Two novel nucleosidyl–peptide antibiotics: Sansanmycin F and G produced by Streptomyces sp SS

Sansanmycins were a group of nucleosidyl–peptide antibiotics, produced by Streptomyces sp SS (CGMCC no. 1764).^{1, 2, 3, 4} During our further studies on minor components of Sansanmycins, two new components, Sansanmycin F and G, were obtained. In this paper, we report the production, isolation, structure elucidation and biological activities of Sansanmycin F and G (Figure 1).

Figure 1

Structures of Sansanmycin F and G.

Streptomyces sp SS wase cultured as described previously.¹ Antibiotic activity was determined by a paper-disk agar diffusion assay using Pseudomonas aeruginosa on Mueller–Hinton medium. The fermentation broth was filtered to remove mycelia; 50 l of filtrate thus obtained was applied on a column of Amberlite XAD-2 (Rohm and Haas Co., Philadelphia, PA, USA) (5 l) and, after washing with 25 l of water and 20 l of 10% aqueous acetone successively, the absorbed materials were eluted with 20 l of 30% aqueous acetone. The active fraction was concentrated in vacuo and lyophilized to obtain a crude powder (30.0 g). Thereafter, 5 g of the crude powder was dissolved in 100 ml 0.1% (w/v) (NH₄)₂CO₃ aqueous solution, and then applied on a column of Amberlite XAD1180 (1.25 l). After washing with 15 l of 8% aqueous acetone, the column was sequentially eluted with 15% (10 l) and 30% (10 l) of aqueous acetone to yield 100 fractions, each of which was analyzed by HPLC-UV (XTerra MS C18 column (Waters, Dublin, Ireland), 150 × 4.6 mm, 5 μm; MeOH/0.1% (w/v) (NH₄)₂CO₃, 3:7 (v/v); flow rate, 1 ml min⁻¹; UV detection at 254 nm and oven temperature at 40 °C). Fractions 60–80, containing Sansanmycin F and G, were combined, concentrated in vacuo and lyophilized to obtain a mixture of Sansanmycin F and G (100 mg). Further separation was achieved by means of preparative RP-HPLC (Shim-pack PREP-ODS column (Shim-pack, Kyoto, Japan), 250 × 20 mm, 10 μm; eluted with a gradient of buffer A (0.1% (w/v) (NH₄)₂CO₃-MeOH, 65:35 (v/v)) to buffer B ((0.1% (w/v) (NH₄)₂CO₃-MeOH, 55:45 (v/v); flow rate, 5 ml min⁻¹; UV detection at 254 nm and oven temperature at 40 °C) to yield Sansanmycin F (15 mg) and G (10 mg).

Sansanmycin F is a white amorphous powder; electrospray ionization (ESI)-MS m/z 904.6 [M+H]⁺, HR-ESI-MS m/z 904.36496 [M+H]⁺ (calcd for C₄₃H₅₄N₉O₁₁S, 904.36635). ¹H and ¹³C NMR spectra of Sansanmycin F (Table 1) were similar to those of Sansanmycin,¹ except that there were new signals in the NMR spectra of Sansanmycin F instead of m-tyrosine residue in Sansanmycin. A series of NMR (¹H NMR, ¹³C NMR, ¹H-¹H COSY, heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond coherence (HMBC)) data indicated that it had the 1,1-dimethyl-6-hydroxy-1,2,3,4-tetradhydro-3-isoquinoline carboxylic acid (TIC) (Figure 2) as the blocked N terminus, similar to Napsamycin B,⁵ which had 1-methyl-6-hydroxy-1,2,3,4-tetradhydro-3-isoquinoline carboxylic acid, and Mureidomycin F,⁶ which had 6-hydroxy-1,2,3,4-tetradhydro-3-isoquinoline carboxylic acid as the blocked N terminus. The proton resonances at δ 6.75 and 7.18 were coupled (¹H-¹H COSY) and corresponded to positions of 7 and 8 of TIC, respectively. The signals at δ 117.6, 137.1 and 156.7 were all coupled (HMBC) to the proton of TIC-7 and were assigned as carbons of 5, 9 and 6 of TIC, respectively. The carbonyl that resonated at δ 176.8 and coupled to the proton of the N-methyl protons was assigned as TIC carbonyl. Also coupled to TIC carbonyl were the protons at δ 4.01 (TIC-3) and δ 2.67 (TIC-4). The signal at δ 136.1, which was coupled to the protons of TIC-4 and TIC-3, was assigned as the carbon of TIC-10. The signal at δ 56.5 showing a cross-peak with protons of TIC-3 and TIC-8 was assigned as the carbon of TIC-1. The methyl proton resonances at both δ 1.46 and 1.35 were coupled to the carbons of TIC-1 and TIC-9, and corresponded to the two methyls of TIC-1. From these results, the structure of Sansanmycin F was elucidated as shown in Figure 1.

Table 1 ¹H and ¹³C NMR Data for Sansanmycin F and G in D₂O^a (pD=8.0, δ in p.p.m.)

Figure 2

Key correlations of TIC moiety in HMBC and 1H-1H COSY spectra of Sansanmycin F.

The total structure of Sansanmycin F was further confirmed by analysis of ESI-MS/MS spectra. The ESI-MS/MS data of the protonated molecular ion, m/z 904 (Figure 3), indicated that sequence selective fragmentations observed for Sansanmycin F showed complete similarity to those of sansanmycin,¹ except those originating from the N terminus. The fragment ion m/z 176 also confirmed that Sansanmycin F had TIC as the N terminus.

Figure 3

ESI-MS/MS data of Sansanmycin F and G.

Sansanmycin G is a white amorphous powder; ESI-MS m/z 886.7 [M+H]⁺, HR-ESI-MS m/z 886.40338 [M+H]⁺ (calcd for C₄₄H₅₆N₉O₁₁, 886.40993). Close examination of the ¹H and ¹³C NMR data of Sansanmycin G suggested that it is similar to Sansanmycin F with a leucine moiety in place of the methionine unit. HPLC analyses of the alkaline hydrolysate (6 N NaOH, 110 °C, 16 h) of Sansanmycin G confirmed the presence of leucine residue. A series of 2D NMR experiments (¹H-¹H COSY, HMQC and HMBC) were performed, which allowed assignment of all ¹H and ¹³C signals associated with each amino-acid unit and pseudo nucleoside residue (Table 1). The total structure of Sansanmycin G (Figure 1) was also further confirmed by analysis of ESI-MS/MS spectra (Figure 3).

Sansanmycin F and G had weak antibacterial activity, as shown in Table 2. Sansanmycin F exhibited inhibitory activity against P. aeruginosa with an MIC value of 16 μg ml⁻¹. Sansanmycin F and G were further evaluated for their inhibitory activity against Mycobacterium tuberculosis H₃₇Rv, on the basis of alarm blue microplate assay,⁷ with MIC values more than 16 μg ml⁻¹.

Table 2 The MIC values of Sansanmycins