Hemiactinomycin, an undescribed intermediate of actinomycin biosynthesis from an actinomycetes strain Streptomyces antibioticus H41-55

Abstract Hemiactinomycin (1), an intermediate derivative of actinomycin biosynthesis, together with three known actinomycins (2-4) , were isolated from the ethanolic extract of Streptomyces antibioticus H41-55 fermentation mycelium by using various column chromatography. The structure of the derivative was established by extensive spectroscopic analysis, including HRESIMS, 1D, and 2D NMR spectroscopy. In addition, the anti-inflammatory activities of all the isolates were tested. The derivative (1) showed inhibiting NO release activities in LPS-induced RAW 264.7 macrophages with the IC50 values of 15.41 ± 0.66 μM. Graphical Abstract


Introduction
Actinomycins are a family of chromopeptides with antitumor, antibacterial, antiviral and other activities produced by various streptomycete strains (Waksman and Woodruff 1941;Davis et al. 1998;Bitzer et al. 2009;Kroon et al. 2014).Studies of actinomycin biosynthesis showed that it is formed through condensation of two actinomycin halves, that is, 3-hydroxy-4-methyl-anthranilic acid-pentapeptide lactones (Crnovcic et al. 2010;Crnovcic et al. 2014;Keller et al. 2010;Liu et al. 2019).Our investigation on the chemical compositions of an Actinomycetes strain Streptomyces antibioticus H41-55 had led to the discovery of Hemiactinomycin (1), an intermediate derivative of actinomycin biosynthesis, together with three known actinomycin compounds (2-4) (Figure 1).The structure of compound 1 is similar to actinomycin halves which has a pentapeptide lactone ring connected to an aryl carboxylic acid through an amide bond.The difference of 1 from actinomycin half is the formation of oxazole ring between o-NH 2 and m-OH groups.It is an undescribed compound and named hemiactinomycin.We discussed its important significance on actinomycin biosynthesis.The anti-inflammatory activity of the compounds 1-4 were evaluated using inhibitory activity of NO release in LPS-induced RAW 264.7 macrophages-assay.
According to the homological biogenesis between the compound 1 and actinomycin D, the absolute configurations of all the chiral residuals in the compound were assumed to be same as those showed in actinomycin D. Compound 1 is a new compound, named hemiactinomycin (Figure 2).
Actinomycin D (2), actinomycin V (3), actinomycin X Ob (4) were identified by comparison of their NMR and MS data with those reported in the literature respectively (Bitzer et al. 2006;Cai et al. 2016).
In the study of actinomycin biosynthesis, there is no report of direct isolation of semi-actinomycin or actinomycin halves compounds from the fermentation broth of actinomycetes strains as a metabolite.This is the first direct isolation as an immediate derivative of actinomycin biosynthesis.Generally, the real immediate of actinomycin biosynthesis should be a more reactive one, that is, 3-hydroxy-4-methyl-anthranilic acid-pentapeptide lactones.It was detected in the culture of actinomycetes with LC-MS, but not isolated (Keller et al. 2010;Crnovcic et al. 2014;Yuan et al. 2014).The isolation of hemiactinomycin (1) in this study may attribute to the formation of oxazole ring between o-NH 2 and m-OH groups, which blocks the dimerization and condensation of two of the semi-actinomycins.The discovery of 1 to some extent give a strong support for this biosynthetic pathway.
In addition to the anti-tumor effects reported in the literature, we also tested the anti-inflammatory activity of compounds 1-4 (Table S28, see supplementary materials).Compound 1 exhibited the inhibitory activity of NO release in LPS-induced RAW 264.7 macrophages with the IC 50 value of 15.41 ± 0.66 lM.

Materials and methods
The actinomycete strains are isolated from the sediments of the mangroves in the South China Sea.According to the characteristics of culture, morphology, physiology and biochemistry of strain H41-55, as well as its 16S rDNA gene sequence and phylogenetic tree, strain H41-55 was finally determined to be Streptomyces antibioticus.And the specimens are preserved at Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences.

Extraction and isolation
The fermented mycelium of Actinomyces strain H41-55 from the bottom of marine mangroves is soaked and extracted with 95% ethanol.The extract was partitioned between EtOAc and H 2 O, and the organic phase was concentrated and then partitioned between methanol and petroleum ether.The methanol part obtained is the active part of actinomycetes.This active part was separated using the combination of silica gel column chromatography, Sephadex LH-20 column chromatography, ODS column chromatography.Finally, compound 1 was isolated using reversed-phase semipreparative HPLC at a proportion of CH 3 CN/H 2 O (50:50, v/v, t R ¼ 23.3 min, 210 nm), compounds 2 and 3 were obtained by reversed-phase semi-preparative HPLC at a proportion of MeOH/H 2 O (85:15, v/v, t R ¼ 14.8 min and t R ¼ 17.5 min respectively, 210 nm), and compound 4 was purified by reversed-phase semi-preparative HPLC eluting with a proportion of CH 3 CN/H 2 O (75:25, v/v, t R ¼ 9.2 min, 210 nm).

Anti-inflammatory activity and antitumor activities
Compound 1 exhibits anti-inflammatory activity, and compounds 2 and 3 exhibit antitumor activity (Table S1 and S2, see supplementary materials).
NO release inhibition assay: The anti-inflammatory activity was investigated based on the inhibition of NO generation.The NO concentration was detected by the Griess reagent.Quercetin was used as the positive control (Li et al. 2021).
RAW 264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator (37 C, 5% CO 2 ) for 24 h.Firstly aspirate the culture medium, next add 100 ng/mL LPS to the culture medium, then add the different concentrations compounds and positive control, and incubate for 24 h.Aspirate the supernatant and add 100 lL of Griess A and B solutions mixed in a 1:1 ratio.Centrifuge at 3 000 rpm for 1 min and continue to react in the dark for 10 min.The OD value of each well was measured with a microplate reader at a wavelength of 540 nm, and the IC 50 value of the compound was calculated.
indicating the molecular formula of C 32 H 44 N 6 O 8 (calcd for C 32 H 45 N 6 O 8 þ , 641.3293; Figure S7, see supplementary materials).The 1 H-NMR spectrum of 1 in MeOH-d 4 exhibited aromatic proton signals ascribed to an ortho-tetrasubstituted benzene group at d H 8.02 (d, J ¼ 7.