Youssoufenes A2 and A3, Antibiotic Dimeric Cinnamoyl Lipids from the ΔdtlA Mutant of a Marine-Derived Streptomyces Strain

Two new dimeric cinnamoyl lipids (CL) featuring with an unusual dearomatic carbon-bridge, named youssoufenes A2 (1) and A3 (2), were isolated from the ΔdtlA mutant strain of marine-derived Streptomyces youssoufiensis OUC6819. Structures of the isolated compounds were elucidated based on extensive MS and NMR spectroscopic analyses, and their absolute configurations were determined by combination of the long-range NOE-based 1H-1H distance measurements and ECD calculations. Compounds 1 and 2 exhibited moderate growth inhibition against multi-drug-resistant Enterococcus faecalis CCARM 5172 with an MIC value of 22.2 μM.


Introduction
The ortho-substituted cinnamoyl lipids (CL) comprise a small class of secondary metabolites, which are attractive due to their broad bioactive properties, including antibacterial [1], antifungal [2], antitumor [3], antiangiogenic [4] and antituberculosis activities [5,6]. To date, only a small number of CL-containing compounds have been discovered [1,3,[7][8][9][10]. Youssoufenes are a series of cryptic compounds which were activated by disruption of aminotransferase family gene dtlA in marine-derived Streptomyces youssoufiensis OUC6819 [1]. Youssoufenes B1-B4 represent a typical ortho-substituted CL skeleton; while youssoufene A1 comprises unique dearomatic carbon-bridged CL dimers [1]. Interestingly, the antibacterial activity of youssoufene A1 against multi-drug-resistant (MDR) Enterococcus faecalis was increased 4-fold compared to its monomer [1]. It attracted our interest in the dimeric CL, potentially as a novel drug scaffold. Thus, to discover new dimeric youssoufene analogs, an LC-MS-directed isolation was conducted towards the ∆dtlA mutant strain, and two new compounds (1 and 2) were obtained ( Figure 1). Herein, we describe the isolation, structural elucidation, as well as biological evaluation of these compounds.

Results and Discussion
The ΔdtlA mutant strain, which was constructed in our previous study [1], was cultured for 50 mL, and the culture broth was extracted with EtOAc followed by HPLC-HRESIMS analysis ( Figure S1). Except for youssoufene A1, two minor peaks (m/z 563 [M + H] + ]) with similar UV-spectra were observed (Figures S1 and S2), which were proposed to be new dimeric youssoufene analogs. Then, large-scale fermentation (50 L) of the ΔdtlA mutant was conducted and afforded compounds 1 and 2.

Results and Discussion
The ∆dtlA mutant strain, which was constructed in our previous study [1], was cultured for 50 mL, and the culture broth was extracted with EtOAc followed by HPLC-HRESIMS analysis ( Figure S1). Except for youssoufene A1, two minor peaks (m/z 563 [M + H] + ]) with similar UV-spectra were observed (Figures S1 and S2), which were proposed to be new dimeric youssoufene analogs. Then, large-scale fermentation (50 L) of the ∆dtlA mutant was conducted and afforded compounds 1 and 2.
Compound 2 was isolated as a yellow amorphous solid. The molecular formula of 2 was established as C 38 H 42 O 4 on the basis of the HRESIMS data ([M + H] + at m/z 563.3167, calcd 563.3161), indicative of an isomer of youssoufene A2. Compound 2 shares similar NMR data with those of youssoufenes A1 and A2. The difference between 2 and youssoufene A1 was the additional C-3 (δ C 125.3)/C-4 (δ C 134.1) double bond in 2, which showed HMBC correlations to H-5 (δ H 3.58). Thus, compound 2 was determined to comprise a dimeric CL skeleton with dearomatic youssoufene B1 connected to youssoufene B3/serpentene unit at C-5. By combination of the NOE correlations ( Figure 3) and 3 J H,H values (Table 1), the double-bond geometries in 2 were determined to be 3-E, 12-Z, 14-E, 16-Z, 18-E, 3 -E, 5 -Z, 10 -Z, 12 -E, 14 -Z and 16 -E, respectively. The absolute configurations of C-5, C-7 and C-9 in 1 were determined by ECD calculations performed on the CAM-B3LYP/6-31G(d) level of theory with Gaussian 09. The calculated ECD curve of (5R,7 R,9 S)-2 was in good agreement with the experimental ECD data of 2 ( Figure 4). Thus, compound 2 was identified as a new dimeric CL, named youssoufene A3. The 1 H and 13 C NMR chemical shift values of 2 are listed in Table 1. Compound 2 was isolated as a yellow amorphous solid. The molecular formu was established as C38H42O4 on the basis of the HRESIMS data ([M + H] + at m/z 56 calcd 563.3161), indicative of an isomer of youssoufene A2. Compound 2 shares s NMR data with those of youssoufenes A1 and A2. The difference between 2 and soufene A1 was the additional C-3 (δC 125.3)/C-4 (δC 134.1) double bond in 2, showed HMBC correlations to H-5 (δH 3.58). Thus, compound 2 was determined t prise a dimeric CL skeleton with dearomatic youssoufene B1 connected to youss B3/serpentene unit at C-5. By combination of the NOE correlations ( Figure 3) an values (Table 1), the double-bond geometries in 2 were determined to be 3-E, 12-Z 16-Z, 18-E, 3'-E, 5'-Z, 10'-Z, 12'-E, 14'-Z and 16'-E, respectively. The absolute con tions of C-5, C-7' and C-9' in 1 were determined by ECD calculations performed CAM-B3LYP/6-31G(d) level of theory with Gaussian 09. The calculated ECD cu (5R,7'R,9'S)-2 was in good agreement with the experimental ECD data of 2 ( Fig  Thus, compound 2 was identified as a new dimeric CL, named youssoufene A3. T and 13 C NMR chemical shift values of 2 are listed in Table 1.

Mar. Drugs 2022, 20, x
In the antibacterial assay, both youssoufenes A2 (1) and A3 (2) showed growt bition against multi-drug-resistant Enterococcus faecalis CCARM 5172 with an MIC of 22.2 μM (Table S2), but not active against Staphylococcus aureus CCARM 3090 or richia coli CCARM 1009. These results were comparable to that of youssoufene A1, displayed over 4-fold-increased activity compared to youssoufenes B1-B4 [1]. This demonstrated that the dimeric CL skeleton endows the youssoufene A-type with n In the antibacterial assay, both youssoufenes A2 (1) and A3 (2) showed growth inhibition against multi-drug-resistant Enterococcus faecalis CCARM 5172 with an MIC value of 22.2 µM (Table S2), but not active against Staphylococcus aureus CCARM 3090 or Escherichia coli CCARM 1009. These results were comparable to that of youssoufene A1, which displayed over 4-fold-increased activity compared to youssoufenes B1-B4 [1]. This result demonstrated that the dimeric CL skeleton endows the youssoufene A-type with notably enhanced antibacterial activities compared to their monomeric B-type structures. While we have demonstrated that monomeric youssoufenes B1-B4 are assembled via an unusual type II polyketide synthetase pathway [11], the diaromatic dimerization to afford youssoufene A-type structures remains unclear.

General Experimental Procedures
Optical rotations were recorded with a JASCO P-1020 digital polarimeter (JASCO, Tokyo, Japan). UV spectra in MeOH were recorded on a PerkinElmer Lambda 35 (PerkinElmer, Waltham, MA, USA). Experimental ECD spectra in MeOH were recorded on a JASCO J-815 spectrometer (JASCO, Tokyo, Japan). IR spectra were measured on a Nicolet NEXUE470 FTIR (Thermo, Waltham, MA, USA). Then, 1D ( 1 H and 13 C) and 2D (COSY, HSQC, HMBC and NOESY) NMR spectra were recorded on a Bruker Avance III 600 spectrometer (Bruker, Billerica, MA, USA). Chemical shifts were reported with reference to the respective solvent peaks and residual solvent peaks (δ H 3.31 and δ C 49.0 for CD 3 OD; δ H 2.50 and δ C 39.5 for DMSO-d 6 ). LC-MS experiments were performed on Agilent 1260 HPLC (Agilent, Santa Clara, CA, USA) system coupled with a Q-TOF Ultima Global GAA076 mass spectrometer (Waters, Milford, MA, USA). Preparative HPLC was performed on a Hitachi Chromaster System (Hitachi, Tokyo, Japan).

Fermentation, Extraction and Isolation of the Compounds
A total of 50 L of fermentation culture of the ∆dtlA mutant strain were prepared and extracted with EtOAc. The EtOAc extract (8.5 g) was partitioned between 90% MeOH and n-hexane to yield two residues. The aqueous MeOH layer (7.1 g) was applied to a reversed-phase (C18) open column (100 × 30 mm) chromatography to give 13 fractions (Fr.1-13) by eluting with gradient from 20% to 100% MeOH. The Fr. 9 (50.4 mg) was further subjected to semipreparative HPLC using a YMC ODS-A column (250 × 10 mm, 5 µm) eluting with 70% ACN to afford compounds 1 (1.1 mg, t R 51.9 min) and 2 (1.2 mg, t R 55.1 min).

Computational Methods
Conformational searches were run by the "Random search" procedure implemented in the SYBYL-X 2.0 program (Certara, Princeton, NJ, USA) using the Molecular Merck force field (MMFF94). Among the generated conformers of each compound, the conformers that well supported the NOESY data were selected and were subjected to geometry optimization with DFT calculations at the TZVP/6-31G(d) level using the Gaussian 09 program (Gaussian, Inc., Pittsburgh, PA, USA). The TD calculations were performed on each optimized conformer using the long-range-corrected hybrid CAM-B3LYP. The number of excited states per molecule was 50. Solvent effects were considered by using the polarizable continuum model (PCM) for MeOH. The ECD spectra were generated by the program GaussView 5.0 (Gaussian, Inc., Pittsburgh, PA, USA).

Antibacterial Activity Assay
The antibacterial activity of compounds 1 and 2 was evaluated using the MIC (minimum inhibitory concentration) assay. The multi-drug-resistant Enterococcus faecalis CCARM 5172, Staphylococcus aureus CCARM 3090 and Escherichia coli CCARM 1009 strains were purchased from Culture Collection of Antimicrobial Resistant Microbes (Seoul Women's University, Seoul, Korea). The strain was grown overnight at 37 • C in LB medium and diluted with LB broth to 10 6 cfu/mL. Then, 10 µL of the compound solutions with different concentrations in MeOH were dispensed into 190 µL of the cell suspension in the 96-well plates. LB broth was used as a blank. MeOH was used as a negative control; ciprofloxacin and tetracycline were used as positive controls. The bacterial growth was measured after 18 h of incubation at 37 • C on a microplate reader at a wavelength of 600 nm. Each assay was performed in triplicate.

Conclusions
In summary, two new dimeric cinnamoyl lipids youssoufenes A2 (1) and A3 (2), which feature with a unique dearomatic carbon-bridge, were isolated from the ∆dtlA mutant strain of marine-derived S. youssoufiensis OUC6819. Compounds 1 and 2 exhibited growth inhibition against multi-drug-resistant E. faecalis CCARM 5172 (MIC = 22.2 µM), which was comparable to the positive controls, and meanwhile, notably higher than its monomeric form. These results indicated that yousoufene A-type could be an interesting new chemical scaffold for the development of next-generation antibacterial drugs.
Author Contributions: J.L. contributed to fermentation, compound isolation, antibacterial activity assay, and manuscript preparation; H.L. contributed to structure elucidation, manuscript writing and editing, and project supervision; Z.L. involved in ECD calculation; T.L. involved in construction of the mutant strain; F.X. involved in manuscript review; W.L. contributed to manuscript review and editing, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.