Production of a New Cyclic Depsipeptide by the Culture Broth of Staphylococcus sp. Isolated from Corallina officinalis L.

A new cyclic depsipeptide (1) has been isolated from culture broth of Staphylococcus sp. (No. P-100826-4-6) derived from Corallina officinalis L., together with the known compounds indol-3-carboxylic acid (2), 1,5-dideoxy-3-C-methyl arabinitol (3), thymine (4), uracil (5), cyclo (L-pro-L-omet) (6) and macrolactin B (7). The structure of (1) was established to be cyclo (2α, 3-diaminopropoinc acid-L-Asn-3-β-hydroxy-5-methyl-tetradecanoic acid-L-Leu1-L-Asp-L-Val-L-Leu2-L-Leu3) by extensive spectroscopic techniques including 1H NMR, 13C NMR, 1H‒1H COSY, HMBC, HSQC, NOESY, and HRFABMS. The antimicrobial activities of compounds 1–7 were evaluated. Compounds 1–5, and 7 showed moderate antimicrobial activity while compound 6 exhibited a potent antimicrobial and antifungal activities.


Introduction
Bioprospecting studies of endophytic microorganisms play a principal part in the discovery of lead compounds for the improvement of drugs for the management of humanity's diseases [1][2][3]. Marine microorganisms are widely recognized as promising sources of secondary metabolites [2,[4][5][6]. These organisms, prosperous in diverse marine environments, have produced a wide variety of structurally exclusive and biologically active compounds that have attracted significant interest for biomedical researches [4,[7][8][9].
In the last decade, a significantly increased interest in the isolation of bioactive secondary metabolites from marine microbes has been reported [10]. Peptides, which were derived from different organisms including marine microbes, represent an important chemical class with diverse structures and significant biological activities. The biological activity of marine-derived peptides has been shown to depend on the composition and sequence of amino acids, their structural properties, as well as on the environmental habitat for producer bacteria [11,12]. These substances are actively synthesized by marine microorganisms during their life cycle [13]. To date, lots of peptide metabolites, which are consisting of 20-40 amino acids, have been separated from various marine microorganisms [11,12,14]. Most of them are capable of quick inhibition or killing of a wide range of microbes. Other antimicrobial peptides (proteins consisting of 100 or more amino acids) disrupt the function or the structure of microbial cell membranes by binding to specific targets [15,16].

Isolation Method of Compounds 1-7
The fermented broth of the marine bacterium Staphylococcus sp. No. [P-100826-4-6] was extracted with different organic solvents. Successive fractionation of the combined extracts was done using silica gel column chromatography based on increasing polarity, Sephadex LH-20, Diaion HP-20, reversed-phase C18 silica gel column; subsequently, final purification on a C18 RP-HPLC column gave seven compounds 1-7 ( Figure 1).
Metabolites 2019, 9, x 2 of 11 organisms including marine microbes, represent an important chemical class with diverse structures and significant biological activities. The biological activity of marine-derived peptides has been shown to depend on the composition and sequence of amino acids, their structural properties, as well as on the environmental habitat for producer bacteria [11,12]. These substances are actively synthesized by marine microorganisms during their life cycle [13]. To date, lots of peptide metabolites, which are consisting of 20-40 amino acids, have been separated from various marine microorganisms [11,12,14]. Most of them are capable of quick inhibition or killing of a wide range of microbes. Other antimicrobial peptides (proteins consisting of 100 or more amino acids) disrupt the function or the structure of microbial cell membranes by binding to specific targets [15,16]. In our pursuit of isolation of natural compounds from marine sources [17][18][19][20], chemical investigation for the antimicrobial extract of Staphylococcus sp. No. [P-100826-4-6] derived, was carried out. This study led to discovery of a new natural cyclic depsipeptide (1) along with six known compounds; indol-3carboxylic acid (2) [21], 1,5-dideoxy-3-C-methyl arabinitol (3) [22], thymine (4) [21], uracil (5) [23], cyclo (L-pro-L-omet) (6) [24], and macrolactin B (7) [25] (Figure 1). Therefore, structure identification of the isolated pure compounds 1-7 and their antimicrobial activity will be discussed.

Isolation Method of Compounds 1-7
The fermented broth of the marine bacterium Staphylococcus sp. No. [P-100826-4-6] was extracted with different organic solvents. Successive fractionation of the combined extracts was done using silica gel column chromatography based on increasing polarity, Sephadex LH-20, Diaion HP-20, reversedphase C18 silica gel column; subsequently, final purification on a C18 RP-HPLC column gave seven compounds 1-7 ( Figure 1).

Biological Activities of the Pure Compounds 1-7
The antimicrobial activities (Table 3) of compounds 1-7 were examined for their growth inhibition of 6 microorganisms including Gram-negative, Gram-positive bacteria and fungi using paper disk method

Biological Activities of the Pure Compounds 1-7
The antimicrobial activities (Table 3) of compounds 1-7 were examined for their growth inhibition of 6 microorganisms including Gram-negative, Gram-positive bacteria and fungi using paper disk method with replication (n = 2). The antimicrobial activities were studied in a concentration of 100 µg/disk. As a result, compounds 1-5, and 7 showed moderate activity against Schizophyllum commune, Staphylococcus aureus subsp. aureus, and Escherichia coli, with inhibition zones between 9 and 13, while compound 6 exhibited a significant antifungal activity against Aspergillus niger, Penicillum crustosum, and Schizophyllum commune, with inhibition zones of 16, 18 and 23. Furthermore, compound 6 showed antibacterial activity against Staphylococcus aureus subsp. aureus, Pseudomonas aeruginosa and Escherichia coli, with inhibition zones of 20, 25 and 21 as shown in Table 3. a positive antibacterial control (50 µg/disc); b positive antifungal control (100 µg/disc).
The minimum inhibitory concentration (MIC) of compound 6 were evaluated. As a result, compound 6 exhibited a potent antifungal activity against Aspergillus niger, Penicillum crustosum, and Schizophyllum commune with MIC value of 50 µg/mL. Additionally, compound 6 showed antibacterial activity against Staphylococcus aureus subsp. aureus, Pseudomonas aeruginosa and Escherichia coli, with MIC value of 100 µg/mL.

Configuration of Amino Acids
Hydrolysis of 1.5 mg of compound 1 was achieved using 1 mL of 6 N HCl for 16 h at 110 • C. Concentration for the resulting hydrolysate was followed by complete dryness under a vacuum to afford a residue. Dissolution of the solid residue was achieved in 50 µL of pure H 2 O and 40 µL of 1 M NaHCO 3 aq. and 100 µL of 1% of (2S)-2-(5-fluoro-2,4-dinitroanilino)-4-methylpentanamide (FDLA) dissolved in acetone. Heating of the formed mixture was performed for 1 h at 37 • C, followed by adding 20 µL of 1 N HCl. A yellow solid was formed after complete drying of the previously prepared solution. The resulting solid was dissolved in 40% MeCN:H 2 O (500 µL) and co-injected with standard d-and l-amino acids using RP-HPLC (UV detector: 340 nm, flow rate: 1 mL/min, mobile phase: 40% MeCN:H 2 O).

Antimicrobial Activity of Compounds 1-7
Antimicrobial activities of the pure compounds were checked using paper disk methods [28,29] against Staphylococcus aureus subsp. aureus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, Schizophyllum commune, and Penicillium crustosum, with concentration 100 and 50 µg/disk. Determination of the MIC for compound 6 was achieved by using tube-dilution method [30].