Nocaviogua A and B: two lipolanthines from root-nodule-associated Nocardia sp.

Nocaviogua A (1) and B (2), two lipolanthines featuring a non-canonical avionin (Avi)-containing macrocycle and a long acyl chain, were identified from the mutualistic actinomycete Nocardia sp. XZ19_369, which was isolated from the nodules of sea buckthorn collected in Tibet. Their planar structures were elucidated via extensive analyses of 1D and 2D NMR, as well as HRMS data. The absolute configurations were fully elucidated by advanced Marfey’s analysis and GIAO NMR calculations, representing the first time that the configurations of this family of lipolanthines have been determined. Nocaviogua A (1) exhibited weak cytotoxicity against human chronic uveal melanoma cells (UM92-1), non-small cell lung cancer (NCI-H2170), and breast cancer (MDA-MB-231). Our work provides valuable information on this burgeoning class of lipolanthines for further investigations.


Introduction
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products that give rise to a potent biological activity and desirable drug-like properties, such as resistance to chemical and enzymatic degradation (Watrous et al., 2012;Montalban-Lopez et al., 2021;Mazo et al., 2023), conformational rigidity (Bobeica et al., 2020), and high target specificity (Ongey et al., 2017;Grant-Mackie et al., 2021). The structural diversity of RiPPs is multiplied by fusions with polyketides or fatty acids (Kozakai et al., 2020). Among them, lipolanthines are a class of polyketide/fatty acid-ribosomally synthesized hybrid lipopeptides (PK/FA-RiPPs) that feature a unique avionin (Avi)containing macrocycle and a long acyl chain (Wiebach et al., 2018;Kozakai et al., 2020). Due to the utilization of class-III-type lanthipeptide synthetases in biosynthesis, these unusual natural lipopeptides were also classified as class-Ⅲ lanthipeptides (Grant-Mackie et al., 2021).
Since the first lipolanthine was reported in 2018 (Wiebach et al., 2018), only 12 lipolanthines, belonging to three groups (microvionin, nocavionin, and goadvionins), have been discovered, and none of their absolute configurations have been fully determined. As an emerging class of natural products, some of these lipolanthines have shown promising antibacterial effects against antibiotic-resistant bacteria, e.g., microvionin has exhibited strong antibacterial effects with MIC values of less than 0.46 μg/mL against MRSA and less than 0.15 μg/mL against Streptococcus pneumoniae (Wiebach et al., 2018); goadvionins have also been shown to inhibit the growth of Gram-positive bacteria, with goadvionin B2 displaying the MIC values of 6.4 μg/mL against Staphylococcus aureus, 3.2 μg/mL against B. subtilis, and 3.2 μg/mL against Micrococcus luteus (Kozakai et al., 2020). Despite their potential, the low yield and difficulties in synthesis have hindered their pharmaceutical commercialization (Mazo et al., 2023).
In our continuing efforts to explore novel bioactive metabolites from unique habitat-derived microbes Luo et al., 2022a;Chang et al., 2022b;Luo et al., 2022b), two lipolanthines (1 and 2) were produced by Nocardia sp. XZ19_369, which was isolated from the nodules of sea buckthorn collected in Tibet, China. The whole structures of 1 and 2 were fully elucidated by the extensive analysis of 1D and 2D NMR data, advanced Marfey's method, and NMR calculations. These compounds represent the first lipolanthines with determined absolute configurations. Herein, the isolation, structural elucidation, and bioactivities of compounds 1 and 2 are described.

Experimental section 2.1 General experimental procedures
The optical rotations were obtained using a JASCO J-815 spectrometer (JASCO International Co. Ltd., Tokyo, Japan). 1D and 2D NMR spectra were measured using a Bruker AVⅢHD spectrometer (Bruker Co. Ltd., Bremen, Germany) at 600 MHz for 1 H and 150 MHz for 13 C in DMSO-d 6 (δ H 2.49 and δ C 34.9). High-resolution mass data and advanced Marfey's analysis were measured using a Waters Xevo G2-XS QTof mass spectrometer (Waters Co., Manchester, United Kingdom). Column chromatography was performed on macroporous adsorption resins (Diaion 4006; Nankai Chemical Co. Ltd., China) and the CombiFlash ® Rf system fitted with an ODS flash column (RediSep Rf C 18 flash column, 130 g). Semi-preparative HPLC was performed using an Agilent 1200 series liquid chromatography system (quaternary pump, autosampler, and diode array detector) using a Reprosil-Pur Basic-C 18 column (5 μm, 250 × 10 mm).

Bacterial materials
Nocardia sp. XZ19_369 was isolated from the nodules of sea buckthorn collected from Tibet, China. Their draft genomes were sequenced on an Illumina HiSeq platform (Illumina, San Diego, CA, United States), assembled using SPAdes V3.13.1, and submitted to the National Center for Biotechnology Information (NCBI) database (accession no. JACVED000000000 for XZ19_369). The phylogenetic tree (Supplementary Figure S1) was constructed using autoMLST (Alanjary et al., 2019).

Fermentation
Nocardia sp. XZ19_369 grown on the ISP2 medium (0.4% glucose, 0.4% yeast extract, 1% malt extract, and 2% agar) was inoculated into 100 mL of the ISP2 liquid medium contained in a 500-mL Erlenmeyer flask and cultured at 28°C for 2 days as the seed culture. For large-scale fermentation, 50 mL of the seed culture was inoculated into 10 Erlenmeyer flasks (5 L), each containing 1 L of the sterile YMS medium (0.4% yeast extract, 1% malt extract and 0.4% starch soluble), and then cultivated at 28°C for 10 days.

Advanced Marfey's method
Each compound 1-2 (50 μg) was dissolved in a 100 μL of 6 N HCl and heated at 110°C for 24 h. After heating, the hydrolysates were divided into two parts and dried under the N 2 flow. The dried hydrolysates and each standard amino acid (L-Ala, L-Val, L-Ser, and L-Asp) were dissolved in 30 μL of the 0.1 M NaHCO 3 solution. To each reaction vial, 30 μL of L-FDAA (N α -(2,4-dinitro-5fluorophenyl)-L-alaninamide, 1% solution in acetone) was added and heated at 40°C for 1 h (Kiyonaga Fujii, 1997a;Kiyonaga Fujii, 1997b). After cooling at room temperature, 30 μL of 0.1 M HCl was added to each vial. The mixtures were diluted with 500 μL of MeOH.
Frontiers in Chemistry frontiersin.org

NMR calculations
In order to simplify the calculation, the bismethylated guanidino fatty acid (MGFA) was replaced with acetic acid, which was not impacted with the accuracy of the result (Daranas and Sarotti, 2021). Conformational analysis was performed using OpenBabel (O'Boyle et al., 2011) with a genetic algorithm at the MMFF94 force field, with energies within the 3.0 kcal/mol energy threshold (Spartan14. Wavefunction, 2019). The conformers were optimized using Gaussian 16 (Gaussian Inc.) (M. J. Frisch, 2010) at the M062X/6-311+G (d, p) level in vacuum. At the same level, the frequencies were calculated to provide the relative thermal free energies (ΔG), which are used to calculate the equilibrium populations. NMR chemical shift calculations for those optimized conformers within their Boltzmann distribution (>1%) were performed using the GIAO method at the mPW1PW91/6-311+G (d, p) level in DMSO with the PCM model. The calculated chemical shifts of conformers for 1-2 were averaged in terms of their relative Gibbs free energy and the  (Grimblat et al., 2015).
The remaining two configurations in Avi 2 and Avi 5 are proving to be a challenge to resolve. However, unlike the NMR spectra of nocavionin and microvionin in D 2 O and H 2 O, respectively, which showed the presence of conformer isomers (Wiebach et al., 2018), the NMR spectra of nocaviogua A (1) in DMSO-d 6 did not exhibit any obvious signals indicating different conformers. This allows us to use the GIAO NMR calculation to determine their stereochemistry. In order to solve the structure of complex, large, and highly flexible molecules using readily available computational resources, the MGFA was replaced with acetic acid and the four possible diastereomers 1a-1d ( Figure 2) were evaluated. Conformational searches were performed at OpenBabel with the MMFF94 force field and an energy cutoff value of 3.00 kcal/mol. Subsequently, the obtained conformers were optimized by the DFT method at the M062X/6-311+G (d, p) level in vacuum, and the NMR chemical shifts were calculated at the PCM/ mPW1PW91/6-311+G (d, p) level. Accordingly, the calculated carbon chemical shifts for 1a showed the highest similarity toward the experimental values with the smallest corrected mean absolute error (CMAE, 1a: 1.5113 vs. 1b-1d: 2.0405, 1.9820, and 1.8977 in 13 C NMR data and 1a: 0.1397 vs. 1b-1d: 0.2068, 0.1813, and 0.2073 in 1 H NMR data, respectively) values (Supplementary Tables S2-S3), particularly for Avi 2 and Avi 5 moieties (Figure 3). In addition, DP4+ analysis based on NMR data provided 100% probability for the isomer 1a (Supplementary Table S4), indicative of the S, S configuration for Avi 2 and Avi 5 moieties in 1. Thus, the whole structure of    (Table 1) for 2 that were recorded in DMSO-d 6 revealed nine amide and/or ester carbonyl carbons (δ C 165.5-172.7) and nine alkenyl carbons (δ C 99.3-158.3 and δ H 7.19-5.38), accounting for 14 DBEs and requiring only one ring incorporation into 2. The 1 H and 13 C NMR (DMSO-d 6 ) data for 2 were closely similar to those with 1 except for three signals of NH (Asn 6 -4-NH 2 : δ H 7.33 and 6.89 and Ser 4 -NH 2 : δ H 8.11) in 2, which is more than 1, indicating that the amide bond between Val 3 and Ser 4 was opened, and the Asp unit in 1 was replaced with the Asn residue in 2. This hypothesis was further confirmed by HMBC and 1 H-1 H COSY correlations (Figure 1). Based on the coupling constant and the ROESY correlation, the geometry of the double bond in the Avi 8 moiety was established as Z and the alkenyls in MGFA were all in the E arrangement, which were the same as in 1. Considering the number of nitrogen atoms and hydrogen atoms, the MGFA unit in 2 was also with the positively charged functional group as in 1.
The absolute configurations of Ala, Val, Ser, and Asn moieties were determined as L by the advanced Marfey's method (Supplementary Figures S11-S14; Supplementary Table S1). The configurations in Avi 2 and Avi 5 units of compound 2 were also determined using GIAO NMR calculations by evaluating the four possible diastereomers 2a-2d (Supplementary Figure S27) Tables S13-S14). In addition, DP4+ analysis based on NMR data with 100% probability for the isomer 2a (Supplementary  Table S15), assigned Avi 2 and Avi 5 units as S, S configurations, respectively, are identical to those determined in compound 1. Thus, the whole structure of compound 2 was fully determined and designated as nocaviogua B (2).

Conclusion
Lipolanthines, featuring unique structures and physicochemical properties, are increasingly attracting wide interests as pharmaceutical leads (Grant-Mackie et al., 2021). In our research, two new lipolanthines, nocaviogua A (1) and B (2), were identified from mutualistic actinomycete Nocardia sp. XZ19_369 that was isolated from the nodules of sea buckthorn collected in Tibet. Their planar structures were elucidated via extensive 1D and 2D NMR, and HRMS data. The absolute configurations were fully elucidated by advanced Marfey's analysis and GIAO NMR calculations.
Structurally, compounds 1 and 2 are analogs of microvionin, nocavionin, and goadvionins. Unlike their analogs, compounds 1 and 2 did not exhibit any antimicrobial activity against the seven tested pathogens at concentrations up to 128 μg/mL. However, compound 1 did show weak cytotoxicity against cancer cell lines UM92-1, NCI-H2170, and MDA-MB-231, with inhibition rates of approximately 15%, 30.0%, and 31.1%, respectively, at a concentration of 50 μM. On the other hand, compound 2, the one-ring-opened derivative of compound 1, did not show any obvious cytotoxic activity, suggesting that the bicycle scaffold in lipolanthines plays a crucial role in their cytotoxicity. To date, 12 lipolanthines have been discovered, but nocaviogua A (1) and B (2) were the first compounds in this family with fully determined configurations, which is of great importance for further stereoselective synthesis and bioactive studies.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and