A Novel Benzo[f][1,7]Naphthyridine Produced by Streptomyces Albogriseolus from Mangrove Sediments

Mangrove Streptomyces represent a rich source of novel bioactive compounds in medicinal research. A novel alkaloid, named 1-N-methyl-3-methylamino-[N-butanoic acid-3′-(9′-methyl-8′-propen-7′-one)-amide]-benzo[f][1,7]naphthyridine-2-one (1) was isolated from Streptomyces albogriseolus originating from mangrove sediments. The structure of compound 1 was elucidated by extensive spectroscopic data analyses and verified by the 13C-NMR calculation at the B3LYP/6-311+G(2d,p) level of theory.


Introduction
Mangrove sediments, which consisting of rich organic matter and salts represent some of the most complex microbial habitats on Earth. Culture independent molecular analysis has been intensively used to detect bacterial communities in mangrove sediments. At least eight different bacterial phyla OPEN ACCESS including actinobacteria were detected in 16 S rRNA gene libraries constructed using a mangrove sediment of Sundarban, India [1]. Groups of actinobacteria strains were isolated from sediments collected from the national mangrove reserve in the Fujian Province of China and at least two novel Streptomyces species, S. xiamenensis [2] and S. avicenniae [3], were identified. The immense and untapped microbial biodiversity in the mangrove ecosystem is a promising resource for natural product studies. The actinobacterial strains that are subjected to the periodical changes in environmental factors seen in mangrove forests, such as salinity and tidal gradients, are believed to be effective selectors for metabolic pathway adaptations that could generate unique metabolites. Previous investigation on secondary metabolites from mangrove endophytes provided a number of new bioactive compounds [4][5][6][7][8]. Streptomyces albogriseolus is well-known for producing novel secondary metabolites and possessing good bioactivity. For instance, echinosporins isolated from marine-derived S. albogriseolus were found to be cell cycle inhibitors, apoptosis inducers and potential anti-tumor agents [9]. Antibiotics, namely cephamycin C, toyocamycin and neomycin complex, were also produced by the strain [10,11]. Thienodolin with its unique chloro-substituted structure was used as a plant growth-regulating substance [12]. A mangrove-derived S. albogriseolus was isolated from the sediments and its potential pharmaceutical applications were surveyed both genetically and chemically. This strain produced a novel benzonaphthyridine alkaloid (see Figure 1). In this paper, we report the structural elucidation of a new alkaloid identified as 1-N-methyl-3-methylamino-[N-butanoic acid-3′-(9′-methyl-8′-propen-7′one)-amide]-benzo[f] [1,7]naphthyridine-2-one (1) by extensive spectroscopic data analyses and the 13 C-NMR calculations at the B3LYP/6-311+G(2d,p) level of theory.
Compound 1 possesses a fused tricyclic heteroaromatic system, which belongs to diazaphenathrene family. Considering such structural character, calculation of 13 C-NMR shifts has been applied as a useful tool for the structural elucidation, e.g. in the example of samoquasine A [13]. Here calculations of the theoretical 13 C-NMR spectrum were carried out with the Gaussian 09 software package [14], using a simplified model in which the aliphatic chain was replaced with aldehyde. Geometry optimizations and frequency calculations were performed at the B3LYP/6-31G(d) level of theory and the optimized structures were verified as true minima with no imaginary frequency [15]. 13 C-NMR chemical shifts were estimated with the GIAO-based method [16] at the B3LYP/6-311 + G(2d,p), using a benzene reference of 128.5 ppm. The calculated results were in good agreement with the experimental carbon resonance assignments and an unambiguous assignment of the 13 C-NMR chemical shifts was achieved by the comparison of the computed 13 C-NMR data with the experimental data (see Table 2). The deviation was found to be less than 5.5 ppm, and averaged 2.3 ppm.  [1,7]naphthyridine part of 1.
The benzonaphthyridines are unique alkaloids with diverse bioactivities. The marine environment seems to be a rich source of such molecules. Aaptamines were isolated from a marine sponge Aaptos sp. [17][18][19], which exhibited different bioactivities, e.g. competitive antagonist of α-adrenoceptors in vascular smooth muscles [20], activator of p21 promoter in a p53-independent manner [21], antineoplastic [22], antivirus and anticancer properties [23], hence triggering major interest in its total synthesis [24]. Another type of 2,7-naphthyridine alkaloids, the lophocladines and subarine were isolated from the marine red alga Lophocladia sp. [25] and a Singaporean ascidian, respectively [26]. In this work, benzo[f] [1,7]naphthyridine was identified as the first example of this class found among mangrove related microbial metabolites. A series of such type of alkaloids was further detected by Dragendorff's reagent on TLC and by HPLC-MS technique. An analogue of 1 possessing the molecular formula of C 24 [1,7]naphthyridine seem to be produced by the S. albogriseolus, but their low productivity in fermentation hindered further chemical and pharmaceutical investigations.
Primary bioinformatics analysis of the draft genome sequence of the producer strain, S. albogriseolus has revealed the existence of at least two putative antibiotic biosynthetic gene clusters, including polyketide synthases and key components in phenazines biosynthesis in the genome (unpublished data). Resolving the biosynthesis pathway of benzonaphthyridine will probably clarify the real source of such compounds in the microorganism. Considering the novelty of structure and the minor amounts isolated, the genetic manipulation of the biosynthetic genes might offer us another opportunity for searching for bioactive novel leading compounds besides synthetic methods and for producing such compounds in large amounts for medicinal research. Organic solvents for HPLC were analytical grade were purchased from Merck KGaA (Darmstadt, Germany).

Strain Isolation, Characterization, and Cultivation
The strain was isolated from the sediment collected in the national mangrove reserve in Fujian province of China, where Avicennia marina grows as the dominant species. The collected sediment was air dried and ground up. Soil samples (1 g) were mixed with sterile water (9 mL) and then diluted to 10 −6 . The diluted suspension (100 μL) was transferred to Gause's synthetic agar [soluble starch 20.0 g/L, K 2 HPO 4 0.5 g/L, MgSO 4 7H 2 O 0.5 g/L, NaCl 0.5 g/L, NaNO 3 1.0 g/L, FeSO 4 0.01 g/L, agar 2%, 25 μg/mL nalidixic acid for inhibiting the grow of bacteria, pH 7.2] then incubated at 28 °C for 10-15 days. Purified single colonies were classified according to colony morphology. One isolated strain MGR072 was assigned as Streptomyces albogriseolus by 16S rRNA gene sequence analysis. The production culture was carried out in a 30 L of ISP3 media (20 g oats per 1 L media, 1.5% agar, pH 7.2-7.4). It was grown for 12 days at 28 ± 1 °C.

Extraction and Isolation
The production culture of S. albogriseolus was extracted at room temperature overnight with a solvent mixture of ethyl acetate/methanol/acetic acid (about 5 L, 80: 15:5, v:v:v). The supernatant was filtered and the residue was then extracted twice more as described above. The supernatants were combined and concentrated under vacuum at 37 o C in order to remove the organic phase. The remaining aqueous solution was partitioned four times with ethyl acetate (about 2 L, 1:1, v:v). The upper layer was collected to afford a crude extract (6 g) which was subjected to column chromatography on RP

Computational Methods
Structure optimization was accomplished with the program package Gaussian 09 using the hybrid DFT functional B3LYP together with Pople's 6-31(d) and 6-311 + G(2d,p) basis sets.