Rifamycin W Analogues from Amycolatopsis mediterranei S699 Δrif-orf5 Strain.

Rifamycin W, the most predominant intermediate in the biosynthesis of rifamycin, needs to undergo polyketide backbone rearrangement to produce rifamycin B via an oxidative cleavage of the C-12/C-29 double bond. However, the mechanism of this putative oxidative cleavage has not been characterized yet. Rif-Orf5 (a putative cytochrome P450 monooxygenase) was proposed to be involved in the cleavage of this olefinic moiety of rifamycin W. In this study, the mutant strain Amycolatopsis mediterranei S699 Δrif-orf5 was constructed by in-frame deleting the rif-orf5 gene to afford thirteen rifamycin W congeners (1-13) including seven new ones (1-7). Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Presumably, compounds 1-4 were derivatized from rifamycin W via C-5/C-11 retro-Claisen cleavage, and compounds 1-3, 9 and 10 featured a hemiacetal. Compounds 5-7 and 11 showed oxygenations at various sites of the ansa chain. In addition, compounds 1-3 exhibited antibacterial activity against Staphylococcus aureus with minimal inhibitory concentration (MIC) values of 5, 40 and 0.5 µg/mL, respectively. Compounds 1 and 3 showed modest antiproliferative activity against HeLa and Caco-2 cells with half maximal inhibitory concentration (IC50) values of about 50 µM.

1. Introduction

Ansamycins are a family of macrolactam antibiotics that are synthesized by type I polyketide synthase (PKS), which are structurally characterized by an aromatic moiety bridged at nonadjacent positions by an aliphatic chain (ansa chain) [1,2]. As the representative members of the ansamycin family, rifamycins were first isolated from Amycolatopsis mediterranei S699 in 1957 [3,4,5]. Semi-synthetic rifamycin derivatives, such as rifampicin, rifapentine and rifambutin, have long been the first-line antituberculosis drugs since the mid-1960s, and are effective in combating leprosy and tuberculosis involved in AIDS-related mycobacterial infections [6,7,8,9]. However, Mycobacterium tuberculosis has developed significantly increased resistance to rifamycin antibiotics due to their extensive clinical use during recent decades [10,11].

The biosynthesis of rifamycins has been continuously studied since the 1980s, which can be divided into three stages. During the first two stages, the biosynthesis of start unit 3-amino-5-hydroxybenzoic acid (AHBA) and the polyketide skeleton were investigated, respectively. The third stage is still in progress, involving exploring hypotheses concerning post-PKS modifications [12,13,14,15,16]. As the most predominant intermediate in rifamycin biosynthesis, rifamycin W must undergo C-12/C-29 double bond oxidative cleavage to form 27-O-demethyl-25-O-deacetyl-rifamycin S (DMDARS) that is the basic rifamycin B polyketide skeleton. However, the mechanism of this putative oxidative cleavage has not been characterized yet [16,17].

In this study, a candidate cytochrome P450 oxidase gene, rif-orf5, which may be responsible for the oxidative cleavage of the rifamycin W ansa chain, was inactivated in A. mediterranei S699. The mutant strain A. mediterranei S699 Δrif-orf5 cultivated for 7 d on YMG agar medium resulted in the isolation of thirteen rifamycin W congeners (1–13) including seven new ones (1–7) (Figure 1).

Figure 1

Structures of rifamycin W congeners 1–7.

2. Materials and Methods

2.1. Bacterial Strains, Plasmids and Culture Media

The Amycolatopsis mediterranei S699 strain, isolated in 1957 in St. Raphael, France [5], was stored in our lab. The A. mediterranei S699 Δrif-orf5 strain was constructed by deleting the rif-orf5 gene through homologous recombination. The A. mediterranei S699 Δrif-orf5::orf5 strain was constructed by transformation of the rif-orf5 gene into the Δrif-orf5 mutant through electroporation. These strains were grown on YMG (yeast extract 4 g, malt extract 10 g, glucose 4 g, 20 g agar, ddH2O 1000 mL, pH 7.2) agar media at 28 °C for the production of rifamycins.

The Escherichia coli DH5α strain was used for plasmid propagation. Suicide vector pOJ260 was used for gene knock-out. Integrating vector pSET152 was used for gene complementation [18]. E. coli strains were maintained in LB (tryptone 10 g, yeast extract 5 g, NaCl 10 g, ddH2O 1000 mL, pH 7.2) media at 37 °C. Apramycin was added into media at a final concentration of 50 μg∙mL−1. Cells were stocked with 20% glycerol and stored at −80 °C.

2.2. Molecular Cloning and Mutant Construction

2.2.1. Construction of the rif-orf5 Gene Knock-Out Mutant Δrif-orf5

First, the rif-orf5 gene knock-out vector pOJ260-orf5 was constructed. Two ca. 2 kb DNA fragments flanking upstream and downstream of the target gene were amplified from the genomic DNA of A. mediterranei S699, and named HF1 and HF2, respectively. The purified homologous fragments HF1 and HF2 were digested with HindIII/XbaI and XbaI/EcoRI, and cloned into linearized HindIII/EcoRI digested pOJ260. The ligation product was transformed into DH5α-competent cells. Positive clones were verified by restriction enzyme digestion and sequencing (Figures S1A and S2). The gene knock-out vector pOJ260-orf5 was introduced into the rifamycin-producing strain A. mediterranei S699 by electrotransformation [19]. Apramycin-resistant (AprR) colonies were selected and confirmed to be single cross-over mutants by PCR amplification (Figures S1B and S3A). Apramycin-sensitive (AprS) colonies were counterselected from the initial AprR single cross-over colonies after several rounds of nonselective growth, and confirmed to be double cross-over gene knock-out mutant Δrif-orf5 by PCR amplification (Figures S1B and S3B).

2.2.2. Construction of the rif-orf5 Gene Complementation Mutant Δrif-orf5::orf5

First, the rif-orf5 gene complementation vector pSET152-orf5 was constructed. The targeted gene rif-orf5 was amplified using the genomic DNA of A. mediterranei S699 as a template. The purified PCR fragment was digested with NdeI and XbaI, and cloned into the downstream of the rifKp promoter in pSET152 through Gibson assembly [20]. Similarly, the assembled product was transformed into DH5α-competent cells, and positive clones were verified by restriction enzyme digestion and sequencing (Figure S4). The gene complementation vector pSET152-orf5 was transformed into the rif-orf5 gene knock-out mutant Δrif-orf5 by electroporation. Apramycin-sensitive (AprS) colonies were selected and confirmed to be the rif-orf5 gene complementation mutant Δrif-orf5::orf5 by PCR amplification (Figure S5).

Primers used in this study are shown in Table S1.

2.3. HPLC Detection of the Metabolites in Mutants

A. mediterranei S699 mutants were inoculated on YMG agar media (100 mL) and cultivated for 7 days at 28 °C. The culture was diced and extracted overnight with EtOAc at room temperature. The concentrated crude extract was dissolved in 1 mL MeOH, and analyzed by high-pressure liquid chromatography (HPLC; Agilent 1200, Santa Clara, CA, USA) in a gradient system consisting of ddH2O + 0.5% formic acid as solvent A and acetonitrile as solvent B. The program of solvent gradient was as follows: 20–35% B in the first 5 min, 35–55% B from 5 to 19 min, 55–65% B from 19 to 23 min, 65–100% B from 23 to 27 min. Flow rate was 1 mL/min, and UV detection was monitored at 254 nm (Figure S6).

2.4. Extraction and Isolation of the Metabolites from the Δrif-orf5 Strain

2.4.1. General Experimental Procedures

The nuclear magnetic resonance (NMR) spectra were recorded on Bruker 400 MHz NMR spectrometer. HRESIMS analyses were carried out on an LTQ-Orbitrap XL (Thermo Scientific, Waltham, MA, USA). HPLC was performed on an Agilent 1200. Semi-preparative HPLC was performed on a Waters 1525 Binary HPLC Pump (Agilent Eclipse XDB-C18, 5 µm, 9.4 × 250 mm) with a Waters 996 Photodiode Array Detector (Milford, MA, USA). Sephadex LH-20 was obtained from GE Amersham Biosciences (Piscataway, NJ, USA). Column chromatography (CC) was performed over reversed-phase (RP) C18 silica gel (Merck, Darmstadt, Germany). Silica gel GF254 for thin-layer chromatography (TLC) was purchased from Qingdao Marine Chemical Ltd. (Qingdao, China). Optical rotations were measured on an Auton Paar MCP200 Automatic Polarimeter. IR spectra (KBr) were obtained on a Thermo Fisher Scientific Nicolet 6700 FT-IR spectrometer (Waltham, MA, USA). Compounds were visualized under UV light and by iodine vapor.

2.4.2. Fermentation, Extraction and Isolation of the Metabolites from the Δrif-orf5 Strain

The fermentation (20 L) was performed on YMG agar Petri dishes for 7 d at 28 °C. The culture was diced and extracted overnight with EtOAc/MeOH (4:1, v/v) at room temperature three times. The crude extract was partitioned between H2O and EtOAc (1:1, v/v) until the H2O layer was colorless. The EtOAc extract was partitioned between 95% aqueous MeOH and petroleum ether (PE) to afford the defatted MeOH extract. The MeOH extract was fractionated by medium-pressure liquid chromatography (MPLC) over RP C18 silica gel (130 g) eluted with gradient aqueous CH3CN (30%, 50%, 70% and 100% CH3CN, 500 mL each) to give Fr. A–J.

Fr. C (1.43 g) was purified by HPLC (4 mL/min, UV 254 nm) eluted with 32% CH3CN to afford 7 (tR 13.4 min, 17.5 mg) and 12 (tR 6.2 min, 29.8 mg). Fr. D (5.62 g) was subjected to CC over silica gel (150 g) eluted with gradient CH2Cl2:MeOH (50:1, 30:1, 15:1 and 5:1, 500 mL each) to afford Fr. D1–4. Fr. D2 (1.85 g) was purified by HPLC (4 mL/min, UV 254 nm) eluted with 35% CH3CN to afford 10 (tR 8.6 min, 33 mg) and 14 (tR 10.3 min, 41 mg). Fr. D3 (2.49 g) was purified by HPLC (4 mL/min, UV 254 nm) eluted with 40% CH3CN to afford 6 (tR 7.9 min, 9.9 mg), 8 (tR 15.3 min, 17.0 mg) and 13 (tR 11.4 min, 179.5 mg). Fr. E (1.74 g) was purified by HPLC (4 mL/min, UV 254 nm) eluted with 43% CH3CN to afford 2 (tR 7.5 min, 10.5 mg), 4 (tR 13.4 min, 6.8 mg) and 5 (tR 6.2 min, 29.7 mg). Similarly, compounds 9 (12.3 mg) and 11 (7.2 mg) were obtained from Fr. F, and 3 (13.5 mg) was purified from Fr. G by HPLC (4 mL/min, UV 254 nm) eluted with 45% CH3CN. Fr. I gave compound 1 (6.6 mg) through HPLC (4 mL/min; UV 254 nm) eluted with 58% CH3CN.

Compound 1: dark brown powder; [α]20 = +10.0 (c 0.10, MeOH); UV (MeOH) λmax (logε) 217 (4.40), 269 (4.30), 325 (4.00) nm; IR (KBr) νmax 3369, 2963, 2925, 1688, 1631, 1496, 1322, 1142, 977, 862 cm−1; 1H NMR data, Table 1; 13C NMR data, Table 2; HRESIMS: m/z 686.3172 [M + H]+ (calcd for C36H48NO12+, 686.3171), and 708.2992 [M + Na]+ (calcd for C36H47NNaO12+, 708.2990).

Table 1

1H NMR spectroscopic data (400 MHz, CD3OD) of compounds 1–3 (δH, J in Hz) *.

Position	1		2		3
	1a	1b	2a	2b	3a	3b
3	7.64 (s)	7.64 (s)	7.64 (s)	7.64 (s)	7.61 (s)	7.61 (s)
5	7.18 (s)	7.18 (s)	6.99 (s)	6.99 (s)	7.02 (s)	7.02 (s)
MeO-6	4.00 (s)	4.00 (s)
13	1.89 (s)	1.87 (s)	1.90 (s)	1.88 (s)	1.90 (s)	1.88 (s)
14	2.09 (s)	2.09 (s)	2.07 (s)	2.07 (s)	2.09 (s)	2.09 (s)
17	6.50 (d, 10.8)	6.50 (d, 10.8)	6.64 (d, 11.3)	6.64 (d, 11.3)	6.50 (d, 10.8)	6.50 (d, 10.8)
18	6.87 (dd, 11.2, 14.9)	6.87 (dd, 11.2, 14.9)	7.21 (dd, 11.1, 15.0)	7.21 (dd, 11.1, 15.0)	6.86 (dd, 12.4, 14.9)	6.86 (dd, 12.4, 14.9)
19	6.08 (m)	6.08 (m)	6.23 (m)	6.23 (m)	6.07 (m)	6.07 (m)
20	2.44 (m)	2.44 (m)	2.52 (m)	2.52 (m)	2.47 (m)	2.47 (m)
21	3.82 (m)	3.82 (m)	3.84 (d, 8.7)	3.84 (d, 8.7)	3.83 (m)	3.83 (m)
22	2.04 (m)	2.04 (m)	1.95 (m)	1.95 (m)	1.96 (m)	1.96 (m)
23	3.62 (m)	3.62 (m)	3.64 (m)	3.64 (m)	3.62 (m)	3.62 (m)
24	1.98 (m)	1.98 (m)	2.03 (m)	2.03 (m)	2.02 (m)	2.02 (m)
25	3.55 (m)	4.21 (d, 10.4)	3.56 (m)	4.22 (d, 10.3)	3.56 (m)	4.21 (m)
26	1.59 (m)	1.63 (m)	1.60 (m)	1.62 (m)	1.60 (m)	1.61 (m)
27	3.18 (t, 9.8)	3.55 (m)	3.20 (t, 9.8)	3.56 (m)	3.17 (m)	3.56 (m)
28	2.42 (m)	2.64 (td, 3.5, 10.0)	2.47 (m)	2.65 (td, 3.5, 10.2)	2.42 (m)	2.61 (m)
29	6.62 (d, 10.7)	6.79 (d, 10.1)	6.61 (d, 11.3)	6.80 (d, 10.0)	6.62 (d, 10.7)	6.78 (d, 10.1)
30	2.08 (s)	2.08 (s)	4.34/4.33 (s)	4.34/4.33 (s)	2.08 (s)	2.08 (s)
31	1.02 (s)	1.02 (s)	1.03 (d, 7.0)	1.03 (d, 7.0)	1.02 (s)	1.02 (s)
32	1.00 (s)	1.00 (s)	1.01 (d, 7.1)	1.01 (d, 7.1)	1.00 (s)	1.00 (s)
33	0.98 (s)	0.98 (s)	0.98 (d, 6.7)	0.98 (d, 6.7)	0.98 (s)	0.98 (s)
34	0.96 (s)	0.96 (s)	0.97 (d, 6.9)	0.97 (d, 6.9)	0.96 (s)	0.96 (s)
34a	4.54 (d, 8.4)	5.08 (d, 3.2)	4.56 (d, 6.9)	5.09 (d, 3.3)	4.54 (d, 6.9)	5.08 (d, 3.3)

* s: singlet, d: doublet, dd: double doublet, t: triplet, m: multiplet.

Table 2

13C NMR spectroscopic data (100 MHz, CD3OD) of compounds 1–7 (δC) *.

Position	1		2		3		4	5	6	7
	1a	1b	2a	2b	3a	3b
1	184.7s	184.7s	184.1s	184.1s	184.1s	184.1s	183.7s	184.1s	184.0s	184.7s
2	143.0s	143.0s	143.2s	143.2s	142.4s	142.4s	142.7s	142.4s	142.4s	143.0s
3	117.3d	117.3d	117.2d	117.2d	117.2d	117.2d	117.2d	118.6d	119.1d	119.2d
4	187.1s	187.1s	186.8s	186.8s	186.5s	186.5s	186.8s	186.6s	186.9s	187.5s
5	103.7d	103.7d	108.6d	108.6d	108.8d	108.8d	108.9d	108.3s	108.1s	108.7s
6	165.9s	165.9s	163.9s	163.9s	164.0s	164.0s	164.0s	161.8s	163.6s	163.1s
MeO-6	57.0q	57.0q
7	120.3s	120.3s	118.2s	118.2s	118.3s	118.3s	118.4s	119.2s	119.3s	119.2s
8	161.7s	161.7s	165.0s	165.0s	165.2s	165.2s	165.6s	164.0s	164.0s	164.7s
9	131.7s	131.7s	130.6s	130.6s	131.1s	131.1s	126.1s	125.5s	124.7s	126.0s
10	132.6s	132.6s	132.3s	132.3s	132.4s	132.4s	132.4s	129.9s	130.8s	130.9s
11	172.3s	172.3s	171.8s	171.8s	172.3s	172.3s	173.0s	200.1s	200.3s	201.1s
12	133.3s	132.4s	133.1s	131.5s	132.4s	131.1s	131.8s	141.6s	142.0s	142.0s
13	13.9q	13.3q	13.9q	13.3q	13.9q	13.3q	13.9q	12.7q	13.0q	13.4q
14	8.3q	8.3q	8.2q	8.2q	8.2q	8.2q	8.1q	8.7q	8.7q	9.3q
15	170.0s	170.0s	169.0s	169.0s	170.0s	170.0s	170.1s	172.2s	172.8s	172.2s
16	129.3s	129.3s	142.6s	142.6s	129.4s	129.4s	129.5s	132.2s	133.1s	133.4s
17	139.0d	139.0d	143.7d	143.7d	139.1d	139.1d	138.9d	135.2d	133.4d	136.2d
18	127.6d	127.6d	128.1d	128.1d	127.8d	127.8d	127.6d	126.3d	127.3d	136.2d
19	146.3d	146.3d	150.1d	150.1d	146.4d	146.4d	146.5d	141.6d	140.7d	126.1d
20	42.5d	42.5d	42.7d	42.7d	42.5d	42.5d	42.5d	39.2d	43.4d	77.0s
21	75.8d	75.8d	75.8d	75.8d	75.8d	75.8d	75.8d	74.9d	78.3d	76.7d
22	36.6d	36.6d	36.7d	36.7d	36.5d	36.5d	36.9d	34.4d	49.7d	35.3d
23	78.5d	78.5d	78.5d	78.5d	78.5d	78.5d	79.3 d	79.0d	211.3s	80.8d
24	37.0d	37.0d	37.1d	37.1d	37.0d	37.0d	36.7d	38.0d	49.9d	39.0d
25	73.4d	72.8d	73.3d	72.8d	73.4d	72.7d	73.3d	71.3d	71.2d	72.3d
26	40.3d	41.3d	40.3d	41.3d	40.4d	41.3d	40.9d	43.9d	42.7d	44.6d
27	77.5d	73.4d	77.1d	73.3d	77.1d	73.4d	72.1d	68.7d	68.3d	70.0d
28	53.9d	50.7d	53.9d	50.7d	53.9d	50.7d	47.0d	46.1d	49.3d	49.1d
29	141.0d	142.4d	141.2d	142.7d	141.1d	142.4d	142.7d	139.0d	140.2d	141.5d
30	20.7q	20.7q	66.0t	66.0t	20.7q	20.7q	20.7q	20.3q	20.4q	21.0q
31	17.3q	17.3q	17.2q	17.2q	17.2q	17.2q	17.5q	18.2q	20.2q	26.7q
32	11.2q	11.2q	11.2q	11.2q	11.2q	11.2q	10.5q	11.3q	14.8q	14.6q
33	10.5q	10.5q	10.7q	10.7q	10.9q	10.9q	10.7q	8.9q	8.4q	9.8q
34	12.9q	12.9q	12.8q	12.8q	12.8q	12.8q	10.8q	11.8q	11.9q	12.4q
34a	98.2d	94.5d	98.0d	94.6d	98.0d	94.5d	64.6t	65.8t	64.4t	65.1t
AcO-34a								21.0q172.9s

* s: quaternary carbon, d: tertiary carbon, t: secondary carbon, q: primary carbon.

Compound 2: dark brown powder; [α]20 = −4.0 (c 0.10, MeOH); UV (MeOH) λmax (logε) 218 (4.38), 274 (4.27), 323 (3.99) nm; IR (KBr) νmax 3359, 2972, 2931, 1687, 1629, 1503, 1326, 1122, 1047, 979, 875, 755 cm−1; 1H NMR data, Table 1; 13C NMR data, Table 2; HRESIMS: m/z 688.2956 [M + H]+ (calcd for C35H46NO13+, 688.2964) and 710.2781 [M + Na]+ (calad for C35H45NNaO13+, 710.2783).

Compound 3: dark brown powder; [α]20 = +12.0 (c 0.10, MeOH); UV (MeOH) λmax (logε) 218 (4.12), 272 (3.94), 323 (3.66) nm; IR (KBr) νmax 3366, 2972, 2932, 1598, 1498, 1377, 1325, 1121, 1069, 981, 758 cm−1; 1H NMR data, Table 1; 13C NMR data, Table 2; HRESIMS: m/z 672.3011 [M + H]+ (calcd for C35H46NO12+, 672.3015), and 694.2835 [M + Na]+ (calcd for C35H45NNaO12+, 694.2834).

Compound 4: maroon powder; [α]20 = +28.0 (c 0.10, MeOH); UV (MeOH) λmax (logε) 214 (3.90), 273 (3.66), 323 (3.37) nm; IR (KBr) νmax 3409, 2948, 2836, 1656, 1451, 1413, 1203, 1114, 1024, 695 cm−1; 1H NMR data, Table 3; 13C NMR data, Table 2; HRESIMS: m/z 674.3172 [M + H]+ (calcd for C35H48NO12+, 674.3171), and 696.2988 [M + Na]+ (calcd for C35H47NNaO12+, 696.2990).

Table 3

1H NMR spectroscopic data (400 MHz, CD3OD) of compounds 4–7 (δH, J in Hz) *.

Position	4	5	6	7
3	7.65 (s)	7.57 (s)	7.56 (s)	7.57 (s)
5	7.07 (s)
13	1.89 (s)	2.08 (s)	2.04 (s)	2.06 (d, 1.0)
14	2.12 (s)	2.18 (s)	2.17 (s)	2.17 (s)
17	6.53 (t, 14.3)	6.25 (d, 10.8)	6.24 (d, 10.8)	6.26 (dd, 0.8, 10.9)
18	6.84 (dd, 10.9, 14.3)	6.51 (dd, 11.0, 15.8)	6.09 (dd, 11.0, 15.1)	5.96 (d, 16.0)
19	6.08 (dd, 8.2, 15.0)	6.09 (dd, 6.6, 15.9)	5.85 (dd, 9.6, 15.2)	6.47 (dd, 10.9, 15.9)
20	2.45 (m)	2.36 (m)	1.89 (m)
21	3.82 (d, 8.8)	4.03 (m)	3.61 (dd, 1.5, 9.2)	3.95 (d, 1.2)
22	1.90 (m)	1.87 (m)	2.86 (dd, 6.8, 9.2)	2.01 (m)
23	3.60 (m)	3.48 (d, 10.2)		3.42 (q, 2.7, 9.4)
24	1.84 (m)	1.80 (m)	2.52 (m)	1.72 (m)
25	4.05 (d, 9.7)	3.98 (m)	3.87 (d, 10.2)	3.94 (dd, 1.9, 10.6)
26	1.80 (m)	1.43 (m)	1.35 (m)	1.40 (m)
27	4.13 (d, 5.5)	4.31 (s)	4.43 (s)	4.37 (br s)
28	2.82 (m)	2.89 (m)	2.58 (m)	2.65 (q, 7.1, 16.0)
29	6.91 (d, 10.4)	6.30 (d, 9.3)	6.28 (d, 9.1)	6.35 (dd, 1.0, 9.5)
30	2.09 (s)	2.09 (s)	2.05 (s)	2.10 (s)
31	1.00 (d, 6.8)	0.91 (d, 6.9)	1.06 (d, 3.1)	1.21 (s)
32	0.90 (d, 6.8)	1.05 (d, 7.0)	1.05 (d, 3.2)	1.17 (d, 7.0)
33	0.96 (d, 6.8)	0.72 (d, 6.8)	1.12 (d, 7.4)	0.74 (d, 6.8)
34	0.83 (d, 6.8)	0.40 (d, 7.0)	0.41 (d, 7.0)	0.41 (d, 7.0)
34a	3.62 (m)3.54 (m)	4.01 (m)4.00 (m)	3.52 (dd, 8.6, 10.9)3.38 (dd, 6.1, 11.0)	3.40 (m)3.58 (dd, 8.0, 10.9)
AcO-34a		2.03 (s)

* s: singlet, d: doublet, dd: double doublet, t: triplet, m: multiplet.

Compound 5: brown powder; [α]20 = +206.3 (c 0.10, MeOH); UV (MeOH) λmax (logε) 225 (4.44), 263 (4.24), 326 (3.93) nm; IR (KBr) νmax 3368, 2970, 2934, 1631, 1607, 1495, 1388, 1197, 1053, 974, 887 cm−1; 1H NMR data, Table 3; 13C NMR data, Table 2; HRESIMS: m/z 698.3170 [M + H]+ (calcd for C37H48NO12+, 698.3171), and 720.2986 [M + Na]+ (calcd for C37H47NNaO12+, 720.2990).

Compound 6: brown powder; [α]20 = +174.2 (c 0.10, MeOH); UV (MeOH) λmax (logε) 222 (4.42), 312 (3.91) nm; IR (KBr) νmax 3347, 2972, 2932, 1629, 1495, 1387, 1323, 1196, 968, 884 cm−1; 1H NMR data, Table 3; 13C NMR data, Table 2; HRESIMS: m/z 654.2914 [M + H]+ (calcd for C35H44NO11+, 654.2909), and 676.2731 [M + Na]+ (calcd for C35H48NNaO11+, 676.2728).

Compound 7: brown powder; [α]20 = +246.3 (c 0.11, MeOH); UV (MeOH) λmax (logε) 222 (4.38), 319 (3.89) nm; IR (KBr) νmax 3362, 2967, 2935, 1631, 1497, 1389, 1318, 1193, 1054, 976, 801 cm−1; 1H NMR data, Table 3; 13C NMR data, Table 2; HRESIMS: m/z 672.3019 [M + H]+ (calcd for C35H46NO12+, 672.3015), and 694.2839 [M + Na]+ (calcd for C35H45NNaO12+, 694.2834).

2.5. Bioactivity

2.5.1. Antimicrobial Assay

Compounds 1–13 were assayed for their antimicrobial activity against Staphylococcus aureus ATCC 25923, Mycobacterium smegmatis mc2 155, Pseudomonas aeruginosa PA01 and Proteusbacillus vulgaris CPCC 160013 with the paper disk diffusion assay as previously described [21]. The tested compounds (20 µg/µL, 2 µL each) were absorbed onto individual paper disks (Ø 6 mm) and placed on the surface of the agar. The assay plates were incubated for 24 h at 37 °C and examined for the presence of inhibitory zones.

The MIC values of active compounds against the growth of Staphylococcus aureus ATCC 25923 were measured through the microbroth dilution method [22]. Microorganisms were cultured in LB media in 96-well plates at a concentration of 1 × 106 CFU/mL. The MIC values were obtained after incubating for 12 h at 37 °C with the tested compounds (concentration ranging from 320 to 0.039 µg/mL).

2.5.2. Cytotoxicity Assay

The in vitro antiproliferative activity against HeLa and Caco-2 cells was measured as previously reported [23,24]. Briefly, cells were seeded in 96-well plates at 7 × 103 cells/well and treated for 24 h with different concentrations of compounds 1–13. Then, 10 µL Cell Counting Kit-8 (CCK-8) was added to each well and incubated for another 4 h. The absorbance was read at 480 nm by Spark 30086376 (TECAN, Männedorf, Switzerland).

3. Results

Compound 1 was determined to have the molecular formula C36H47NO12 on the basis of the quasi molecular ion peaks at HRESIMS m/z 686.3172 [M + H]+ (calcd for C36H48NO12+, 686.3171) and 708.2992 [M + Na]+ (calcd for C36H48NNaO12+, 708.2990). The presence of a naphthaquinone chromophore was indicated by the HMBC correlations from H-3 (δH 7.64) to C-1 (δC 184.7), C-2 (δC 143.0) and C-10 (δC 132.6), and from H-14 (δH 2.09) to C-6 (δC 165.9), C-7 (δC 120.3) and C-8 (δC 161.7). The MeO-6 (δH 4.00) was supported by the HMBC correlations from MeO-6 to C-6 and NOE correlations from MeO-6 to H-5 (δH 7.18) (Table 1 and Table 2, Tables S2 and S3, Figure 2). The twenty-four-carbon fragment from C-15 (δC 170.0) to C-11 (δC 172.3) was established on the basis of 1H-1H COSY correlations, along with the HMBC correlations from the H-30 (Me), H-31 (Me), H-32 (Me), H-33 (Me), H-34 (Me) and H-34a to the corresponding carbons (green, Figure 2). The hydroxylation of C-34a and oxidization to an aldehyde group followed by hemiacetal formation with the hydroxyl group at C-25 were determined based on the 1H NMR of H-34a (δH 4.54/5.08) (Table 1, Figure 2). The ansa chain was determined to undergo retro-Claisen cleavage between C-5 and C-11 on the basis of the chemical shift of C-11 downfield, the presence of the extra aromatic proton H-5 compared to that of normal rifamycins and HMBC from H-5 to C-7 (Tables S2 and S3). Hence, the planar structure of 1 was established. The stereochemistry of the hemiacetal existed as a pair of epimers (1a and 1b) at C-34a, and 1a was determined to be α-form on the basis of the coupling constants J34a,28 = 8.4 Hz and the NOE correlations from H-34a (δH 4.54) to H-25 (δH 3.55) and H-27 (δH 3.18), and between H-25 and H-27. Accordingly, 1b was determined to be β-form on the basis of the coupling constants J34a,28 = 3.2 Hz (Figure 2). The stereochemistry of other carbons was assumed to be the same as that of rifamycin W-hemiacetal [25] based on biosynthetic logic [12]. Thus, compound 1a was named 34a-α-6-O-methyl-rifamycin W-M1-hemiacetal and 1b was named 34a-β-6-O-methyl-rifamycin W-M1-hemiacetal.

Figure 2

Selected HMBC (→), COSY (▬) and NOESY (↔) correlations of 1.

Compound 2 was confirmed to have the molecular formula C35H45NO13 on the basis of the HRESIMS quasi molecular ion peaks at m/z 688.2956 [M + H]+ and 710.2781 [M + Na]+. The NMR spectroscopic data of 2 were similar to that of 1, except that C-34 was a hydroxymethyl (δH 4.33, 4.34, δC 66.0) instead of a methyl group, and the 6-hydroxyl group was free (Table 1 and Table 2). The relative configuration of 2 was proposed to be identical to that of 1, and the hemiacetal existed as a pair of epimers (2a and 2b) (Tables S4 and S5) as well. Thus, compound 2 was determined to be 34a-α-30-hydroxyrifamycin W-M1-hemiacetal (2a) and 34a-β-30-hydroxyrifamycin W-M1-hemiacetal (2b), respectively.

Similarly, the NMR (Tables S6 and S7) and HRESIMS (m/z 672.3011 [M + H]+ and 694.2835 [M + Na]+) comparison determined compound 3 to be 34a-α-rifamycin W-M1-hemiacetal (3a) and 34a-β-rifamycin W-M1-hemiacetal (3b), respectively.

The molecular formula of 4 was elucidated as C35H47NO12 on the basis of the HRESIMS quasi molecular ion peaks at m/z 674.3172 [M + H]+ and 696.2988 [M + Na]+. Similar to that of compounds 1, 2 and 3, the ansa chain of 4 also underwent retro-Claisen cleavage between C-5 and C-11 due to the presence of the extra aromatic proton H-5 (δH 7.07), the chemical shift of C-11 (δC 173.0) and the HMBC from H-5 to C-7 (Table 2 and Table 3, Table S8). Thus, compound 4 was determined to be rifamycin W-M1 [26].

The molecular formula of 5 was confirmed to be C37H47NO12 by the HRESIMS quasi molecular ion peaks at m/z 698.3170 [M + H]+ and 720.2986 [M + Na]+. A close NMR comparison with that of rifamycin W (12) (Tables S9 and S12) [27] revealed that 5 was 34a-O-acetyl-rifamycin W, which was confirmed by the HMBC correlations between H-34a (δH 4.01, 4.00) and the acetyl carbon (δC 172.9).

The molecular formula of 6 was elucidated as C35H43NO11 on the basis of the HRESIMS quasi molecular ion peaks at m/z 654.2914 [M + H]+ and 676.2731 [M + Na]+. NMR comparison with rifamycin W (12) (Tables S10 and S12) revealed that 6 was 23-ketorifamycin W on the basis of the downfield chemical shifts of C-22 (δC 49.7), C-23 (δC 211.3) and C-24 (δC 49.9).

Compound 7 was determined to have the molecular formula of C35H45NO12 on the basis of HRESIMS quasi molecular ion peaks at m/z 672.3019 [M + H]+ and 694.2839 [M + Na]+, revealing one more oxygen atom than that of rifamycin W. NMR comparison (Tables S11 and S12) determined 7 to be 20-hydroxyrifamycin W, which was supported by the chemical shift of C-30 (δC 77.0).

Based on the 1D and 2D NMR data, HRESIMS data and spectroscopic comparisons with those reported in the literature, compounds 8–13 were determined to be rifamycin Z (8) [28], 30-hydroxyrifamycin W-hemiacetal (9) [29], rifamycin W-hemiacetal (10) [25,30], 30-hydroxyrifamycin W (11) [30], rifamycin W (12) [25,27] and protorifamycin I (13) [31] (Figure S7).

Compounds 1−13 were assayed for their antimicrobial activity against Staphylococcus aureus ATCC 25923, Mycobacterium smegmatis mc2 155, Pseudomonas aeruginosa PA01 and Proteusbacillus vulgaris CPCC 160013. The results showed that new compounds 1–3 and known compounds 11 and 13 exhibited inhibitory activity against S. aureus ATCC 25923, while other compounds showed no antimicrobial activity (Figure S56). Thus, new compounds 1–3 were further tested for their antibacterial activity against S. aureus ATCC 25923 using the microbroth dilution method [22], and their MIC values were determined to be 5, 40 and 0.5 µg/mL, respectively (Table S14).

In view of no evident bactericidal activity, compounds 1–13 were evaluated for their antiproliferative activity against HeLa and Caco-2 cells using Cell Counting Kit-8 (CCK-8) (Bimake, Houston, TX, USA) and etoposide (VP-16) as a positive control. Compounds 1 and 3 showed modest activity in inhibiting the proliferation of HeLa and Caco-2 cells with IC50 values of about 50 µM (Table S15, Figures S57 and S58).

4. Discussion

Post-PKS modifications play an important role in increasing the structural diversity and improving the biological activity of rifamycins. As the proposed earliest macrocyclic intermediate in rifamycin post-PKS biosynthesis, proansamycin X tended to undergo dehydration to form putative protorifamycins (without C-8 hydroxyl group) or undergo dehydrogenation to form rifamycin W [24,32,33]. Rifamycin W undergoes a rearrangement of the polyketide backbone to produce rifamycin B via the oxidative cleavage of the C-12/C-29 double bond. The mechanism of this oxidative cleavage has not been characterized yet. For the rif-orf5 gene, when cloned and heterologously expressed in E. coli, the recombinant protein showed spectra typical of P450 cytochromes [34]. Thus, the rif-orf5 gene was confirmed to code for a cytochrome P450 enzyme, which is the key step for oxygen incorporation in rifamycin B biosynthesis and may be involved in the cleavage of the olefinic moiety of rifamycin W [16,17].

In this study, systematic isolation of the fermentation products of the mutant strain Δrif-orf5 afforded thirteen rifamycin W derivatives besides the main product rifamycin W (12), indicating that the rif-orf5 gene was probably involved in the oxidative cleavage of the C-12/C-29 double bond. Compounds 1–4 all undergo C-5/C-11 retro-Claisen cleavage, as observed in the biosynthesis of proansamycin B-M1 and protorifamycin I–M1 [24,35], hygrocins I and J [36], divergolides R and S [37] and microansamycins G–I [38]. This C-5/C-11 cleavage probably occurred due to an over-accumulation of rifamycin W, which serves as a detoxification mechanism. Compounds 1–3, 9 and 10 featured a hemiacetal, in which 9 and 10 existed in β-form according to the chemical shift of C-34a and the coupling constants between C-34a and C-28, while 1–3 existed as epimer pairs (Table S13), which may be due to the feasibility of polyketide chain cleavage in C-5/C-11. Additionally, the hemiacetal containing compounds 1–3, 9 and 10, as well as the lactone-containing rifamycin Z (8), indicated that the oxidation of C-34a alcohol to the carboxyl group may occur before the C-12/C-29 olefinic bond cleavage. In addition, compared to 8-deoxy rifamycins [24], compounds 5, 6, 7 and 11 also oxygenated at C-34a, C-23, C-20 and C-30, which suggested that the rifamycin ansa chain is prone to oxidization in these specific sites during fermentation (Figure 3).

Figure 3

Proposed biosynthetic pathway of compounds from mutant Δrif-orf5.

5. Conclusions

In this study, the cytochrome P450 monooxygenase gene rif-orf5 was confirmed to be involved in the oxidative cleavage of the ansa chain of rifamycin W through in vivo gene inactivation and isolation of the main product rifamycin W. Systematic isolation of the fermentation products of the mutant strain Δrif-orf5 afforded seven new rifamycin W congeners, from which 1–3 featured two epimeric forms of hemiacetal at C-34a, and C-5/C-11 retro-Claisen cleavage. Compounds 1–3 exhibited antibacterial activity against Staphylococcus aureus, and 1 and 3 showed modest antiproliferative activity against HeLa and Caco-2 cells.