Thiopeptides are members of the ribosomally synthesized and post-translationally modified peptide family of natural products, which are widely distributed in the genera of Streptomyces, Nocardia, Micromonospora, Micrococcus, Actinoplanes, Amycolatopsis, Planobispora, and so on [1,2,3,4,5,6,7,8]. Thiopeptides are well known for their potent antibacterial activities against clinically-problematic Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), penicillin-resistant Streptococcus pneumonia (PRSP), and Clostridium difficile [1, 9, 10].

Thiopeptides are characterized by a highly modified macrocycle possessing a central pyridine, hydroxypyridine, or dehydropiperidine. Based on the size of macrocycle core, thiopeptides were divided into four classes: 26 atoms (thiostrepton and nosiheptide), 29 atoms (GE2270A), 32 atoms (lactazoles), and 35 atoms (berninamycins) [11, 12]. Among members of 35-atom subclasses, geninthiocins are variations on the core scaffolds via amino acid substitutions compared to berninamycins, their differences only consist in one amino acid at the genome sequence level. Our previous chemical investigation of Streptomyces sp. CPCC 203702 affforded six berninamycin analogs with excellent antiviral activities [13]. Meanwhile, three geninthiocin derivatives were isolated from Streptomyces sp. CPCC 200267 by Wu Linzhuan’s Group [12], however, the isolated geninthiocin analogs were not evaluated for their antiviral activities.

In our continuing search for antiviral thiopeptide derivatives, the one strain many compounds (OSMAC) strategy was applied to maximize the chemical diversity of strain CPCC 200267. When the strain was separately cultured by eleven medium fermentation, high performance liquid chromatography (HPLC) analyses of its culture extracts displayed different secondary metabolite profiles (Fig. S1). Subsequently, the follow-up fermentations of optional two mediums led to the isolation of six geninthiocin derivatives, including two new cyclic thiopeptides, geninthiocins E and F (1 and 2), along with four known geninthiocin analogs (36) (Fig. 1). Herein, we describe details of the isolation, structural elucidation, and bioactivities of all compounds.

Fig. 1
figure 1

Chemical structures of 16. Pyr (pyridine); Thz (thiazole); Thr (threonine); Oxa (oxazole); Dha (dehydroalanine); Hyval (hydroxyvaline); Val (valine); Ala (alanine)

Geninthiocin E (1) was obtained as white amorphous powder. Its molecular formula was determined as C44H43N13O12S by HRESIMS with m/z 978.29828 [M + H]+, requiring 30 degrees of unsaturation. The 1H and 13C NMR spectroscopic data (Table 1) of 1 closely resembled those of geninthiocin D [12], except for the presence of a methane (δH 2.18, δC 29.8) in 1 instead of one oxygenated non-protonated carbon (δC 71.4) in geninthiocin D, indicating the loss of the OH group in the Hyval residue. This assignment was further confirmed by the 1H–1H COSY correlations of Val-NH (δH 8.69)Val-Hα/(δH 4.40)/Val-Hβ (δH 2.18)/Val-Hγ (δH 0.96, 0.95) and the HMBC correlations of Val-Hβ (δH 2.18)/Val-Cα (δC 60.0) and Val-Cγ (δC 19.2, 18.9). The NOESY correlation (Fig. 2) between methyl protons (δH 1.73) and amide proton (δH 9.63) indicated the Z configuration of the vinyl methyl group in Oxa1 residue. The configurations of Thr and Val were established as l using Marfey’s method (Fig. S2) [14].

Table 1 1H NMR (600 MHz) and 13C NMR (150 MHz) data of 1 and 2 in DMSO-d6
Fig. 2
figure 2

Key COSY, HMBC and NOESY correlations of 1 and 2

Geninthiocin F (2) was isolated as white amorphous powder. Its molecular formula was determined as C47H46N14O13S by HRESIMS with m/z 1047.31873 [M + H]+, which is 69 amu more than that of 1. This suggests the introduction of a Dha residue in 2 compared with 1. Comparison of 1H and 13C NMR spectroscopic data (Table 1) of 2 with those of 1 indicated that 2 displayed the additional presence of one carbonyl (δC 164.9), one double bond [δH 6.55, 5.82, δC 103.0; δC 133.7], constructing a Dha unit. The location of Dha moiety was determined by the HMBC correlations (Fig. 2) from terminal -NH2 (δH 8.14, 7.64) to Dha3-C=O (δC 164.9) and Dha3-Cα (δC 133.7), from Dha3-H2β (δH 6.55, 5.82) to Dha3-Cα (δC 133.7) and Dha3-C=O (δC 164.9), and from Dha3-NH (δH 10.63) to Pyr-C=O (δC 161.1) and Dha3-C=O (δC 164.9). The NOESY correlation (Fig. 2) between methyl protons (δH 1.74) and amide proton (δH 9.66) estblished the Z configuration of the vinyl methyl group in Oxa1 residue. The configurations of Thr and Val were determined to be l using Marfey’s method (Fig. S2) [14].

The known cyclic thiopeptides were identified as val-geninthiocin (3) [15], geninthiocin A (4) [12], geninthiocin B (5) [16], geninthiocin C (6) [12] by comparison of their NMR and MS data with those reported in the literatures.

Compounds 16 were evaluated for anti-influenza A virus (IAV) activities [13]. Compounds 1, 3, 4, and 5 exhibited significant anti-IAV (H1N1) activities with the IC50 values of 28.7, 15.3, 7.3, and 18.3 μM, respectively (Table 2). Compounds 1–6 were tested for antibacterial activities against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 [13]. Compound 3 and 4 displayed moderate antimicrobial activity against S. aureus ATCC 29213 with MIC of 16 and 4 μg ml–1, respectively.

Table 2 Anti-IAV activities of 16

In summary, two new geninthiocin derivatives, geninthiocins E (1) and F (2), together with four known geninthiocin analogs were isolated from Streptomyces sp. CPCC 200267 using OSMAC approach. Compounds 1, 3, 4, and 5 showed significant anti-IAV activities, and compounds 3 and 4 displayed moderate antibacterial activities.