2–6.3), and two ester carbinol protons (δ H 5.14 and 5.24). Furthermore, two ortho-coupled aromatic protons [δ H 6.62 (d, H-8) and 7.11 (d, H-7)], four NMe singlets [δ H 2.75 (s, 6H) and 3.19 (s, 6H)], as well as 12 additional methyl singlets or doublets (δ H 0.7–2.3) were also observed in the 1H NMR spectrum. Analysis of the 13C NMR and DEPT spectra revealed the presence of 13 carbonyl signals (δc 165–173) and 10 α-amino acid carbon signals (δc 51–70), indicating that 1 was a peptide. Additional carbon NMR data showed the presence of 16 methyl groups, 8 methylenes, 2 sp2 methines, 6 sp3 methines, and 11 sp2 quaternary carbons (δc 100–165), in agreement with spectral features observed for actinomycins[2, 15]. Comparison of NMR data of 1 with actinomycin D (Tables S1 and S2) revealed similar resonances in the two peptidolactone rings and differences in the chromophore moiety, indicating that the chromophore moiety of 1 was significantly modified. This conclusion was corroborated by the UV spectrum, which showed a bathochromic shift (absorption maximum at 410 nm) compared to actinomycin D.

Table 1

NMR Data for Neo-actinomycin A (1) in DMSO-d 6 a . a1H and 13C NMR data were recorded at 600 and 150 MHz, respectively.

no.	pentapeptidolactone (α-ring)		no.	pentapeptidolactone (β-ring)
	δ H, m (J in Hz)	δ C, type		δ H, m (J in Hz)	δ C, type
Thr 1		169.2, C	Thr 1		169.4, C
2	4.97, dd (9.0, 2.4)	53.9, CH	2	4.84, dd (8.4, 2.4)	54.9, CH
3	5.14, qd (6.0, 2.4)	72.3, CH	3	5.24, qd (6.0, 2.4)	71.9, CH
4	1.17, d (6.0)	16.3, CH3	4	1.20, d (6.0)	16.7, CH3
NH	7.25, d (9.0)		NH	9.66, d (8.4)
Val 1		170.6, C	Val 1		170.7, C
2	3.50, m	57.4, CH	2	3.49, m	57.4, CH
3	1.88, m	30.1, CH	3	1.88, m	30.2, CH
4	0.92, d (6.6)	18.6, CH3	4	0.94, d (6.6)	18.6, CH3
5	0.69, d (7.2)	19.1, CH3	5	0.70, d (7.2)	19.1, CH3
NH	8.42, d (6.0)		NH	8.29, d (5.4)
Pro 1		173.0, C	Pro 1		173.0, C
2	6.24, dd (9.0,3.0)	54.8, CH	2	6.21, dd (9.0,3.0)	54.8, CH
3	2.10, m; 1.74, m	31.1, CH2	3	2.10, m; 1.74, m	31.2, CH2
4	1.91, m; 1.67, m	22.7, CH2	4	1.91, m; 1.67, m	22.8, CH2
5	3.49, m; 3.27, m	46.4, CH2	5	3.50, m; 3.27, m	46.4, CH2
Sar 1		167.0, C	Sar 1		167.1, C
2	4.80, d (18.0); 4.08, d (18.0)	51.2, CH2	2	4.78, d (18.0); 4.08, d (18.0)	51.2, CH2
NMe	2.75, s	34.4, CH3	NMe	2.75, s	34.4, CH3
MeVal 1		168.1, C	MeVal 1		168.2, C
2	3.23, d (9.6)	69.5, CH	2	3.23, d (9.6)	69.6, CH
3	2.54, m	26.5, CH	3	2.54, m	26.5, CH
4	0.97, d (6.6)	21.0, CH3	4	0.98, d (6.6)	21.1, CH3
5	0.79, d (6.6)	18.8, CH3	5	0.80, d (6.6)	18.8, CH3
NMe	3.19, s	38.6, CH3	NMe	3.19, s	38.8, CH3
chromophore
2		165.8, C	10a		139.5, C
3a		132.9, C	11		111.8, C
4		100.2, C	11a		143.5, C
4a		132.0, C	12	2.13, s	14.8, CH3
5	11.74, s		13	2.27, s	8.9, CH3
5a		131.3, C	14		166.6, C
6		113.4, C	15		165.4, C
7	7.11, d (8.4)	122.0, CH	16	3.17, m	23.3, CH2
8	6.62, d (8.4)	122.0, CH	17	2.89, m	30.3, CH2
9		127.5, C	18		173.1, C
9a		140.7, C

NMR Data for <pan class="Chemical">spn>an class="Chemical">Neo-actinomycin Apan> (1) in <sppan>an class="Chemical">DMSO-d 6 a . a1H and 13C NMR data were recorded at 600 and 150 MHz, respectively.

Extensive analysis of 2D NMR (HSQC, COSY, and HMBC) pan class="Chemical">spn>ectroscopic data revealed 10 amino acid residues in 1: 2 × <pan class="Chemical">span class="Chemical">Thrpan>, 2 × <spn>n>an class="Chemical">Val, 2 × Pro, 2 × sarcosin (Sar), 2 × <span class="Chemical">MeVal. Amino acid sequences in the two peptidolactone units in 1 were determined to be identical with those of actinomycin D using HMBC correlations (Fig. 2). The chromophore moiety of 1 was established as follows. The ortho-coupled aromatic protons H-7 and H-8 correlated with two overlapped carbons at δ C 122.0 (C-7 and C-8) in the HSQC spectrum. In the HMBC spectrum, H-8 correlated with C-6 (δ C 113.4), C-9a (δ C 140.7), and the methyl carbon C-12 (δ C 14.8), whereas H-7 showed cross-peaks with C-5a (δ C 131.3), C-6, C-9 (δ C 127.5), and the carboxyl carbon C-14 (δ C 166.6). Further, the exchangeable NH proton at δ H 11.74 correlated with C-4 (δ C 100.2), C-4a, C-6, C-9a, and C-10a (δ C 139.5), whereas the methyl protons at δ H 2.27 (s, H3–13) showed correlation with C-10a, C-11 (δ C 111.8), and C-11a. These HMBC correlations, combined with the chemical shift of the carbon resonances, indicated the presence of the 9,11-dimethyl-5H-phenoxazine moiety. The chemical shift of C-11a (δ C 143.5) suggested that this carbon participated in a C-O single bond in 1, instead of the C=O double bond found in conventional actinomycins. Carbon resonance at δ C 173.1 (C-18) indicated the presence of a free carboxylic acid group. HMBC correlation of the mutually coupled methylene protons H2-16 (δ H 3.17) and H2-17 (δ H 2.89) with C-18 and the remaining downfield quaternary carbon at δ C 165.8 (C-2) revealed the presence of N=CCH2CH2COOH substructure moiety. This structure fragment, together with two peptidolactone units and the 11a-oxy-9,11-dimethyl-5H-phenoxazine moiety, accounted for 27 degrees of unsaturation. To fulfill 28 degrees of unsaturation, an additional ring in 1 was required, indicating that the quaternary carbon C-2 was connected with C-3 and C-11a through a nitrogen and an oxygen atom, respectively, forming an oxazole ring and completing the backbone of 1.

Figure 2

Key HMBC and 1H-1H COSY correlations of 1.

Key HMBC and pan class="Chemical">1Hn>-pan class="Chemical">1H COSY correlations of 1.

The absolute configurations of amino acids were determined using advanced Marfey’s method after acid hydrolysis[17, 18]. Comparison of retention times between l- and d/<n class="Chemical">pan class="Chemical">span class="Chemical">l-FDLAclass="Chemical">n>n> (<sppan>an class="Chemical">1-fluoro-2,4-dinitrophenyl-5-leucine amiden>) adducts (Supplementary Figure S2) assigned l configurations for <span class="Chemical">Thr, <span class="Chemical">MeVal, and Pro residues and d configuration for Val. With its structure fully resolved, 1 was established as a new member of the actinomycin family and named neo-actinomycin A.

The molecular formula of <pan class="Chemical">spn>an class="Chemical">neo-actinomycin Bpan> (2) was elucidated as <sppan>an class="Chemical">C64H88N12O16 by HRESIMS, with one <span class="Chemical">C2H2O2 unit less than 1. The UV and NMR data (Supplementary Tables S3 and S4) of 2 were similar to those of 1. The major difference between the NMR data of 1 and 2 was the absence of signals corresponding to the carboxyethyl group in 2 that were present in 1. A new methyl signal was observed at δ H/δ C 2.63/14.4 in 2, suggesting that the <span class="Chemical">carboxyethyl group in 1 was replaced by methyl in 2, which was confirmed by 2D NMR data analysis (Supplementary Figure S3). This is the first report on naturally occurring compound 2, which was previously synthesized as a derivative of actinomycin D[14]. It should be noted that prior to this study, no NMR data have been reported for this compound. Compound 2 was assigned the trivial name neo-actinomycin B since it had not been described from nature previously.

A plausible biosynthetic pathway (Fig. 3) for <n class="Chemical">pan class="Chemical">span class="Chemical">neo-actinomycins A and Bclass="Chemical">n>n> (1 and 2) is <sppan>an class="Chemical">pron>posed, starting from <span class="Chemical">actinomycin D (3) and the <span class="Chemical">tricarboxylic acid (TCA) cycle intermediates[19], α-ketoglutarate (α-KG) and pyruvate (PA), respectively. Nucleophilic addition of the free 2-amino group of 3 to the α-keto function of α-ketoglutarate (or pyruvate), followed by a cyclization, would give rise to 1 (or 2). The cyclization process might proceed by a concerted intramolecular hydrogen shift mechanism as proposed previously[20, 21]. To test the biosynthetic hypothesis we investigated the possibility of precursor-directed in situ synthesis of 1 and 2 by adding the proposed precursors α-ketoglutaric acid and pyruvic acid (1 mg/mL) after cultivation of Streptomyces sp. IMB094. LC-MS analysis indicated 12-fold increase in the production of 1 in α-ketoglutaric acid-supplemented cultures compared to unsupplemented control (Figure S4). It is interesting to note that the yield of 1 and 2 both increased about 6-fold 24 h after pyruvic acid was added into the cultures. A possible explanation is that the exogenous pyruvic acid is converted into α-ketoglutaric acid through the in vivo tricarboxylic acid (TCA) cycle biosynthesis pathway during cultivation, but this remains to be demonstrated.

Figure 3

Plausible biosynthesis pathway for neo-actinomycin A (1).

Plausible biosynthesis pathway for <n class="Chemical">pan class="Chemical">span class="Chemical">neo-actinomycin Aclass="Chemical">n>n> (1).

We further explored the possibility of in vitro transformation of the precursors in a variety of solvents, including the fermentation <pan class="Chemical">spn>an class="Chemical">M8 mediapan>, <sppan>an class="Chemical">H2O, and <span class="Chemical">MeOH (Figures S5 and S6). After incubation of 3 and α-ketoglutaric acid at 28 °C for 36 h, we observed approximately 10% conversion of 3 to 1 in <span class="Chemical">H2O and in M8 media, but no production of 1 in MeOH (Figure S5). Incubation with pyruvic acid under the same conditions lead to about 50% conversion of 3 to 2 in H2O and M8 media, and only 5% conversion in MeOH (Figure S6). Further investigation revealed that the conversion rates in MeOH and H2O varied slightly under pH 1.0 and 2.0 conditions, but dramatically decreased under pH 4.0 (Table S6). The low conversion rate in MeOH could be explained by the strong hydrogen bond between the 2-amino group and the pentapetidolactone[14, 22]. These results suggest that 1 and 2 were formed by a condensation of actinomycin D with α-ketoglutarate and pyruvate, respectively.

In a preliminary investigation of the biosynthetic origin of α-ketoglutarate and <pan class="Chemical">spn>an class="Chemical">pyruvatepan>, strain IMB094 was cultured in parallel in the <sppan>an class="Chemical">M8 medium and in the chemically defined medium, the <span class="Chemical">galactose-glutamate-mineral salts (GGM) medium, which was <span class="Chemical">proved to be good for actinomycin production for S. antibioticus 3720[23]. After cultivation at 28 °C for six days, the marine-derived strain IMB094 grew considerably better in the M8 medium containing 3.0% artificial sea salts than in the GGM media. LC-MS analysis of the metabolites showed that 1 and 2 were not present in the GGM fermentation broth. Compounds 3 and 4 were produced in the cultures grown in GGM medium; but their yield rates were only 7% of those in the M8 medium (Figure S7 and Table S7). Although α-ketoglutarate and pyruvate could not be detected in both media prior to cultivation by LC-MS analysis, they were found to be present in the extracts of both cultures grown in the M8 and GGM media (Figure S8 and Table S7). This suggested that α-ketoglutarate and pyruvate were formed during cultivation. Since galactose and l-glutamic acid were the only two organics in the GGM medium, the presence of pyruvate in the cultures (21.4 μg/mL, even higher than in the M8 cultures) indicated that α-ketoglutarate and pyruvate in the cultures did not directly derive from the exogenous alanine and glutamate in the medium by deamination. A plausible origin for the two precursors was from the TCA cycle biosynthesis pathway of the strain. Further biosynthesis studies of neo-actinomycins A and B were being undertaken.

Compounds 1 and 2 were e<pan class="Chemical">spn>an class="Chemical">valpan>uated for their in vitro antibacterial activity against a series of Gram-positive and Gram-negative drug-resistant pathogens (Table 2) and for <sppan>an class="Disease">cytotoxicity against two <span class="Species">human cancer cell lines (Table 3) using <span class="Chemical">actinomycin D (3) as a control. Compound 1 showed moderate antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) strains with MIC values of 16–64 μg/mL, 64–256-fold less active than 3. No antibacterial effects were observed for 2 (MIC > 128 μg/mL). Neo-actinomycin A (1) exhibited potent cytotoxicities against HCT116 and A549 cancer cell lines at nanomolar concentrations (IC50: 38.7 and 65.8 nM), approximately 800-fold decrease in activity relative to 3. This could be explained by the loss of planarity of the chromophore and the 2-amino group, which interacts with the DNA backbone by forming an additional hydrogen bond with cytosine residues[9, 24]. Compound 2 differs from 1 only in the substituent at C-2 of the oxazole ring (methyl vs. carboxyethyl), which results in a significant 10-fold decrease in activity. A possible reason for the higher potency of 1 than 2 might be that the carboxyl group in 1 forms an additional interaction with the DNA as the 2-amino group in 3 [24].

Table 2

Antimicrobial Bioassay Results (MIC, μg/mL) for compounds 1–4. aMethicillin-susceptible Staphylococcus aureus. bMethicillin-resistant S. aureus. cMethicillin-susceptible S. epidermidis. dMethicillin-resistant S. epidermidis. eVancomycin-susceptible Enterococcus. fVancomycin-resistant Enterococcus. gExtended-spectrum beta-lactamase-producing strain. hNew Delhi metallo-beta-lactamase 1.

microorganism	strain no.	phenotype	1	2	3	4	levofloxacin
Staphylococcus aureus	ATCC 29213	MSSAa	16	>128	0.25	0.25	0.125
Staphylococcus aureus	15	MSSA	32	>128	0.25	0.125	0.125
Staphylococcus aureus	13–17	MSSA	32	>128	0.25	0.125	0.125
Staphylococcus aureus	ATCC 33591	MRSAb	16	>128	0.25	0.25	0.125
Staphylococcus aureus	13–18	MRSA	64	>128	0.25	0.25	32
Staphylococcus epidermidis	ATCC 12228	MSSEc	64	>128	0.5	0.25	0.125
Staphylococcus epidermidis	13–1	MSSE	64	>128	0.5	0.125	0.25
Staphylococcus epidermidis	13–3	MRSEd	32	>128	0.5	0.25	64
Enterococcus faecalis	ATCC 29212	VSEe	16	>128	0.25	0.125	1
Enterococcus faecalis	13–4	VSE	16	>128	0.25	0.06	1
Enterococcus faecalis	ATCC 51299	VREf	16	>128	0.25	0.125	1
Enterococcus faecalis	ATCC 51575	VRE	16	>128	0.25	0.25	1
Enterococcus faecium	ATCC 700221	VRE	32	>128	0.125	0.125	64
Enterococcus faecium	13–7	VSE	32	>128	0.25	0.25	64
Enterococcus faecium	12–1	VRE	32	>128	0.25	0.25	64
Escherichia coli	ATCC 25922		>128	>128	64	128	≤0.03
Escherichia coli	1515		>128	>128	64	128	≤0.03
Escherichia coli	14–10		>128	>128	32	128	2
Escherichia coli	14–11	ESBLsg	>128	>128	64	64	16
Klebsiella pneumoniae	ATCC 700603	ESBLs	>128	>128	>128	>128	0.5
Klebsiella pneumoniae	7		>128	>128	>128	>128	0.06
Klebsiella pneumoniae	ATCC BAA-2146	NDM-1h	>128	>128	128	128	>128
Klebsiella pneumoniae	14–4		>128	>128	>128	>128	0.125
Klebsiella pneumoniae	14–15	ESBLs	>128	>128	>128	>128	0.25
Pseudomonas aeruginosa	ATCC 27853		>128	>128	>128	>128	1
Pseudomonas aeruginosa	PAO1		128	>128	>128	>128	4
Pseudomonas aeruginosa	13–46		>128	>128	>128	>128	0.25
Acinetobacter calcoacetious	ATCC 19606		>128	>128	32	32	0.125
Enterobacter cloacae	ATCC 43560		>128	>128	32	16	≤0.03
Enterobacter aerogenes	ATCC 13048		>128	>128	>128	>128	0.125
Serratia marcescens	ATCC 21074		>128	>128	>128	>128	0.125
Morganella morganii	ATCC 25830		>128	>128	128	>128	0.125
Providentia rettgeri	ATCC 31052		>128	>128	>128	>128	≤0.03
Proteus vulgaris	ATCC 29905		>128	>128	>128	>128	≤0.03
Proteus mirabilis	13–1		>128	>128	>128	128	0.125
Stenotrophomonas maltophilia	ATCC 13636		>128	>128	32	16	1
Citrobacter freundii	ATCC 43864		>128	>128	>128	>128	0.125

Table 3

Cytotoxicity of Compounds 1–4.

Compound	IC50 (nM)
	A549	HCT116
1	65.8	38.7
2	952.3	339.1
3	0.095	0.045
4	0.0025	0.0075

Antimicrobial Bioassay Results (MIC, μg/mL) for compounds 1–4. <pan class="Chemical">spn>an class="Chemical">aMethicillinpan>-susceptible <sppan>an class="Species">Staphylococcus aureus. b<span class="Chemical">Methicillin-resistant S. aureus. <span class="Chemical">cMethicillin-susceptible S. epidermidis. dMethicillin-resistant S. epidermidis. eVancomycin-susceptible Enterococcus. fVancomycin-resistant Enterococcus. gExtended-spectrum beta-lactamase-producing strain. hNew Delhi metallo-beta-lactamase 1.

<pan class="Chemical">spn>an class="Disease">Cytotoxicitypan> of Compounds 1–4.

Conclusion

In summary, we have isolated two new natural <pan class="Chemical">spn>an class="Chemical">actinomycinspan>, <sppan>an class="Chemical">neo-actinomycins A and B (1 and 2) from a marine-derived <span class="Species">Streptomyces sp. strain. Neo-actinomycin A represents the first reported instance of natural <span class="Chemical">actinomycins possessing an unusual 5H-oxazolo[4,5-b]phenoxazine ring system. Investigation of biosynthesis indicated that neo-actinomycins A and B are formed by a condensation of α-ketoglutarate and pyruvate, with the actinocin chromophore of actinomycin D.

Methods

General Experimental Procedures

Optical rotations were determined using a Perkin-Elmer model 343 polarimeter. UV and CD pan class="Chemical">spn>ectra were recorded on an Applied Photophysics Chirascan pan class="Chemical">spectropolarimeter. IR pan class="Chemical">spectra were recorded on a Nicolet 5700 FT-IR microscope spectrometer (FT-IR microscope transmission). 1D- and 2D-NMR spectra were obtained at 600 MHz for 1H and 150 MHz for 13C, respectively, on a Bruker AVANCE III HD 600 MHz spectrometers in DMSO-d 6 (δ H 2.500 and δ C 39.520), and MeOH-d 4 (δ H 3.310 and δ C 49.000) with solvent peaks used as references. HRESIMS data were obtained using a Thermo LTQ Orbitrap XL mass spectrometer. LC-MS analysis was performed using an Agilent 1290 Infinity II LC coupled with a 1100 LC/MSD Model G1946D mass spectrometer. Preparative HPLC was conducted on a Shimadzu LC-20AP pump with a SPD-M20A photodiode array detector. TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 7% H2SO4 in 95% aqueous EtOH followed by heating.

Producing Microorganism and Fermentation

The <pan class="Chemical">spn>an class="Chemical">propan>ducing microorganism, <sppan>an class="Species">Streptomyces sp. IMB094, was isolated from a marine sediment sample collected at a depth of ca. 40 m from Heishijiao Bay, Dalian, China. On the basis of the 16S rDNA gene sequence (GenBank accession no. KY111376) analysis, strain IMB094 is most closely related to <span class="Species">Streptomyces antibioticus NBRC 12838 (GenBank no. AB184184). Strain IMB094 was grown on ISP4 <span class="Chemical">agar plates prepared with 3.0% artificial sea salt at 28 °C for 10 days and then inoculated into 300 replicate 500 mL Fernbach flasks each containing 100 mL of sterile M8 medium (composed of 10 g of starch, 25 g of glucose, 10 g of cottonseed flour, 3 g of peptone, 5 g of CaCO3, 0.1 g of KH2PO4, 0.1 g of MgSO4, and 30 g of artificial sea salt in 1L of H2O) and cultured on rotary shakers (200 rpm) at 28 °C for 5 days.

Isolation

The culture broth (30L) was separated into the mycelia and the supernatant by centrifugation. The mycelial cake was extracted four times with <n class="Chemical">pan class="Chemical">span class="Chemical">acetoneclass="Chemical">n>n>. The supernatant was subjected to an XAD-7HP macroporous adsorbent resin column (6L). The column was washed with <sppan>an class="Chemical">H2On>, and eluted successively with 50% and 90% aqueous <span class="Chemical">acetone. The two latter fractions were combined with the mycelial <span class="Chemical">acetone extracts and then concentrated under reduced pressure to afford a crude extract (25 g). The extract was applied to silica gel column chromatography (CC) eluting with a step-gradient of CH2Cl2−MeOH (50:1–0:100, v/v) to give 10 fractions (F1–F10) on the basis of TLC results. The fractions F5 (20:1, 0.36 g), F6 (9:1, 0.54 g), and F7 (9:1, 0.65 g) were separately applied to Sephadex LH-20 CC eluting with 90% aqueous MeOH and then separated on a preparative reversed-phase (RP) C18 HPLC column (Capcell MGII 5 μm, 20 mm × 250 mm, 10 mL/min) using a linear gradient of 75–100% aqueous MeOH over 60 min to yield impure neo-actinomycins A and B as major components (retention times: A, 23.96–30.60 min; B, 34.25–41.26 min). Final purification were achieved by repeated RP-HPLC on a 10 mm × 250 mm C-18 column (Nacalai Cosmosil MGII) using 58% MeCN in 0.5% formic acid for 1 and 75% MeCN in 0.5% formic acid for 2 as a mobile phase (4 mL/min flow rate, 400 nm detection) to obtain 28 mg of 1 (t R: 28.41 min) and 23 mg of 2 (t R: 15.42 min). Typical recoveries of compounds 1–4 from 1 L cultures were 0.9, 0.8, 23, 1.8 mg, respectively.

Neo-actinomycin A (1)

Red amorphous powder; [α]20 D –97.7 (c 0.35, <pan class="Chemical">spn>an class="Chemical">MeOHpan>); UV (<sppan>an class="Chemical">MeOH) λ max (logε) 251 (4.50), 410 (4.12) nm; ECD (c 1.89 × 10−4 M, MeOH) λ max (Δε) 210 (−27.4), 242 (+1.1), 277 (−7.0), 405 (−2.0) nm; IR v max 3397, 2922, 1743, 1647, 1505, 1467, 1259, 1194, 1098 cm−1; 1H NMR (<span class="Chemical">DMSO-d 6, 600 MHz) and 13C NMR (DMSO-d 6, 150 MHz) data, Table 1; HRESIMS m/z 1339.6542 [M+H]+ (calcd for C66H91N12O18, 1339.6569), 1361.6355 [M+Na]+ (calcd for C66H90N12O18Na, 1361.6388).

Neo-actinomycin B (2)

Red amorphous powder; [α]20 D −105.2 (c 0.28, <pan class="Chemical">spn>an class="Chemical">MeOHpan>); UV (<sppan>an class="Chemical">MeOH) λ max (logε) 251 (4.57), 409 (4.13) nm; ECD (c 1.95 × 10−4 M, MeOH) λ max (Δε) 210 (−43.4), 242 (+1.1), 277 (−10.4), 403 (−3.0) nm; IR v max 3394, 2922, 1745, 1647, 1504, 1467, 1255, 1193, 1126, 1098 cm−1; 1H NMR (<span class="Chemical">DMSO-d 6 and CD3OD, 600 MHz) data, Supplementary Table S4; 13C NMR (DMSO-d 6 and CD3OD, 150 MHz) data, Supplementary Table S3; HRESIMS m/z 1298.6775 [M+NH4]+ (calcd for C64H92N13O16, 1298.6779), 1303.6327 [M+Na]+ (calcd for C64H88N12O16Na, 1303.6333).

Marfey’s Analysis of Compound 1

Compound 1 (0.9 mg) was dissolved in 0.5 mL of 6 M <pan class="Chemical">spn>an class="Chemical">HClpan> and heated to 110 °C for 16 and then the hydrolysate was evaporated to dryness, redissolved in <sppan>an class="Chemical">H2O (120 μL) and divided into two portions. To each portion (60 μL) was added 100 μL of a 1% solution of 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (l-FDAA) or 1% <span class="Chemical">d-FDAA in acetone and 20 μL of 1 M NaHCO3. The reaction mixture was heated at 40 °C for 1 h, cooled to room temperature, neutralized with 2 M HCl (10 μL), and diluted with MeCN (100 μL). The analyses of l- and l/d-FDLA derivatives were carried out with ESI-LC/MS using the following conditions: column, Cosmosil MG II C18 column (5 μm, 4.6 × 150 mm); flow rate, 1.0 mL/min; solvent A, 0.1% formic acid; solvent B, MeCN; linear gradient elution from 25–55% of B in A over 60 minutes; UV detection at 340 nm; column temperature, 40 °C.

The retention times of the <pan class="Chemical">spn>an class="Chemical">l-FDLApan> derivatives for compound 1 were as follows: <sppan>an class="Chemical">d-Val (44.35 min), l-Thr (17.79 min), l-Pro (24.75 min), and <span class="Chemical">l-MeVal (37.35 min). The retention times of the l/d-FDLA derivatives for compound 1 were as follows: d-Val (44.29, 32.04 min), l-Thr (17.75, 25.87 min), l-Pro (24.78, 30.07 min), and l-MeVal (37.31, 44.80 min). These results established the d-configuration for Val and l-configurations for other amino acid residues.

Cytotoxicity Assay

The <pan class="Chemical">spn>an class="Disease">cytotoxicitiespan> of the tested compounds against the <sppan>an class="Species">human <span class="Disease">cancer cells A549 (<span class="Disease">lung adenocarcinoma) and HCT116 (colon carcinoma) were evaluated by the sulforhodamine B (SRB) assay using a standard protocol developed by the NCI[25]. A549 and HCT116 cells were maintained in dulbecco’s modified eagle medium(DMEM, Hyclone) and RPMI 1640 medium (Hyclone), respectively. All media contained 100 units/mL of penicillin, 100 mg/mL of streptomycin and 10% fetal bovine serum. For the cytotoxicity assays, cells were inoculated into 96-well plates at a concentration of 4000 cells per well. After incubation at 37 °C under a humidified atmosphere containing 5% CO2 for 24 h, cells were treated with various concentrations of test compounds in triplicate and further incubated for 48 h. Cell proliferation was determined by the SRB assay. The IC50 value was defined as the compound concentration which produces 50% inhibition of cell growth during 2 days of compound treatment and calculated using GraphPad Prism 5 software with nonlinear regression fit analysis.

Procedure for precursor-directed in situ synthesis

<pan class="Chemical">spn>an class="Chemical">Sppan>ore suspn>ensions of <span class="Species">Streptomyces sp. IMB094 were inoculated into 100 mL of <n class="Chemical">span class="Chemical">M8 medium in 500 mL flasks. After incubation at 28 °C for 4 days, the cultures (each 30 mL) were transferred to two new 100 mL flasks and then α-ketoglutaric and <spn>an class="Chemical">pyruvic acids were separately added at a final concentration of 1 mg/mL. The remaining cultures (30 mL) without supplemental precursors were grown in parallel as negative control. The cultures were further grown at 28 °C for 1 day at 200 rpm. 30 mL samples of culture broth were concentrated in vacuum to dryness. The mixtures were then extracted with 30 mL of MeOH in an ultrasonic bath at 40 °C for 30 minutes. The mixture was spun down and the clear methanol extract was evaporated to dryness and redissolved in 300 μL of MeOH-EtOAc (5:1) solvents. 5 μL of the extract was analyzed by HPLC-ESI-MS (Capcell MGII C-18 5 μM, 4.6 mm × 150 mm; mobile phase A: 5 mM ammonium formate aqueous solution; B: MeCN; gradient conditions: 0–20 min, linear gradient 40–80% B; 25 min, 80% B; flow rate: 1 mL/min). If not mentioned otherwise, LC-MS analysis in this study was performed using the conditions described above.

Comparison analysis of the metabolites in M8 and GGM media

<pan class="Chemical">spn>an class="Chemical">Sppan>ores of strain IMB094 were inoculated into 500 mL Erlenmeyer flasks containing 100 mL of <sppan>an class="Chemical">TCG medium (3 g of tryptone, 5 g of casitone, 4 g of glucose, and 30 g of artificial sea salt in 1 L of <span class="Chemical">H2O)[26]. The cultures were grown at 28 °C for 3 days. 5 mL of seed medium was inoculated to 500 mL Erlenmeyer flasks containing 100 mL of the M8 and GGM (10 g of galactose, 2 g of l-glutamic acid, 1 g of K2HPO4, 0.025 g of MgSO4·7H2O, 0.025 of ZnSO4·7H2O, 0.025 of FeSO4·7H2O, 0.025 of CaCl2·2H2O in 1 L of H2O, pH 7.2) media in 500 mL Erlenmeyer flasks. Fermentation was carried out 28 °C for 6 days with agitation at 180 rpm. 50 mL samples of culture broth were concentrated in vacuum to dryness. The mixtures were then extracted with 50 mL of MeOH in an ultrasonic bath at 40 °C for 30 minutes. The mixture was spun down and the clear methanol extract was evaporated to dryness and redissolved in 1200 μL of MeOH-EtOAc (5:1) solvents. 5 μL of the extract was analyzed by LC-MS (LC-MS analysis conditions for α-ketoglutarate and pyruvate: Tosoh TSKgel Amide-80, 5 μM, 4.6 mm × 250 mm; mobile phase A: 0.1% formic acid aqueous solution; B: MeCN containg 0.1% formic acid; gradient conditions: 0–15 min, linear gradient 99–90% B; 16 min, 10% B; 26 min, 10% B; flow rate: 1 mL/min).

General procedure for conversion of 3 to 1 and 2 in the fermentation medium, H2O and MeOH

Compound 3 (4 mg), α-ketoglutaric acid (46.7 mg), and <pan class="Chemical">spn>an class="Chemical">pyruvic acidpan> (28.2 mg) were separately dissolved in 0.5 mL of <sppan>an class="Chemical">MeOH to make 6.4 mM, 640 mM, and 640 mM stock solutions, respectively. For conversion of 3 to 1, stock solutions of 3 and α-ketoglutaric acid (50 μL each) were separately added to 200 μL of the M8 medium, H2O, and MeOH. Similarly, for conversion of 3 to 2, stock solutions of 3 and <span class="Chemical">pyruvic acid (50 μL each) were added to 200 μL of the M8 medium, H2O, and MeOH. After shaking at 200 rpm at 28 °C for 36 h, 0.4 mL of MeOH was added to the reaction mixture to get a clear solution. Aliquots (0.2 mL) were taken from each reaction and analyzed by LC-MS.

Antibacterial Assay

The antibacterial assay was performed by using the <pan class="Chemical">spn>an class="Chemical">agarpan> dilution method as described previously[13]. Organisms used in this study included strains from the ATCC collection and clinical isolates. The test medium was Mueller-Hinton broth, and the inoculum was 10,000 colony forming units (CFU)/sppan>ot. The final concentrations of compounds ranged from 0.03 to 128 μg/mL. Culture plates were incubated at 35 °C for 18 h and MICs were then recorded. The MIC was defined as the lowest concentration that prevented visible growth of the bacteria.

Data Availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files).