Note

Polyketides are one of the most commercially successful classes of natural products ranging from metabolites for the treatment of infectious disease to numerous cancers [1]. This useful class of secondary metabolites are synthesized through a specific series of proteins known as polyketide synthases (PKSs) [2]. The biogenetic engineering study on this class of the natural products was facilitated by PKSs, which consist of separate modules. The introduction of new building blocks is required as a substitution for PKSs modules, which can be employed easily.

The genus Nocardiopsis is a phylogenetically independent group within the order Actinomycetales, which can be found in a wide range of environment [3]. Various biologically active secondary metabolites, such as the cytotoxic apoptolidins [4], the antimicrobial nocardiamides [5], the antimicrobial nocarimidazoles [6], and the antiphotoaging-active compounds nocariones [7]. have been reported from this fertile genus.

During our investigations of new secondary metabolites from marine microorganisms, we encountered a Nocardiopsis sp., strain CNQ-115, which we now report produces a new 14-membered macrolactam, named fluvirucin B6 (1). This strain was also known to produce rare 4-aminoimidazole natural products [6]. Herein we report the detailed production, isolation, structure elucidation, and biological activity of fluvirucin B6 (1) (Fig. 1).

Fig. 1
figure 1

a Chemical structure of fluvirucin B6 (1). b Key COSY and HMBC correlations of 1

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Fluvirocin B6 (1) was isolated as an optically active white oil ([α]25D −12.6, c 0.34, DMSO). The molecular weight of 1 was obtained from the HRESITOF mass spectrum, which showed pseudomolecular ions at m/z 485.3596 [M + H]+ and at m/z 507.3533 as a sodium ion adduct [M + Na]+. Interestingly, the sodium ion adduct was the major MS peak observed. (Figure S8) On this and upon NMR data, the molecular formula was defined as C26H48N2O6. The UV/vis spectrum of 1 showed only end absorption, while the IR (KBr) spectrum of 1 showed the presence of an amide (3436 and 1638 cm−1) functionality.

The 1H NMR spectrum of 1 included two overlapping methyl triplets at δH 0.89, methyl doublets at δH 0.92 and 1.22, and a methyl singlet at δH 2.05. Numerous signals between δH 3.4–4.2 p.p.m. as well as a doublet at δH 4.87 p.p.m., which indicated an overlapping with methanol solvent peak, revealed signals that were ascribable to a monosaccharide. The NMR DEPT spectrum indicated the presence of five methyl groups, ten methylene carbons, and nine methines. Two quaternary signals at δC 173.3 and 179.2, indicated the presence of two carbonyl groups. A proton at δH 8.16 that by HSQC analysis showed no correlation in the HSQC spectrum consists of an amide proton.

Analysis of COSY and HMBC spectroscopic data revealed two fragments. COSY NMR cross-peaks (H-6/H2-5/H2-4/H2-3/H-2/H2-15/H3-16, H-14/H2-13/H2-12/H2-11/H-10) and HMBC correlations (H-6/C-8, H3-19/C-6, C-5, H2-7/C-9, H2-13/ C-1 (δC 179.2)) permitted the establishment of a branched 14-membered lactam moiety as one fragment. Further COSY cross-peaks (H-20/H-3′/H-2′/H-1′, H3-6′/H-5′) and HMBC correlations (H-1′/C-2′ and C-3′, H-3′/C-5′, H-5′/C-1′, and C-3′) suggested the presence of amino sugar moiety for the second fragment.

The positions of a methyl and two ethyl groups were assigned by analysis of HMBC NMR correlations. The HMBC correlations from H3-16 (δH 0.89) to C-2 (δC 51.4) and C-15 (δC 27.8), and form H-2 (δH 2.10) to C-16 (δC 12.7) and C-15 (δC 27.8) permitted establishing an ethyl group at C-2. The HMBC correlations from H3-18 (δH 0.89) to C-10 (δC 42.4) and C-17 (δC 22.5), and from H-10 (δH 1.6) to C-18 (δC 9.7) and C-17 (δC 22.5) allowed another ethyl group to be positioned at C-10. The connectivity of C-19 methyl with C-6 was established from the observation of long-range HMBC correlations from H3-19 (δH 0.92) to C-6 (δC 32.6) and C-5 (δC 35.7; Fig. 1b).

The amino sugar moiety of compound 1 was determined as an epi-mycosamine based on the small coupling constant values for H-2′ (t, J = 3.0 Hz), H-3′ (t, J = 3.0 Hz), H-4′ (t, J = 3.0 Hz), suggesting the equatorial orientation of these protons [8]. Furthermore, an equatorial orientation of H-1′ and H3-6′ were deduced by analysis of NOESY NMR correlations with H-9 and H-4′. The acetyl group was located at C-3′ based on the long-range HMBC correlations of H-3′ (δH 4.17) and H3-22 (δH 2.05) to a carbonyl carbon C-21 (δC 173.3). Finally, connectivity of the two fragments was achieved on the basis of a long-range HMBC correlation from H-1′ (δH 4.87) to C-9 (δC 78.8), thus completing the structure assignment of N-acetyl-2,5-epi-mycosamine (1) (Table 1 for NMR data).

Table 1 NMR spectroscopic data for fluvirucin B6 (1) in MeOD
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To determine the absolute configuration of 1, numerous attempts to crystalize the compound were employed. Unfortunately, a crystal of suitable size could not be obtained for X-ray analysis. Therefore, we carefully compared the NMR spectroscopic data of 1 to those of the previous reported natural product, Sch 38516/fluvirucin B1. Most of the 1H, and 13C NMR signals of 1 and Sch 38516/fluvirucin B1 were very similar, except for the substitution of one amino proton with an acetyl group in the sugar moiety. The average carbon chemical shift differences in conserved areas are 0–0.7 p.p.m., whereas the differences around the attached acetyl group are 1.1–2.8 p.p.m. (Table S1) Moreover, the negative optical rotation values of compound 1 ([α]25D −12.6, c 0.34, DMSO), which compared favorably with the reported value for Sch 38516 ([α]26D −6.7, c 0.50, DMSO) [9] suggested these compounds could have the same configurations.

Compound 1 belongs to the fluvirucins class of polyketides, which possess a 14-membered macrolactam attached to a sugar. The fluvirucin A series have a sugar at the C-3 position whereas the B series have a sugar at the C-9 position [10,11,12]. Compound 1 is the acetylated derivative of fluvirucin B1, thus we have named this compound fluvirucin B6.

Fluvirucins are known to have various bioactivities such as antiviral, antibacterial, anthelmintic, and antifungal [10, 13,14,15]. Fluvirucin B6 (1) was tested against six pathogenic bacteria including three Gram-positive bacteria (Bacillus subtillis ATCC 6644, Kocuria rhizophila ATCC 9341, and Staphylococcus aureus ATCC 6538 P) and three Gram-negative bacteria (Escherichia coli ATCC 11775, Salmonella typhimurium ATCC 14028, and Klebsiela pneumoniae ATCC 4352). Compound 1 was found to exhibit weak antibacterial activities against B. subtillis, K. rhizophila, and S. aureus with MIC values of 64, 32, and 32 μg/L, respectively. Fluvirucin B6 (1) only showed an antibacterial activity against Gram-positive bacteria, any significant activity against Gram-negative bacteria was not observed up to 128 μg/L. The weak antibacterial activity of compound 1 could be due to the impact of the modified sugar moiety within 14-membered macrolactone/lactam class PKS products [16].

Lactam substitution for a lactone moiety has been employed to improve the bioactivity or pharmacological profiles of known-macrolactone structure [17], which was demonstrated with the azalides [18]. However, macrolactam structures are mainly acquired from low-yield synthetic methods which limit a more facile approach for these PKS products. For example, most improved enantioselective synthetic efforts for fluvirucin B1 aglycon–fluvirucinin B1 resulted in 12 linear synthetic steps with a 11% overall yield [19].

In combination with the recently revealed fluvirucin B1 biosynthetic pathway [20], Nocardiopsis sp. strain CNQ-115 may provide new macrolactam biosynthesis building block for this class of PKS product. This possibility can be employed to increase the range of novel 14-membered macrolactam analogs.