Antimicrobial Chlorinated 3-Phenylpropanoic Acid Derivatives from the Red Sea Marine Actinomycete Streptomyces coelicolor LY001

The actinomycete strain Streptomyces coelicolor LY001 was purified from the sponge Callyspongia siphonella. Fractionation of the antimicrobial extract of the culture of the actinomycete afforded three new natural chlorinated derivatives of 3-phenylpropanoic acid, 3-(3,5-dichloro-4-hydroxyphenyl)propanoic acid (1), 3-(3,5-dichloro-4-hydroxyphenyl)propanoic acid methyl ester (2), and 3-(3-chloro-4-hydroxyphenyl)propanoic acid (3), together with 3-phenylpropanoic acid (4), E-cinnamic acid (5), and the diketopiperazine alkaloids cyclo(l-Phe-trans-4-OH-l-Pro) (6) and cyclo(l-Phe-cis-4-OH-d-Pro) (7) were isolated. Interpretation of nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESIMS) data of 1–7 supported their assignments. Compounds 1–3 are first candidates of the natural chlorinated phenylpropanoic acid derivatives. The production of the chlorinated derivatives of 3-phenylpropionic acid (1–3) by S. coelicolor provides insight into the biosynthetic capabilities of the marine-derived actinomycetes. Compounds 1–3 demonstrated significant and selective activities towards Escherichia. coli and Staphylococcus aureus, while Candida albicans displayed more sensitivity towards compounds 6 and 7, suggesting a selectivity effect of these compounds against C. albicans.


Introduction
The marine actinomycetes represent a vital source of biologically active secondary metabolites and a promising future source for drug discovery. It is well known that marine tunicates and sponges are highly associated with symbiotic microbes [1][2][3]. There are very few reports about investigation of Red Sea actinomycetes for their chemical diversity and biomedical importance [4].
Streptomycetes represent a group within the actinomycetes with an economical importance and represent a vigorous source of different bioactive secondary metabolites [5]. More than 75% of the reported sequences in the GenBank showed that the LY001 isolate belongs to the genus Streptomyces and displayed 100% similarity with the strain Streptomyces coelicolor AB588124.
Further, the assignment of the substituents on the aromatic moiety was supported by HMBC The structure of 2 was assigned by interpretation of its NMR spectra. The NMR data of 2 are identical with those of 1 (Table 1), suggesting similar structures. Furthermore, the existence of an additional three-proton singlet at δ H 3.67 correlated to the signal at δ C 51.9 (CH 3 , C-10) in the HSQC, which supports the existence of a terminal methoxyl group in 2. The HMBC from H 3 -10 (δ H 3.67) to C-9 (δ C 173.3) supported the existence of a methyl ester group in 2 instead of a free carboxylic acid in 1.
Interpretation of the NMR (Figures S19-S23) and MS spectra of 4 ( Figure 2) supported its structure. The NMR are identical with those reported for 3-phenylpropanoic acid from a terrestrial Streptomyces strain [37]. Accordingly, 4 was assigned as 3-phenylpropanoic acid and this is its first occurrence from a marine Streptomyces.
The structure of 5 ( Figure 2) was assigned, from the NMR (Figures S24 and S25) and MS spectra, as E-cinnamic acid. Its NMR spectra exhibited signals for a monosubstituted benzene ring. The J value (J 7,8 = 16.1 Hz) supported E configuration in 5.
The structures of 6 and 7 ( Figure 2) were identified from interpretation of the corresponding NMR ( Figures S26-S30 for 6 and Figures S31-S35 for 7) and MS spectra. Both compounds (Figure 2) displayed the same molecular formula of (C 14 H 16 N 2 O 3 ). The diketopiperazine nature of 6 and 7 are obvious from the corresponding 1 H and 13 C resonating signals ( Table 2). Those include the resonances of two amidic carbonyls (C-2 and C-5) as well as the resonances for the methine signals at C-3 and C-6 ( Table 2). The remaining of the signals are characteristic for the presence of a hydroxylated proline and phenylalanine moieties in 6 and 7 [38][39][40][41]. The NMR signals at δ H/C 4.60/68.4 (in 6) and δ H/C 4.40/68.1 (in 7) are characteristic for the hydroxylated C-8 supporting the hydroxylation of the proline moieties in both compounds [38][39][40][41]. The NMR data of 6 and 7 (Table 2) are similar to those of cyclo(l-Phe-trans-4-OH-l-Pro) [38,39] and cyclo(l-Phe-cis-4-OH-d-Pro) [40,41], respectively. Furthermore, the 2D experiments (HSQC, COSY, HMBC) ( Figure 3) supported the assignment of all signals of the compounds (Table 2 and Figure 3). Accordingly, compounds 6 and 7 were assigned as cyclo(l-Phe-trans-4-OH-l-Pro) and cyclo(l-Phe-cis-4-OH-d-Pro). The derivatives of 3-phenylpropanoic acid are rarely reported from microbial organisms. We believe that there is only one report about the occurrence of 3-phenylpropanoic acid from a terrestrial Streptomyces strain [37]. Further, it is worth pointing out that this is the first report about the natural existence of chlorinated derivatives of 3-phenylpropanoic acid. These results give an understanding about the biosynthetic potential and structural diversity of the cultured marine-derived microbes and the future application of these metabolites in drug discovery.
Phenylpropanoids area ubiquitous group of organic compounds including flavonoids, coumarins, phenolic acids, stilbenes, and lignins. They originate from phenylalanine and tyrosine [43]. The name phenylpropanoid is derived from a six-carbon forming a phenyl moiety connected to a three-carbon propene unit. The coumaric acid represents the key biosynthetic intermediate in the biosynthesis of all phenylpropanoids. Biosynthetically, a variety of natural products including flavonoids, phenylpropanoids, isoflavonoids, catechins, coumarins, stilbenes, aurones, and lignols originates from 4-coumaroyl-CoA [44], which is originated from cinnamic acid [44].
Three pathogens were used to determine the antimicrobial effects and the minimum inhibitory concentration (MIC) values of 1-7. Compound 1 showed the greatest inhibitory effects towards S. aureus and E. coli with inhibition zones of 17 and 23 mm (Table 3). In addition, compounds 2 and 3 were less active than 1 against these pathogens with inhibition zones of 12-20 mm (Table 3). Compound 4 displayed the lowest activity against these pathogens with inhibition zones of 15 and 11 mm. These findings suggest the importance of the substitution with a "3,5-dichloro-4-hydroxy" moiety on the phenyl moiety as well as the presence of a free terminal carboxylic acid moiety for a maximum antibacterial activity (as in 1). On the other hand, 1-4 showed modest effect towards C. albicans (ATCC 14053) with 6-9 mm inhibition zone (Table 3), suggesting selective antibacterial effects of these compounds against E. coli and S. aureus. Finally, the diketopiperazine alkaloids 6 and 7 displayed better activities towards C. albicans with 12-14 mm inhibition zones, while they were less active towards E. coli and S. aureus with 7-11 mm inhibition zones, suggesting their selective antifungal activity against C. albicans. To determine the MIC values of the compounds, a microdilution method was carried out (Table 3). Compound 1 displayed the highest activity towards E. coli with an MIC of 16 µg/mL, while compounds 6 and 7 displayed the highest antifungal activities with an MIC of 32 µg/mL towards C. albicans. On the other hand, compounds 2 and 3 displayed lower activities with MIC values of 32-64 µg/mL towards E. coli and S. aureus. Other compounds were weakly active with MIC values of 64-250 µg/mL.

General Experimental Procedures
Optical rotations, ultraviolet (UV), infra-red (IR), NMR, and HRESIMS were acquired as previously reported [35,36]. Fractionation of the extracts and successive fractions were performed on SiO 2 and Sephadex LH-20. The purification of the compounds was carried out on an analytical Shim-Pack C18 (250 × 4.6 mm, Shimadzu, Kyoto, Japan).

The Host Organism, C. siphonella
The marine sponge C. siphonella was harvested in May 2016 using scuba at a depth up to 20 m off Jizan at the Saudi Red Sea. The pink tubular sponge is dichotomously divided with a smooth thin-walled surface. It possesses a soft compressible consistency, which is difficult to tear. The voucher specimen measures up to 10 cm, while the branching tubes measure up to 5 cm in height and up to 2.5 cm in diameter. A comprehensive description of the sponge and the specimen's codes are previously reported [45,46].

Isolation of the Actinomycete Streptomyces coelicolor LY001
After surface sterilization, about 1 cm 3 of the internal tissue of the sponge was finely mixed in sterile seawater (10 mL), diluted, and spread on International Streptomyces Project-2 (ISP2) medium. The medium was amended with 3% NaCl. Afterwards, cultured plates were incubated at 30 • C and checked after actinomycetes growth regularly. The actinomycete LY001 was obtained in a pure state after several purification steps.

Characterization of the Actinomycete, Streptomyces coelicolor
The LY001 strain was identified by analysis of its 16S rRNA sequence. DNA preparation was used for 16S rRNA gene PCR amplification using 27f and 1492r primers. The reaction mixture composed of 50 µL included 1000 ng of gDNA, primers (each 20 pmol), and GoTaq Master Mixes (25 µL). The polymerase chain reaction (PCR) thermocycler initiated with denaturation at 95 • C (2 min), 30 cycles at 95 • C (30 s for denaturation), 30 s at 58 • C (for annealing), 60 s at 72 • C (for extension), and at 72 • C for 5 min for final completion of DNA extension. Purification of PCR products was accomplished on Agarose Gel DNA Purification Kit (Biocompare, South San Francisco, CA, USA) as supported by the supplier. 16S rRNA sequence displayed 100% similarity with Streptomyces coelicolor (Accession No. AB588124). The sequence of the Red Sea Streptomyces coelicolor LY001 was placed in the NCBI GenBank under the Accession Number MN883509 on 30 December 2019 (http://getentry.ddbj.nig.ac.jp/).

Large-Scale Culture of Streptomyces coelicolor
Spores of Streptomyces coelicolor were cultured in 2.0 L flasks, each containing ISP2 media (500 mL) [47] including 10 g of malt extract, 4.0 g of yeast extract, 4.0 g of dextrose, and 3% NaCl (w/v) in 1 L distilled water at pH of 7. Incubation of the culture was accomplished by shaking at 180 rpm at 28 • C for 14 days. The combined culture broth (10 L) was shaken against EtOAC three times (each 3 L). The resulted EtOAc extracts dried to give 1.3 g.

Determination of the MIC of 1-7
The MIC values of the compounds was evaluated using a macrodilution method [51]. Briefly, MeOH was used to dissolve the compounds at a final concentration of 2000 µg/mL, while distilled water was used to dissolve ciprofloxacin and ketoconazole at final concentrations of 100 µg/mL. All solutions were sterilized using syringe filters (0.2 µm). A two-fold serial dilution of the solutions was used in Mueller Hinton Broth (MHB) to afford concentrations between 1.0 and 1000 µg/mL for the compounds and between 0.125 and 64 µg/mL for ciprofloxacin and ketoconazole. From the 10 6 colony-forming units (CFU)/mL microbial suspensions, 500 µL were added in sterile tubes giving inoculua of 5 × 10 5 CFU/mL. Additional 100 µL of each stock solution of the compounds and antibiotics were added into the tubes. A control tube, which contains only the test microorganisms and methanol was prepared. The MeOH displayed no antimicrobial effect. Incubation of the tubes was accomplished at 37 • C for 48 h. The lowest concentrations of the compounds/antibiotics, which show no microbial growth were considered as MIC.