Four new meridamycin congeners from Streptomyces sp. SR107

Abstract

Meridamycin and its naturally occurring analog normeridamycin are non-immunosuppressive macrocyclic polyketides with potent neuroprotective activity in dopaminergic neurons. Although the biosynthetic gene cluster of meridamycin has been cloned and therefore generated the analogue, C36-keto-meridamycin, the diversity of this type of unique macrolide is still undiscovered. In this paper, four meridamycin congeners, namely meridamycin A (2), meridamycin B (3), meridamycin C (4) and meridamycin D (5), together with meridamycin (1), were isolated from the agar fermentation of the strain Streptomyces sp. SR107. The structures of these compounds were elucidated on the basis of 1D-, 2D-NMR, and HRESIMS data analysis. The antibacterial activities of compounds 1–5 were also evaluated.

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Introduction

Meridamycin, a 27-membered macrolide, was firstly isolated from a strain of Streptomyces hygroscopicus.¹ It shares the structural motif common to the macrocyclic polyketide immunosuppressants rapamycin,² FK506 ^3–5 and FK520.^6–8 However, it differs from rapamycin, FK506 and FK520 due to the absence of the substituted cyclohexane ring characteristic to these macrolides (Fig. 1). Meridamycin competitively inhibits the binding of FK506 and rapamycin to FK506-binding protein-12 (FKBP-12), IC₅₀ 1.0 ng mL⁻¹. Nevertheless, it is a non-immunosuppressant FKBP-binding macrocyclic polyketide and can antagonize the immunosuppressive activity of both FK506 and rapamycin. Consequently, meridamycin has a potential use as antidote in the treatment of overdoses of immunosuppressants rapamycin and FK506, and also can be used as a neuroprotectant in a range of neurodegenerative disorders including dementia, Parkinson's disease and ischaemic stroke.⁹ However, only two meridamycin analogues, including 3-normeridamycin¹⁰ and C36-keto-meridamycin,¹¹ have been reported till now. In our continuous searching for new secondary metabolites from Streptomyces sp. LZ35, a series of compounds have been identified.^12–16 In the present study, meridamycin (1) and four new congeners, namely meridamycin A (2), meridamycin B (3), meridamycin C (4) and meridamycin D (5) (Fig. 2), were isolated and identified from the agar fermentation extract of the mutant strain SR107 of Streptomyces sp. LZ35.¹² The antibacterial activities of compounds 1–5 were also evaluated.

Fig. 1 Structures of meridamycin (1), rapamycin, FK506 and FK520.

Fig. 2 Structures of compounds 2–5.

Results and discussion

Five compounds (1–5) were isolated from the extract of strain SR107 by column chromatography over Sephadex LH-20, reversed-phase (RP-18) silica gel and normal phase silica gel.

Meridamycin (1) was obtained as a light yellow oil. Its molecular formula was determined to be C₄₅H₇₅NO₁₂ on the basis of HRESIMS (m/z 822.5361 for [M + H]⁺, calcd. 822.5362, 844.5190 for [M + Na]⁺, calcd. 822.5181, C₄₅H₇₅NO₁₂Na⁺) and NMR data (ESI Table S1†). The ¹H, ¹³C NMR and HSQC spectra revealed 45 signals, corresponding to nine CH₃, twelve CH₂, seventeen CH and seven quaternary C-atoms. The NMR comparison with that of literature readily revealed that compound 1 was identical to meridamycin.¹

Meridamycin A (2) has a molecular formula of C₄₅H₇₇NO₁₃ with 18 amu more than that of meridamycin (1). A close NMR comparison (Table 1) with that of 1 revealed that the only evident difference was the upfield shift of H-6 from δ 5.38 in 1 to δ 3.99 in 2. The HMBC showed no ¹H–¹³C long-range correlations from H-6 to C-36, which indicates a structure of seco-meridamycin (Fig. 2).

Table 1 ¹H and ¹³C NMR spectroscopy data for compounds 2–5 (in CD₃OD δ in ppm, J in Hz)

Pos.	2		3		4		5
	^1H	^13C	^1H	^13C	^1H	^13C	^1H	^13C
1	1.15 d (6.1)	21.1 (q)	1.15 d (6.3)	21.1 (q)	1.15 d (6.3)	21.2 (q)	1.15 d (6.4)	21.2 (q)
2	3.52 d (6.5)	72.9 (d)	3.54 f (6.4, 13.0)	72.9 (d)	3.51 m	72.9 (d)	3.53 f (6.4, 13.0)	72.9 (d)
3	2.41 m	41.0 (d)	2.42 m	41.0 (d)	2.43 m	41.0 (d)	2.43 m	41.0 (d)
3a	0.99 d (6.4)	17.4 (q)	0.99 d (6.7)	17.4 (q)	0.99 d (6.7)	17.4 (q)	0.99 d (6.7)	17.4 (q)
4	5.24 d (10.5)	131.0 (d)	5.25 d (9.8)	130.9 (d)	5.25 m	130.9 (d)	5.25 d (9.8)	130.9 (d)
5		137.9 (s)		137.9 (s)		137.9 (s)		137.9 (s)
5a	1.651 s	11.9 (s)	1.66 s	12.0 (q)	1.653 s	11.9 (q)	1.653 s	12.0 (q)
6	3.99 t (6.9)	79.0 (d)	3.99 t (7.0)	79.0 (d)	4.00 t (6.7)	79.0 (d)	3.99 t (7.1)	79.0 (d)
7	2.31 m	34.0 (t)	2.30 m	34.1 (t)	2.34 m	34.1 (t)	2.31 m	34.1 (t)
	2.36 m		2.37 m				2.36 m
8	5.27 d (6.6)	123.9 (d)	5.28 t (7.0)	123.9 (d)	5.29 m	123.8 (d)	5.28 t (7.0)	123.8 (d)
9		145.0 (s)		145.0 (s)		145.1 (s)		145.0 (s)
9a	2.07 m	21.8 (t)	2.08 m	21.8 (t)	2.06 m	21.8 (t)	2.10 m	21.9 (t)
9b	1.06 t (7.1)	14.8 (q)	1.06 t (7.6)	14.8 (q)	1.06 t (7.5)	14.8 (q)	1.06 t (7.6)	14.8 (q)
10	3.68 d (6.4)	82.3 (d)	3.69 d (6.6)	82.2 (d)	3.69 m	82.3 (d)	3.68 d (6.0)	82.3 (d)
11	2.12 m	35.2 (d)	1.61 m	35.2 (d)	1.59 m	35.3 (d)	1.61 m	35.3 (d)
11a	0.88 d (6.5)	15.4 (q)	0.88 d (6.5)	15.4 (q)	0.89 m	15.4 (q)	0.88 d (6.5)	15.4 (q)
12	1.25 m	43.3 (t)	1.10 m	43.2 (t)	1.09 m	43.3 (t)	1.09 m	43.3 (t)
	1.48 m		1.27 m		1.25 m		1.27 m
13	2.54 m	30.8 (d)	2.56 m	30.8 (d)	2.56 m	30.8 (d)	2.56 m	30.8 (d)
13a	0.92 d (6.5)	22.4 (q)	0.92 d (6.2)	22.4 (q)	0.93 m	22.4 (q)	0.92 d (6.8)	22.4 (q)
14	5.12 d (10.1)	132.3 (d)	5.13 d (10.2)	132.3 (d)	5.13 d (7.5)	132.3 (d)	5.13 d (9.7)	132.3 (d)
15		137.9 (s)		137.9 (s)		137.9 (s)		137.9 (s)
15a	1.650 s	12.6 (q)	1.65 s	12.5 (q)	1.650 s	12.6 (q)	1.651 s	12.6 (q)
16	4.16 m	74.5 (d)	4.18 m	74.4 (d)	4.18 m	74.4 (d)	4.18 m	74.4 (d)
17	1.55 m	44.0 (t)	1.78 m	42.4 (t)	1.64 m	44.3 (t)	1.53 m	44.3 (t)
	1.65 m
18	3.91 br s	68.6 (d)	3.92 m	68.8 (d)	3.83 m	69.1 (d)	3.82 m	69.1 (d)
19	1.62 m	44.3 (t)	1.61 m	44.4 (t)	1.57 m	42.7 (t)	1.47 m	42.6 (t)
	1.66 m		1.64 m
20	4.18 m	71.7 (d)	4.24 m	71.8 (d)	4.17 m	71.3 (d)	4.17 m	71.5 (d)
21	2.15 m	36.2 (d)	1.58 m	44.3 (d)	1.55 m	44.3 (d)	1.69 m	44.2 (d)
21a	0.89 m	11.2 (q)	0.88 d (6.5)	11.0 (q)	0.91 m	10.8 (q)	0.91 d (7.1)	10.8 (q)
22	3.82 t (8.6)	71.3 (d)	3.83 m	71.1 (d)	3.86 m	71.8 (d)	3.85 m	71.9 (d)
23	1.80 m	42.5 (t)	3.35 m	46.1 (t)	1.69 m	42.5 (t)	1.55 m	43.1 (t)
			3.58 br d (3.4)		1.85 m
24	4.13 m	68.0 (d)	4.15 m	67.8 (d)	3.93 m	68.7 (d)	3.94 m	68.4 (d)
25	1.42 m	33.1 (t)	1.44 m	33.2 (t)	1.48 m	36.8 (t)	1.43 m	36.7 (t)
	1.61 m		1.62 m
26	1.62 m	28.3 (t)	1.76 m	28.2 (t)	1.81 m	31.3 (t)	1.78 m	31.2 (t)
	1.70 m
27	1.61 m	36.4 (d)	2.23 m	36.2 (d)	2.51 m	37.2 (d)	37.2 (d)	40.6 (d)
27a	0.85 t (7.4)	16.2 (q)	0.84 t (6.5)	15.9 (q)	1.27 m	17.7 (q)	1.16 d (7.1)	17.6 (q)
28		100.9 (s)		100.8 (s)		180.8 (s)		178.9 (s)
28-OMe							3.66 br s	52.1 (q)
29		200.3 (s)		200.1 (s)
30		168.8 (s)		169.4 (s)
31	4.26 m	40.1 (t)	1.49 m	42.5 (t)
	4.37 d (12.0)
32	1.48 m	26.1 (t)	1.67 m	26.0 (t)
	1.69 m
33	1.44 m	25.7 (t)	1.77 m	22.3 (t)
	1.77 m
34	1.70 m	27.7 (t)	2.26 m	27.6 (t)
	2.29 m
35	3.58 d (13.4)	46.1 (d)	5.14 m	52.8 (d)
36		169.3 (s)		171.8 (s)
36a-OMe			3.77 m	53.0 (q)

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Meridamycin B (3) has the molecular formula of C₄₆H₇₉NO₁₃ with 14 amu more than that of 2. The ¹H, ¹³C NMR spectra were similar to that of 2, except for an additional signal at δ 3.77 (s, MeO–)] attributed to a Me ester, as confirmed by the comparison of ¹H and ¹³C NMR spectra with that of 2, and corroborated by the HMBC correlations between H-36a (δ 3.33) and C-36 (δ 171.8). Thus, the structure of 3 was determined to be 36-methyl-seco-meridamycin (Fig. 2).

Meridamycin C (4) was determined to have the molecular formula C₃₇H₆₈O₁₀ based on its HRESIMS and NMR data. The ¹H, ¹³C NMR and HSQC spectra revealed 37 signals, corresponding to nine CH₃, eight CH_2, sixteen CH (eight being oxygenated) and four quaternary C-atoms. The direct connectivity between protons and carbons were established by HSQC experiments (Table 1). Further NMR study on ¹H–¹H COSY and HMBC correlations (ESI Table S4 and Fig. S3.†) revealed that 4 was partially identical to 1 (from C-1 to C-28). Moreover, we found that the chemical shift of C-28 was downfield shifted (δ 180.8 in 4 and 101.1 in 1) and the chemical shift of C-24 was upfield shifted (δ 68.7, 3.93 in 4; and δ 72.8, 4.88 in 1), which confirmed the breakage between C-24 and C-28.

Meridamycin D (5) has the molecular formula of C₃₈H₇₀O₁₀ with 14 amu more than that of 4. The ¹H, ¹³C NMR spectra were similar to that of 4, except for an additional signal at δ 3.66 (s, MeO–)] attributed to a Me ester, which was further corroborated by the HMBC between H-28a (δ 3.33) and C-28 (δ 178.9). Thus, the structure of 5 was determined to be 28-methyl meridamycin C (Fig. 1).

The absolute stereochemistry of meridamycin has been reported. It is biosynthetically logical to assume the identical configurations for 2–5, while experiments are underway to obtain crystalline samples of 3 and 5 for X-ray diffraction analysis.

The seco-meridamycin derivatives 2–5 showed no inhibitory activity against tested microbes, whereas 1 exhibited appropriate activity against Bacillus subtilis 86315 with the inhibitory zone of 16 mm at 20 μg per disc.

Polyketide macrolides constitute a large family of bioactive natural products and include many compounds with important clinical applications in medicine. Meridamycin and normeridamycin are non-immunosuppressive macrocyclic polyketides with potent neuroprotective activity in dopaminergic neurons.¹⁰ The biosynthetic gene cluster of meridamycin (1) has been cloned from Streptomyces sp. NRRL 30748 and Streptomyces sp. DSM 4137 and is located on several overlapping cosmids.^9,11 The discovery of the meridamycin congeners is important from a biosynthetic point of view because their structures grant valuable insights into the assembly of this diverse family. Meridamycins C (4) and D (5) from SR107 strain were obtained as the main components because the chain propagation in PKS module 13 stalled and led to an accumulation of the tetradecyketides, which represented pathway intermediates that were prematurely released from the modular assembly line. Although, it has been 20 years since the discovery of meridamycin,^1,2 only three analogues were identified.^10,11

Experimental section

General experimental procedures

The optical rotation was carried out using an Anton Paar MCP200 polarimeter. NMR spectra were recorded on Bruker DRX-600 MHz NMR spectrometer with tetramethylsilane (TMS) as an internal standard. HRESIMS were carried out on an LTQ-Orbitrap XL. Sephadex LH-20 (25–100 μm; Pharmacia Biotek, Denmark) and LiChroprep RP-18 (40–63 μm; Darmstadt, Germany) were used for column chromatography (CC). Silica gel (200–300 mesh) for CC were purchased from Qingdao Marine Chemical, Ltd. (Qingdao, China). All solvents used were of analytical grade.

Strain and fermentation

SR107 strain, a mutant of Streptomyces sp. LZ35, was constructed by our previously work.¹³ This strain was cultured for 14 d on Petri dishes with ca. 20 mL ISP3 agar medium (1.5% agar, 2% oatmeal, 0.1% trace element solution, pH 7.2) with a total volume of 100 L at 28 °C.

Extraction and isolation

The culture (100 L) was extracted three times with EtOAc–MeOH–AcOH (80 : 15 : 5 v/v/v) to obtain the extract. Then the extract was partitioned between H₂O and EtOAc until the EtOAc layer was colorless. Then, the EtOAc solution was dried over Na₂SO₄, and the solvent was removed under vacuum at 38 °C. The EtOAc extract was partitioned with petroleum ether (PE) and 95% aqueous MeOH until the PE layer was colorless. The MeOH solution was concentrated under vacuum at 38 °C to obtain MeOH extract. The MeOH extract (40 g) was subjected to column chromatography over Sephadex LH-20 (120 g) eluted with MeOH to obtain 5 fractions (Fr. 1–5).

Fr. 2 (15 g) was chromatographed over Sephadex LH-20 (120 g) eluted with MeOH to obtain 3 fractions, Fr. 2a–2c. Fr. 2b (7.2 g) was subjected to CC over Sephadex LH-20 (120 g) eluted with MeOH to obtain 4 fractions, Fr. 2b1–2b4. Fr. 2b3 (3.4 g) was further subjected to MPLC over RP-18 silica gel (80 g), and 14 subfractions with 200 mL for each gradient were obtained from the elution with 30%, 50%, 55%, 60%, 70% and 100% MeOH, respectively. In accordance with TLC results, 5 fractions was obtained, Fr. 2b3a–2b3e. Fr. 2b3c (995.6 mg) was chromatographed over Sephadex LH-20 (60 g) eluted with acetone to obtain 4 fractions, Fr. 2b3c1–2b3c4. Fr. 2b3c2 (343.8 mg) was further purified by CC over silica gel (4 g) eluted with gradient CH₂Cl₂–MeOH (75 : 1, 60 : 1, 50 : 1, 40 : 1) to yield 1 (34 mg). Fr. 2b3c3 (120.5 mg) was further purified by CC over silica gel (2.5 g) eluted with gradient CH₂Cl₂–MeOH (50 : 1, 40 : 1, 30 : 1, 25 : 1, 20 : 1, 8 : 1) to yield 4 (1.4 mg), 5 (2.8 mg) and 2 (8.5 mg). Fr. 2 b3d (728.6 mg) was chromatographed over Sephadex LH-20 (60 g) eluted with acetone to obtain 3 fractions, Fr. 2b3d1–2b3d3. Fr. 2b3d2 (91.2 mg) was further purified by CC over silica gel (2 g) eluted with gradient petroleum ether–acetone (20 : 1, 10 : 1, 5 : 1, 4.5 : 1, 4 : 1, 3 : 1) to yield 3 (14.8 mg).

Meridamycin (1). Light yellow oil; [a]²⁰_D = + 3.98 (c 0.92, MeOH); ¹H and ¹³C NMR data, see ESI Table S1;† HRESIMS: m/z 822.5361 [M + H]⁺ (calcd. for C₄₅H₇₆NO₁₂⁺, 822.5362), and 844.5190 [M + Na]⁺ (calcd. for C₄₅H₇₅NO₁₂Na⁺, 844.5181).

Meridamycin A (2). Light yellow oil; [a]²⁰_D = + 2.89 (c 0.35, MeOH); ¹H and ¹³C NMR data, see ESI Table S2;† HRESIMS: m/z 840.5469 [M + H]⁺ (calcd. for C₄₅H₇₈NO₁₃⁺, 840.5468), and 862.5296 [M + Na]⁺ (calcd. for C₄₅H₇₇NO₁₃Na⁺, 862.5287).

Meridamycin B (3). Light yellow oil; [a]²⁰_D = + 6.75 (c 0.305, MeOH); ¹H and ¹³C NMR data, see ESI Table S3;† HRESIMS: m/z 876.5449 [M + Na]⁺, (calcd. for C₄₆H₇₉NO₁₃Na⁺, 876.5444).

Meridamycin C (4). Light yellow oil [a]²⁰_D = + 3.26 (c 0.515, MeOH); ¹H and ¹³C NMR data, see ESI Table S4;† HRESIMS: m/z 673.4881 [M + H]⁺ (calcd. for C₃₇H₇₀O₁₀⁺, 673.4885).

Meridamycin D (5). Light yellow oil; [a]²⁰_D = −1.45 (c 0.33, MeOH); ¹H and ¹³C NMR data, see ESI Table S5; † HRESIMS: m/z 709.4879 [M + Na]⁺ (calcd. for C₃₈H₇₀O₁₀Na⁺, 709.4871).

Antimicrobial assay

The antimicrobial activities of compounds 1–5 against Candida albicans 5314, Staphylococcus aureus ATCC 25923, Mycobacterium smegmatis mc² 155, Pseudomonas aeruginosa PA01, Bacillus subtilis 86315 or Salmonella enterica serovar Typhimurium UK-1 χ8956 were measured with a paper disc diffusion assay. Kanamycin and amphotericin were used as positive control. Test compounds were absorbed onto individual paper disks (6 mm diameter) at 20 μg per disc and placed on the surface of the agar. The assay plates were incubated at 28 °C for 48 h for antifungal activity and at 37 °C for 24 h for antibacterial activity and examined for the presence of a zone of inhibition.

Conclusions

In summary, we have isolated and characterized four new meridamycin congeners from the SR107 strain. In these compounds, only meridamycin showed certain antimicrobial activity against B. subtilis 86315, but the seco-derivatives (2–5) showed no activity against tested microbe. However, the immunosuppressive and neuroprotective activities of the seco-derivatives of meridamycin (2–5) should be evaluated in the next studies. This work further demonstrates that the solid state fermentation could be a useful strategy to increase antibiotic production.

Acknowledgements

This work was supported in part by the National Natural Science Foundation of China (81373304, 81530091, U1405223), and the Independent Innovation Foundation of Shandong University (IIFSDU, 2014JC027) and Program for Changjiang Scholars and Innovative Research Team in University (IRT13028).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Spectroscopic data and other relevant information for compounds 1–5. See DOI: 10.1039/c6ra09772c

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