Glycosylated and Succinylated Macrocyclic Lactones with Amyloid-β-Aggregation-Regulating Activity from a Marine Bacillus sp.

Abstract

Two new glycosylated and succinylated macrocyclic lactones, succinyl glyco-oxydifficidin (1) and succinyl macrolactin O (2), were isolated from a Bacillus strain collected from an intertidal mudflat on Anmyeon Island in Korea. The planar structures of 1 and 2 were proposed using mass spectrometric analysis and NMR spectroscopic data. The absolute configurations of 1 and 2 were determined by optical rotation, J-based configuration analysis, chemical derivatizations, including the modified Mosher’s method, and quantum-mechanics-based calculation. Biological evaluation of 1 and 2 revealed that succinyl glyco-oxydifficidin (1) inhibited/dissociated amyloid β (Aβ) aggregation, whereas succinyl macrolactin O (2) inhibited Aβ aggregation, indicating their therapeutic potential for disassembling and removing Aβ aggregation.

1. Introduction

Glycosylation is an important biological process in enhancing the structural and biological diversity of metabolites [1,2]. In many cases, the biological function of natural products is altered after glycosylation; glycosylation is thus a frequently adopted route to the functional modification of molecules in nature [3]. For example, a study on the effects of glycosylation on the biological activity of the microbial immunosuppressive drug rapamycin revealed that glycosylation improved water solubility and reduced cytotoxicity depending on its positions [4]. A chemical investigation of Bacillus sp. reported glycosylated macrolactins, which displayed inhibitory activity against Staphylococcus aureus peptide deformylase along with antibacterial activity differing from that of previously reported aglycone macrolactins, indicating that the discovery of glycosylated natural products would lead to the diversification of their biological functions [5].

Marine bacteria are also fruitful sources of structurally and biologically diverse natural product discovery [6]. Since the 21st century, more and more drug candidates were discovered from Gram-positive bacteria such as Streptomyces sp., Bacillus sp. and so on [7]. Our chemical studies on marine bacteria discovered pulvomycins B–D, new macrolides incorporating sugar, and pulvomycin D showed potent cytotoxic effects against cancer cell lines [8]. Suncheonosides A–D, hexasubstituted benzothioate glycosides, were reported to be promotors of adiponectin production from a marine sediment-derived Streptomyces sp. [9]. Our recent genomic and spectroscopic analysis of a marine sand-beach-derived Streptomyces strain resulted in the discovery of a jejucarboside bearing an unusual amino sugar [10]. Bacillus in marine habitats has also been a chemically prolific bacterial clade since new antiviral and cytotoxic macrolides, macrolactins, were reported from deep-sea Bacillus strain [11]. Continuous chemical investigation led to discover antimicrobial glycopeptides, ieodoglucomides, from marine B. licheniformis [12], algicidal thiazole-bearing compounds, bacillamides, from marine Bacillus sp. [13], disulfide-bearing antimicrobial lipoamides from marine B. pumilus [14], and antifungal basiliskamide from tropical marine B. laterosporus [15], respectively.

In our continuous research, we have chemically profiled a marine strain of Bacillus sp. AMD05, which was isolated from an intertidal mudflat in Anmyeondo, Republic of Korea, and discovered two new glycosylated and succinylated macrolides, named succinyl glyco-oxydifficidin (1) and succinyl macrolactin O (2). Combinational analysis of mass, NMR, and UV spectroscopic data enabled us to elucidate the structures of 1 and 2. We further applied chiral derivatization, the modified Mosher’s method, and quantum mechanics-based DP4 probability calculation to determine the absolute configuration of succinyl glyco-oxydifficidin (1), which had previously been unknown. In this study, we report the structure elucidation of 1 and 2 and their biological evaluation in Alzheimer’s-disease-related assays.

2. Results and Discussion

2.1. Structure Elucidation

Succinyl glyco-oxydifficidin (1) was isolated as a yellow powder. The molecular formula of 1 was deduced as C₄₁H₅₈O₁₂ based on its HRESIMS data. Its molecular formula revealed 13 degrees of unsaturation. Its UV spectrum (λ_max: 234, 274, and 284 nm) indicated the existence of a diene and a triene chromophore in 1 [16]. All the ¹H–¹³C one-bond correlations in succinyl glyco-oxydifficidin (1) were assigned by combined analysis of ¹H, ¹³C, and HSQC NMR spectra. Eleven sp² methine protons (δ_H 6.584–4.969), four sp² methylene protons, one proton (δ_H 4.285) bound to a dioxygenated carbon (δ_C 105.91), seven carbinol methine protons (δ_H 4.802, 4.680, 3.693, 3.420, 3.315, 3.267, and 3.188), and two oxygenated methylene protons (δ_H 4.405 and 4.220) were identified. One methine proton (δ_H 2.369), eighteen protons belonging to nine methylene groups (δ_H 3.444, 3.059, 2.732, 2.601, 2.596 (2H), 2.546 (2H), 2.512, 2.403, 2.311, 2.102 (2H), 1.945, 1.817, 1.803, 1.739, 1.703), and three methyl groups (δ_H 1.802 (3H), 1.779 (3H), 0.979 (3H)) were also assigned in the aliphatic region. However, four hydroxy protons and one carboxyl acid proton were not observed in CD₃OD. The ¹³C NMR data revealed three ester or carboxylic acid group carbons (δ_C 177.20, 174.37, and 174.03), sixteen double-bond carbons (δ_C 148.67–113.99), one dioxygenated carbon (δ_C 105.91), eight mono-oxygenated carbons, including seven oxygenated methine carbons (δ_C 86.94–68.14), and one oxygenated methylene carbon (δ_C 64.93), one aliphatic methine carbon (δ_C 41.19), nine aliphatic methylene carbons (δ_C 47.03, 36.75, 34.10, 33.45, 32.29, 32.03, 30.90, 30.64, and 29.18), and three methyl carbons (δ_C 17.76, 17.40, and 16.62) in the structure of 1 (Figure 1).

Via analyzing the COSY NMR data of 1, several spin systems were identified. The spin system from C-4 (δ_C 41.19) to C-15 (δ_C 68.14), including the C-39 methyl group (δ_C 17.40) at C-4, was straightforwardly assembled by a series of COSY correlations from H₃-39 to H-15. The second spin system of C-17 (δ_C 123.89) to C-23 (δ_C 36.75) was revealed by consecutive ¹H-¹H couplings from H-17 (δ_H 6.232) to H₂-23 (δ_H 2.102 (2H)) through H-18 (δ_H 6.208), H-19 (δ_H 5.257), H₂-20 (δ_H 2.601 and 2.403), H-21 (δ_H 4.802), and H₂-22 (δ_H 1.817 and 1.703). A three-carbon connection in the tail of the carbon backbone, C-25 (δ_C 127.37)–C-26 (δ_C 134.48)–C-27 (δ_C 115.53), was also elucidated using the COSY correlations of H-25 (δ_H 5.861), H-26 (δ_H 6.584), and H₂-27 (δ_H 5.071 and 4.969). Another spin system of the hexose was connected based on the ¹H-¹H couplings from H-28 (δ_H 4.285) to H₂-33 (δ_H 4.405 and 4.260) via H-29 (δ_H 3.188), H-30 (δ_H 3.315), H-31 (δ_H 3.267), and H-32 (δ_H 3.420). C-35 (δ_C 30.90) and C-36 (δ_C 30.64) were linked by the H₂-35 (δ_H 2.546 (2H))/H₂-36 (δ_H 2.596 (2H)) COSY correlation (Figure 2).

These partial structures were assembled together by interpretation of HMBC NMR data (Figure 2). The ²J_CH and ³J_CH HMBC correlations from H-4 (δ_H 2.369), H₃-39 (δ_H 0.979), and H₂-2 (δ_H 3.059 and 3.444) to C-3 (δ_C 148.67) constructed the C-2–C-3–C-4 linkage, while the HMBC correlation of olefinic protons, H₂-38a (δ_H 5.026) and H₂-38b (δ_H 5.046), to C-3 (δ_C 148.67) connected the olefinic methylene to C-3. HMBC correlations from H-15 (δ_H 4.680) and H-17 (δ_H 6.232) to C-16 (δ_C 140.85) constructed the C-15–C-16–C-17 connectivity, leading to the two spin systems from C-2 to C-23 merging together. A methyl group was attached to the olefinic C-16 by an HMBC correlation from H₃-40 (δ_H 1.802) to C-16 (δ_C 140.83). A four-olefinic-carbon tail was connected to C-23 by HMBC correlations from H-23 (δ_H 2.102) and H-26 (δ_H 6.582) to C-24 (δ_C 139.27). The HMBC correlation of the methyl protons H₃-41 (δ_H 1.779) to C-24 (δ_C 139.27) revealed the connection between C-41 and C-24. The macrocyclic lactone skeleton was constructed by the HMBC correlations of H₂-2 (δ_H 3.059 and 3.444) and H-21 (δ_H 4.802) to C-1 (δ_C 174.03). Furthermore, the glycosyl group was assigned to the macrocycle by the H-5 (δ_H 3.693)/C-28 (δ_C 105.91) HMBC correlation. The succinate moiety, which was revealed by the HMBC correlations of H₂-35 (δ_H 2.546 (2H)) and H₂-36 (δ_H 2.596 (2H)) to C-34 (δ_C 174.37) and C-37 (δ_C 177.20), was linked to the sugar moiety by the HMBC correlation of H₂-33 (δ_H 4.405 and 4.220) to C-34 (δ_C 174.37). Therefore, the planar structure of succinyl glyco-oxydifficidin (1) was determined to be a new succinyl glycosyl macrolactone, as shown in Figure 1.

The double-bond geometry configurations of 1 were determined by ¹H-¹H coupling constants and ROESY correlations. The ³J_H7H8 value (11.0 Hz) determined the 7Z configuration, which was also supported by the H-7 (δ_H 5.639)/H-8 (δ_H 6.440) and H-6b (δ_H 2.732)/H-9 (δ_H 6.162) ROESY correlations. H-9 (δ_H 6.162)/H-10 (δ_H 5.998) and H-11 (δ_H 6.439)/ H-12 (δ_H 5.707) ROESY correlations assigned 9Z and 11Z configurations. 16Z and 18Z geometries were established by H₃-40 (δ_H 1.802 (3H))/ H-17 (δ_H 6.232) and H-18 (δ_H 6.208)/ H-19 (δ_H 5.257) ROESY correlations (Figure 2). Lastly, the H₃-41/H-26 ROESY correlation determined the 24E configuration.

The relative configuration of the sugar moiety was also assigned by ¹H-¹H coupling constants and ROESY correlations [16]. H-28 (δ_H 4.285) and H-29 (δ_H 3.188) were located at axial positions by the large coupling constant value of ³J_H28H29 (8.0 Hz). The coupling constant of 9.0 Hz between H-29 (δ_H 3.188) and H-30 (δ_H 3.315) also indicated their axial–axial relationship. The large value (9.4 Hz) of both ³J_H30H31 and ³J_H31H32 revealed the assignment of H-30 (δ_H 3.315), H-31 (δ_H 3.267), and H-32 (δ_H 3.420) at axial positions of the sugar. Furthermore, the ROESY correlations of H-28 (δ_H 4.285)/H-30 (δ_H 3.315), H-30 (δ_H 3.315)/H-32 (δ_H 3.420), and H-28 (δ_H 4.285)/H-32 (δ_H 3.420) assigned these three protons in the same plain, and thus the sugar was determined to be β-glucose. In addition, ³J_H32H33a (2.2 Hz), ³J_H32H33b (6.5 Hz), and ³J_H33aH33b (12.0 Hz) supported β-glucose (Figure 3).

The relative configuration between C-4 and C-5 was revealed by J-based configuration analysis [17]. Long-range ¹³C−¹H coupling constants were measured by hetero-half-filtered TOCSY (HETLOC) NMR experiments [18]. The large coupling constant of ³J_H4H5 (8.0 Hz) indicated an anti-relationship between H-4 (δ_H 2.369) and H-5 (δ_H 3.693). The small value of ³J_C39H5 (2.0 Hz) assigned a gauche position of the C-39 (δ_C 17.40) methyl group to H-5 (δ_H 3.693). Furthermore, the small coupling constant (2.2 Hz) between C-6 (δ_C 32.03) and H-4 (δ_H 2.369) established their gauche relationship. In addition, the coupling constant of ²J_C5H4 (6.6 Hz) and ROESY correlations of H-4 (δ_H 2.369)/H-6a (δ_H 2.512), H₃-39 (δ_H 0.979)/H-6b (δ_H 2.732), and H₃-39 (δ_H 0.979)/H-5 (δ_H 3.693) revealed the 4R*,5S* relative configuration (Figure 3).

Once the assignment of β-glucose was established, chemical derivatization was conducted for the absolute configuration of the glucose [19]. We used acid hydrolysis to break the connection between the corresponding aglycone with glucopyranose. β-glucopyranose was assigned as β-d-glucopyranose by chiral derivatization with l-cysteine methyl ester hydrochloride and σ-tolyl isothiocyanate and subsequent LC/MS analysis (Figure S16).

The absolute configuration of the stereogenic center at C-15, which bears a secondary alcohol, was determined using the modified Mosher’s method [20]. The hydroxy groups at C-15 were esterified with R- and S-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride (MTPA-Cl) to the tri-S- and R-MTPA esters (1a and 1b). The calculated ¹H-NMR spectroscopic Δδ_S−R values established the absolute configuration as 15R (Figure 4 and Figures S8–S11).

When it is limited to deduce the configurations of organic compounds by application of NMR spectroscopic analysis and chemical derivatization methods, quantum mechanics-based computational approaches, including the advanced probabilistic methods including CP3 and DP4 calculations, can be utilized [21]. DP4 calculations were then applied to establish the absolute configuration for the remaining chiral centers of C-4, C-5, and C-21 [22]. Four possible diastereomers of 1 simplified without the succinic acid (1c (4R, 5S, and 21S), 1d (4R, 5S, and 21R), 1e (4S, 5R, and 21S), and 1f (4S, 5R, and 21R)) were constructed using Avogadro 3D modeling program. Then, the ¹H and ¹³C NMR chemical shifts of the four conformers whose relative potential energy was below 10 kJ/mol were calculated and averaged with their Boltzmann populations. The computational NMR shielding of 1c, 1d, 1e, and 1f was calculated using TmoleX 4.2.1. 1c (4R, 5S, and 21S) achieved 100.0% probability based on statistical comparison of the calculated and experimental chemical shifts using DP4 calculation (https://www-jmg.ch.cam.ac.uk/tools/nmr/DP4/, accessed on 13 September 2020). (Tables S1 and S2, Figure S17), completing the structure elucidation of succinyl glyco-oxydifficidin (1).

Succinyl macrolactin O (2) was isolated as a yellow powder. The molecular formula of 2 was deduced as C₃₄H₄₈O₁₃ based on its HRESIMS data. Its molecular formula revealed 11 degrees of unsaturation. The UV spectrum (λ_max: 236 and 260 nm) of 2 indicated at least two chromophores in the structure. By careful analysis of ¹H, ¹³C, and HSQC NMR spectra, all the ¹H–¹³C one-bond correlations in 2 were identified. Ten olefinic protons (δ_H 7.229–5.411), eight oxygen-bound methine protons (δ_H 5.001, 4.370, 4.309, 4.118, 3.405, 3.328, 3.300, 3.239), and two oxygenated methylene protons (δ_H 4.423 and 4.250) were identified. Twenty protons belonging to ten methylene groups (δ_H 2.654 (2H), 2.605 (2H), 2.580, 2.555 (2H), 2.463 (2H), 2.461 (2H), 2.373, 2.197 (2H), 2.051, 1.959, 1.630, 1.531, 1.428, 1.393) and one methyl group (δ_H 1.232 (3H)) were also identified using the ¹H NMR and HSQC data. The ¹³C NMR data revealed one ketone carbon (δ_C 211.82), three ester or carboxyl acid groups (δ_C 176.15, 174.21, and 167.97), ten olefinic carbons (δ_C 145.37–117.83), nine oxygenated carbons, including eight oxygenated methine carbons (δ_C 101.37–68.66), and one oxygenated methylene carbon (δ_C 64.94), ten methylene carbons (δ_C 49.42–26.19), and one methyl carbon (δ_C 20.26) in the structure of 2.

The spin system from C-2 (δ_C 117.83) to C-14 (δ_C 49.42) could be straightforwardly connected by a series of COSY correlations from H-2 to H₂-14 through H-3 (δ_H 6.628), H-4 (δ_H 7.229), H-5 (δ_H 6.214), H₂-6 (δ_H 2.560, 2.456), H-7 (δ_H 4.370), H-8 (δ_H 5.636), H-9 (δ_H 6.581), H-10 (δ_H 6.146), H-11 (δ_H 5.545), H₂-12 (δ_H 2.463, 2.373), and H-13 (δ_H 4.118). The second spin system of C-16–C-24 was revealed by the consecutive COSY correlations from H-16 (δ_H 2.461) to H₂-24 (δ_H 1.232) via H-17 (δ_H 2.197), H-18 (δ_H 5.411), H-19 (δ_H 5.411), H₂-20 (δ_H 2.052, 1.959), H₂-21 (δ_H 1.428, 1.393), H₂-22 (δ_H 1.630, 1.531), H-23 (δ_H 5.001), and H₃-24 (δ_H 1.232). The third spin system, C-25–C-30, was also constructed based on COSY correlations from H-25 (δ_H 4.309) to H₂-30 (δ_H 4.423, 4.250). C-32 and C-33 were linked as the last spin system by their COSY correlation H₂-32 (δ_H 2.605 (2H))/H₂-33 (δ_H 2.654 (2H)) (Figure 5). These substructures were connected by key HMBC correlations; the H₂-14 (δ_H 2.580, 2.555)/C-15 (δ_C 211.82) and H-16 (δ_H 2.461)/C-15 (δ_C 211.82) correlations connected the piece C-14–C-15–C-16, therefore constructing the C-2–C-24 chain skeleton. H-2 (δ_H 5.544)/C-1 (δ_C 167.97) and H-3 (δ_H 6.628)/C-1 (δ_H 167.97) heteronuclear correlations revealed the C-1–C-2 linkage, while an ester bond was established by H-23 (δ_H 5.001)/C-1 (δ_H 167.97) coupling, completing the 24-membered macrocyclic skeleton. The glucoside, which was confirmed by H-29 (δ_H 3.402)/C-25 (δ_C 101.37) correlation, was connected to C-7 based on the HMBC correlation of H-25 (δ_H 4.309) to C-7 (δ_H 78.73). A succinate moiety was revealed by H₂-32 (δ_H 2.605 (2H))/C-31 (δ_C 174.21), H₂-33 (δ_H 2.654 (2H))/C-31 (δ_C 174.21), H₂-32 (δ_H 2.605(2H))/C-34 (δ_C 176.15), and H₂-33 (δ_H 2.654 (2H))/C-34 (δ_C 176.15) correlations. This succinate moiety was attached to the glucoside by H₂-30 (δ_H 4.423, 4.250)/C-31 (δ_C 174.21) HMBC correlation (Figure 5). Therefore, the planar structure of 2 was proposed, as shown in Figure 1.

Analogously to 1, the sugar moiety in 2 was assigned as β-glucose by ¹H-¹H coupling constants and ROESY correlations. Its absolute configuration was revealed as β- d-glucopyranose by the chiral derivatization and LC/MS analysis (Figure S28). As many macrolactin derivatives were discovered and their absolute configurations are conserved in the family, we compared the [α]_D values of succinyl macrolactin O (2) ([α]_D = −36.8 (c 0.1, MeOH)) with those of the most closely related compound, macrolactin O (−56.8 (c 0.1, MeOH)), and proposed that succinyl macrolactin O (2) shared the same absolute configuration with macrolactin O [5].

2.2. Biological Activity

According to the previous studies, the antimicrobial activities of difficidin [23] and macrolactin [11] families were reported. However, we could not find any activity of 1 and 2 in our antimicrobial assays (Tables S4–S6). Therefore, we searched for unreported activity of the difficidin and macrolactin families and targeted amyloid-β-aggregation-regulating activity for these two compounds. Amyloid-β (Aβ) aggregates in the brain of patients with Alzheimer’s disease (AD) are considered the pathological and biological hallmarks of this neurodegenerative disorder [24]. Thus, drug candidates have been discovered to inhibit and reverse the Aβ aggregation process [25]. To investigate whether our compounds, succinyl glyco-oxydifficidin (1) and succinyl macrolactin O (2), inhibit and/or reverse Aβ aggregation, we performed two different types of assays, inhibition and dissociation, utilizing a high throughput screening platform we recently developed [26]. For both assays, we immobilized Aβ_1-42 with an additional C-terminal cysteine on the maleimide-coated 96-well plate and added fluorescent Aβ_1-42 to induce on-plate oligomer and fibril formation of Aβ (Figure 6A). First, we assessed 1 and 2, in Aβ aggregation inhibition assay (Figure 6A). Each compound, at concentrations of 5 and 50 µM, was added to the Aβ_1-42-immobilized plate with the fluorescent Aβ_1-42 (10 µM) and incubated for 24 h at room temperature (RT) to observe in situ aggregation inhibition. We used a previously reported Aβ aggregation inhibitor, (1r,2r,3r,4r,5r,6r)-cyclohexane-1,2,3,4,5,6-hexol (scyllo-inositol), as a positive control [27]. The plate was washed after the incubation step, and we measured the levels of the remaining fluorescent Aβ_1-42. Data were then normalized to the signal of fluorescent Aβ_1-42-free wells as 100% inhibition of Aβ aggregation, and the inhibition rate (%) was analyzed as previously reported [26]. As a result, both compounds inhibited Aβ aggregation significantly; 1 by 35.54% (at 5 µM) and 54.35% (at 50 µM), and 2 by 18.88% (at 5 µM) and 40.08% (at 50 µM), when the control compound was inhibited by 43.00% (at 5 µM) and 52.22% (at 50 µM) (Figure 6B).

We further examined the compounds ability to dissociate pre-formed Aβ aggregates. In this assay, compounds were added to the plate after on-plate oligomer and fibril formation was induced [26]. Briefly, fluorescent Aβ_1-42 was added to the Aβ_1-42-immobilized plate and incubated for 8 h at 37 °C. Then, each compound in two concentrations, 5 and 50 µM, was treated to the plate and incubated for additional 24 h at RT (Figure 6A). Previously reported Aβ aggregate dissociators, 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS) [28] and 5-(1H-indol-3-ylmethyl)-3-methyl-2-thioxo-4-imidazolidinone (Necrostatin-1, Nec-1) [29], were used as positive controls. The plate was washed after the incubation step, and we measured the levels of the remaining fluorescent Aβ_1-42. Fluorescent signal of wells without fluorescent Aβ_1-42 treatment was regarded as 100% disassociation rate [26]. As a result, both compounds reversed Aβ aggregation significantly; 1 by 28.46% (at 5 µM) and 49.27% (at 50 µM), and 2 by 30.04% (at 5 µM) and 31.02% (at 50 µM), when the control compounds dissociated pre-formed aggregates by 46.42% (EPPS at 5 µM) and 59.94% (Nec-1 at 5 µM) (Figure 6C).

Next, we docked compounds 1 and 2 to the U-shaped oligomeric Aβ_1-42 structure (PDB ID: 2BEG) to predict the potential binding interactions between them (Figure 7). The docking score of 1 (−9.9 kcal/mol) was slightly better than that of 2 (−9.3 kcal/mol). The docking models suggested that the branched carbon chain of 1, which is not present in 2, contributes to forming extensive hydrophobic contacts with the core of Aβ aggregate. Also, the additional contacts of 1 to the edge strand of Aβ aggregate seem to be a primary factor for inhibiting or dissociating Aβ aggregation. In contrast, 2 showed similar but relatively unfavorable interactions with the Aβ hydrophobic core through the sugar moiety (Figure 7).

Overall, we observed that succinyl glyco-oxydifficidin (1) dose-dependently inhibited/dissociated Aβ aggregation, whereas succinyl macrolactin O (2) dose-dependently inhibited Aβ aggregation. In these assays, we assumed that our scaffold had the therapeutic potential to disassemble and remove Aβ aggregation.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations were measured by a Jasco P-2000 polarimeter with a 1.0 cm cell (Tokyo, Japan). UV and CD spectra were recorded using an applied photophysics Chirascan plus spectrometer (Leatherhead, UK). IR spectra were acquired with a JASCO FT/IR-4200 spectrometer (Tokyo, Japan). ¹H, ¹³C, and 2D NMR spectra were obtained on Bruker Avance 800 MHz NMR spectrometers (Billerica, MA, USA), all the signals being referenced to ¹³C (49.045 ppm) and ¹H (3.306 ppm) signals of CD₃OD [30]. Electrospray ionization source (ESI) low-resolution LC/MS data were collected on an Agilent Technologies 6130 quadrupole mass spectrometer (Santa Clara, CA, USA) coupled with an Agilent Technologies 1200 series HPLC using a reversed-phase C₁₈(2) column (Phenomenex Luna, 5 μm, 4.6 × 100 mm, Torrance, CA, USA). High-resolution electrospray ionization source (ESI) LC/MS data were collected on an AB SCIEX Q-TOF 5600 high-resolution mass spectrometer at the National Instrumentation Center for Environmental Management (NICEM, Seoul, Republic of Korea).

3.2. Isolation and Identification of the Bacterial Strain Bacillus sp. AMD05

The strain, AMD05, was isolated from a mudflat sample collected from the intertidal mudflat on Anmyeon Island, Republic of Korea, using a sterilized 40 mL plastic tube. Various strain isolation media were applied for single-strain isolation, while AMD05 was isolated on a YEME-based agar medium (10 g/L of malt extract, 4 g/L of yeast extract, 4 g/L of glucose, and 18 g/L of agar) incubated at 25 °C for 7 days. The AMD05 strain was most closely related to Bacillus velezensis F-30 (97% identity, accession # MF988699) according to 16S rDNA sequence analysis (AMD05 16S rDNA GenBank deposit #OM319625).

3.3. Cultivation and Extraction

The spores of the bacterial strain Bacillus sp. AMD05 were inoculated into 50 mL of YEME liquid medium in a 125 mL flask. The culture was incubated at 200 rpm at 30 °C for two days. After incubation, 10 mL of the AMD05 liquid culture was inoculated into a 500 mL Erlenmeyer flask containing 200 mL of YEME medium and shaken at 170 rpm and 30 °C for two days. Then, 15 mL of the medium culture was transferred into 1 L of YEME medium in a 2.8 L Fernbach flask for four days at 170 rpm and 30 °C (24 ea × 1 L, total volume 24 L). The entire culture was extracted with 36 L of ethyl acetate (EtOAc). The EtOAc layer was separated using a separation funnel (capacity 3 L), and the residual water in the EtOAc layer was removed by adding anhydrous sodium sulfate. The extract was concentrated in vacuo to yield dry material. This procedure was repeated 3 times (72 L of culture in total) to yield extracted material.

3.4. Isolation of Succinyl Glyco-Oxydifficidin (1) and Succinyl Macrolactin O (2)

The dried extract material was dissolved in methanol (MeOH), adsorbed with Celite 545 (DaeJung Chmicals and Metals Co., Ltd., Siheung-si, Republic of Korea), and loaded onto a reversed-phase flash chromatography column (YMC C₁₈ resin, 60 × 40 mm). Five MeOH/H₂O concentrations (20%, 40%, 60%, 80%, and 100% of aqueous MeOH, each for 400 mL) were used for fractionation. Each fraction was analyzed by LC/MS, which indicated that 1 and 2 were eluted in the 80% aqueous MeOH fraction. Succinyl glyco-oxydifficidin and succinyl macrolactin O were further purified using semipreparative high-performance liquid chromatography (HPLC). The dried 80% MeOH fraction was subjected to reversed-phase HPLC (Kromasil C₁₈ column, 5 μm, 10 × 250 mm) under a step gradient solvent system using 35–65% aqueous CH₃CN from 0 to 30 min, which was continued with an isocratic 80% aqueous CH₃CN method from 30 to 60 min (UV 230 nm detection, flow rate: 2 mL/min). Succinyl glyco-oxydifficidin was eluted at 32 min while 2 was eluted at 22 min. Succinyl glyco-oxydifficidin was further purified using a Kromasil C₁₈ column at a retention time of 23 min (15 mg) under an isocratic condition (54% aqueous CH₃CN). Succinyl macrolactin O was also purified by the same column at a retention of 33 min (10 mg) under an isocratic condition (39% aqueous CH₃CN).

Succinyl glyco-oxydifficidin (1): Yellow powder, [α]_D = −19.2 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 234 (3.80), 274 (3.39), 284 (3.30) nm; IR (neat) ν_max 3414, 2926, 1725, 1424, 1266, 1166, 1081 cm⁻¹; CD (MeOH) λ_max (log ε) 232 (−4.65), 272 (+4.15), 282 (+3.96) nm; for ¹H and ¹³C NMR data, see

Table 1. NMR data for 1 and 2 in CD₃OD.

	1		2
C/H	δC, Type	δH, Mult (J in Hz)	C/H	δC, Type	δH, Mult (J in Hz)
1	174.03, C		1	167.97, C
2	47.03, CH2	3.059, d (14.5)	2	117.83, CH	5.544, d (11.0)
		3.444, d (14.5)	3	145.37, CH	6.628, d (11.0)
3	148.67, C		4	130.23, CH	7.229. dd (15.0, 11.0)
4	41.19, CH	2.369, m	5	141.73, CH	6.214, ddd (15.0, 8.0, 6.0)
5	86.94, CH	3.693, m	6	40.97, CH2	2.560, m
6	32.03, CH2	2.512, m			2.456, m
		2.732, m	7	78.73, CH	4.370, dd (13.0, 7.0)
7	128.61, CH	5.639, td (11.0, 4.0)	8	134.17, CH	5.636, dd (15.0, 7.0)
8	128.72, CH	6.440, t (12.5)	9	129.45, CH	6.581, dd (15.0, 11.0)
9	123.86, CH	6.162, m	10	131.71, CH	6.146, t (11.0)
10	130.46, CH	5.998, t (11.0)	11	128.85, CH	5.545, m
11	126.55, CH	6.439, t (12.5)	12	35.85, CH2	2.463, m
12	135.80, CH	5.707, m			2.373, dt (14.0, 7.0)
13	29.18, CH2	1.945, m	13	68.66, CH	4.118, m
		2.311, m	14	49.42, CH2	2.580, d (6.5)
14	34.10, CH2	1.803, m			2.555, d (6.5)
		1.739, m	15	211.82, C
15	68.14, CH	4.680, dd (9.0, 6.0)	16	44.40, CH2	2.461, m
16	140.83, C		17	27.95, CH2	2.197, m
17	123.89, CH	6.232, d (11.0)	18	130.30, CH	5.411, m
18	126.58, CH	6.208, d (11.0)	19	132.01, CH	5.411, m
19	126.47, CH	5.257, m	20	33.00, CH2	2.051, ddd (17.0, 12.0, 5.5)
20	32.29, CH2	2.403, m			1.959, dt (13.0, 7.0)
		2.601, m	21	26.19, CH2	1.428, m
21	75.89, CH	4.802, m			1.393, m
22	33.45, CH2	1.703, m	22	36.29, CH2	1.630, m
		1.817, m			1.531, m
23	36.75, CH2	2.102, t (8.0)	23	71.75, CH	5.001, m
24	139.27, C		24	20.26, CH3	1.232, m
25	127.37, CH	5.861, broad d (11.0)	25	101.37, CH	4.309, d (8.0)
26	134.48, CH	6.584, dtd (14.5, 10.5, 4.0)	26	75.07, CH	3.239, d (8.5)
27	115.53, CH2	4.969, dd (10.0, 2.0)	27	78.00, CH	3.328, dd (18.0, 9.0)
		5.071, dd (17.0, 2.0)	28	71.78, CH	3.300, m
28	105.91, CH	4.285, d (8.0)	29	75.28, CH	3.402, ddd (9.0, 6.0, 2.0)
29	75.50, CH	3.188, dd (9.0, 8.0)	30	64.94, CH2	4.423, dd (12.0, 2.0)
30	78.09, CH	3.315, m			4.250, dd (12.0, 2.0)
31	71.92, CH	3.267, d (9.0)	31	174.21, C
32	75.19, CH	3.420, ddd (9.0, 6.5, 2.5)	32	29.97, CH2	2.605, t (6.5)
33	64.93, CH2	4.405, dd (12.0, 2.0)	33	30.17, CH2	2.654, t (6.5)
		4.220, dd (12.0, 6.5)	34	176.15, C
34	174.37, C
35	30.90, CH2	2.546, m
36	30.64, CH2	2.596, m
37	177.20, C
38	113.99, CH2	5.026, s 5.046, s
39	17.40, CH3	0.979, d (7.0)
40	17.76, CH3	1.802, s
41	16.62, CH3	1.779, s

¹H 800 MHz, ¹³C 200 MHz.

, HRESIMS m/z 765.3814 [M + Na]⁺ (calcd for C₄₁H₅₈O₁₂Na, 765.3826).

Succinyl macrolactin O (2): Yellow powder, [α]_D = −36.8 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 236 (3.86), 260 (3.70) nm; IR (neat) ν_max 3402, 2927, 1705, 1571, 1413, 1187, 1050 cm⁻¹; CD (MeOH) λ_max (log ε) 233 (+5.26), 258 (−5.19) nm; for ¹H and ¹³C NMR data, see Table 1, HRESIMS m/z 687.2977 [M+Na]⁺ (calcd for C₃₄H₄₈O₁₃Na, 687.2992).

3.5. Determination of the Configuration of the Sugar in 1 and 2

Succinyl glyco-oxydifficidin was hydrolyzed with 6 N HCl at 115 °C for 1 h to yield the free glucopyranose. After drying in vacuo, the acid hydrolysate was reacted with l-cysteine methyl ester hydrochloride and σ-tolyl isothiocyanate at 60 °C, each for 1 h. The authentic β-l-glucose and β-d-glucose were also reacted with l-cysteine methyl ester hydrochloride and σ-tolyl isothiocyanate at 60 °C, each for 1 h. The β-glucopyranose reaction product from 1 was co-injected with the products of the authentic β-l-glucose and β-d-glucose using LC/MS analysis (gradient solvent conditions: 10-100% aqueous CH₃CN (0.1% formic acid) for 20 min). The reaction products of β-glucopyranose from 1 had the same retention time as the reaction product of authentic β-d-glucose (only one peak in the LC/MS spectrum). This method was also applied for succinyl macrolactin O (2), identifying β-d-glucose (Figure S28).

3.6. MTPA Esterification of Succinyl Glyco-Oxydifficidin (1)

Succinyl glyco-oxydifficidin was transferred to two 40 mL vials (2 mg of 1 in each vial) and dried completely under high vacuum overnight. A total of 1 mL of distilled anhydrous pyridine was added to each vial under argon gas. The mixtures were stirred at room temperature for approximately 5 min. Then, R- and S-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride (MTPA-Cl) (50 μL) were added into one of the two vials. The reactions were terminated after 30 min by adding 50 μL of MeOH. The reaction mixtures were dried in vacuo and subjected to reversed-phase HPLC (Kromasil C₁₈ column, 5 μm, 10 × 250 mm). An isocratic solvent system (94% aqueous CH₃CN for 40 min, flow rate: 2 mL/min, detection: UV 230 nm) was used. The S-MTPA ester (1a) and the R-MTPA ester (1b) of 1 were both eluted at a retention time of 30 min. Low-resolution LC/MS analysis was carried out for 30 min using from 70% to 95% aqueous CH₃CN (0.1% formic acid) with an IB chiral column. The Δδ_S-R values around the stereogenic centers were assigned by analyzing their ¹H NMR and ¹H-¹H COSY NMR spectra (Figures S8–S11).

S-MTPA ester of 1 (1a): ¹H NMR (800 MHz, CD₃CN) δ_H 7.57–7.37 (14H overlapped), 6.59 (1H, dt, J = 16.5, 11.0), 6.48 (1H, m), 6.46 (1H, m), 6.35 (1H, d, J = 11.5), 6.32 (1H, d, J = 11.0), 6.21 (1H, t, J = 11.0), 6.05 (1H, m), 5.86 (1H, d, J = 11.0), 5.72 (1H, m), 5.67 (1H, m), 5.37 (1H, m), 5.06 (1H, m), 5.03 (2H, s), 4.96 (2H, m), 4.84 (1H, m), 4.57 (2H, s), 4.43 (1H, m), 4.37 (1H, d, J = 8.0), 4.17 (1H, m), 4.14 (1H, m), 4.00 (1H, t, J = 6.0), 3.78 (1H, m), 3.74 (1H, m), 3.52 (3H, s), 3.50 (3H, s), 3.41 (1H, d, J = 14.0), 3.40 (1H, m), 3.38 (3H, s), 3.08 (1H, d, J = 14.0), 2.73 (1H, m), 2.60 (1H, m), 2.54 (1H, m), 2.44 (1H, m), 2.42 (1H, m), 2.30 (1H, m), 2.11 (1H, m), 2.01 (1H, m), 1.93 (2H, m), 1.82 (1H, m), 1.79 (6H, s), 1.70 (1H, m), 1.34–1.24 (24H overlapped), 1.03 (3H, d, J = 7.0).

R-MTPA ester of 1 (1b): ¹H NMR (800 MHz, CD₃CN) δ_H 7.57–7.37 (14H overlapped), 6.58 (1H, dt, J = 16.5, 11.0), 6.47 (1H, m), 6.45 (1H, m), 6.40 (1H, d, J = 11.5), 6.33 (1H, d, J = 11.0), 6.19 (1H, t, J = 11.0), 6.03 (1H, t, J = 11.0), 5.86 (1H, d, J = 11.0), 5.69 (1H, m), 5.65 (1H, m), 5.37 (1H, m), 5.06 (1H, m), 5.05 (2H, s), 4.96 (1H, m), 4.83 (2H, m), 4.57 (2H, s), 4.43 (1H, m), 4.34 (1H, d, J = 8.0), 4.18 (1H, m), 4.10 (1H, m), 4.00 (1H, t, J = 6.0), 3.83 (1H, m), 3.75 (1H, m), 3.70 (1H, m), 3.60 (1H, m), 3.63 (3H, s), 3.47 (3H, s), 3.41 (1H, d, J = 14.0), 3.38 (3H, s), 3.07 (1H, d, J = 14.0), 2.69 (1H, m), 2.60 (1H, m), 2.50 (1H, m), 2.45 (1H, m), 2.39 (1H, q, J = 7.6, 7.2), 2.21 (1H, m), 2.11 (1H, t, J = 8.0), 1.98 (1H, m), 1.83 (2H, m), 1.82 (1H, m), 1.80 (3iiH, s), 1.79 (3H, s), 1.70 (1H, m), 1.34-1.24 (24H overlapped), 1.01 (3H, d, J = 7.0).

3.7. Conformational Search and DP4 Calculations

For the determination of the configurations of C-4, C-5, and C-21, 1c (4R, 5S, and 21S), 1d (4R, 5S, and 21R), 1e (4S, 5R, and 21S), and 1f (4S, 5R, and 21R) were generated by Avogadro 1.2.0. A conformational search for these diastereomers was performed by MacroModel with the Merck Molecular Force Field to find the stable conformers (with 10 kJ/mol energy limit) of the diastereomers: twelve conformers for 1c (4R, 5S, and 21S), nine conformers for 1d (4R, 5S, and 21R), six conformers for 1e (4S, 5R, and 21S), and twenty-nine conformers for 1f (4S, 5R, and 21R) (Table S1). The Boltzmann populations of the conformers were also calculated by MacroModel. Ground-state geometry optimization was performed by density functional theory (DFT) modeling of Turbomole X 4.3.2. All calculations were performed at the B3LYP/def-SV(P) level in the gas phase. This basis set, taken from the work [31], was used for all atoms. Calculated chemical shifts were calculated based on this equation: ${\mathsf{\delta }}_{calc.}^X=\frac{{\mathsf{\sigma }}^0-{\mathsf{\sigma }}^X}{1-{\mathsf{\sigma }}^0/{10}^6}$. ${\mathsf{\delta }}_{calc.}^X$ is the calculated chemical shifts of nucleus x (e.g., ¹H or ¹³C), while σ^X and σ⁰ are the calculated isotropic constants of nucleus x and tetramethylsilane (TMS) [33]. The calculated NMR chemical shifts of each conformer were averaged by the Boltzmann populations. By comparing these Boltzmann-population-averaged chemical shifts with the experimental chemical shifts of 1 (Table S2), the DP4 calculation result proposed 1c (4R, 5S, and 21S) configurations with 100.0% probability using both carbon and proton data (71.0% probability using only the carbon data and 100.0% probability using only the proton data) (Figure S17).

3.8. Peptide Synthesis

Full-length Aβ_1-42 with a C-terminal cysteine, Aβ_1-42-cys, was synthesized via solid-phase peptide synthesis, as previously reported. Fluorescent Aβ_1-42 was synthesized with the conjugation of Flamma-552 carboxylic acid on N-terminus [26,32].

3.9. Aβ Aggregation Assay Plate Preparation

A maleimide-activated microplate was used to immobilize the peptide. The bovine serum albumin coated on the maleimide-activated microplate was removed after washing the peptide three times with 200 µL of wash buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, 0.05% Tween-20; pH 7.2) in each well on the plate. A full-length Aβ_1-42-cys solution (50 µg/mL, 5% DMSO) was made in binding buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, 10mM EDTA; pH 7.2). A total of 100 µL of 10 µM peptide solution was added to each well and reacted with maleimide for 24 h at RT. After peptide immobilization, the unbounded peptides were washed three times with 200 µL of wash buffer. To inactivate additional maleimide groups, 200 µL of cysteine solution was added to each well and incubated at RT for 1 h. After cysteine capping, all the wells in the plate were washed three times with 200 µL of wash buffer.

3.10. Aβ Aggregation Assay

To examine the inhibition effect of succinyl glyco-oxydifficidin and succinyl macrolactin O, 50 µL of succinyl macrolactin and succinyl glyco-oxydifficidin was prepared in binding buffer (1% DMSO), and 50 µL of Flmma 552-labeled full-length Aβ_1-42 peptide solution (20 µM) was prepared in binding buffer (1% DMSO). Each solution was made with two concentrations: 5 µM and 50 µM. The solution containing two compounds and Flamma-552-labeled full-length Aβ_1-42 peptide was added to the plate and incubated for 24 h at RT. After the incubation, all the wells were washed three times with 200 µL of wash buffer. For the comparison, we only added 100 µL of Flamma-labeled full-length Aβ_1-42 peptide solution (10 µM) on the other wells and incubated them for 24 h at RT. The intensity of the dug-treated well and control well was measured by the microplate reader.

3.11. Aβ Disociationn Assay

Before treating succinyl glyco-oxydifficidin and succinyl macrolactin O, 100 μL of fluorescent Aβ_1-42 peptide solution (10 μM) was added to the wells and incubated for 8 h at 37 °C. After the incubation, all the wells were washed three times with 200 μL of the wash buffer. Succinyl macrolactin (5, 50 µM) and succinyl glyco-oxydifficidin (5, 50 µM) were prepared in a binding buffer (1% DMSO). EPPS and necrostatin-1 (5 µM each) were prepared as positive controls. We added 100 μL of each compound solution to the wells and incubated them for 24 h at RT. After the incubation, all the wells were washed three times with 200 μL of the wash buffer, and 100 μL of binding buffer was added to each well prior to reading. Fluorescent scanning was carried out with a microplate reader (555/580 nm, ex/em).

3.12. Docking Model Generation

The 500 three-dimensional conformers were generated using RDKit (https://www.rdkit.org/, accessed on 15 December 2022) with 0.2 Å RMSD threshold for succinyl glyco-oxydifficidin and succinyl macrolactin O, respectively. Each conformer was used to define potential binding sites by applying global docking with PatchDock program [34] without a predefined binding region. The U-shaped Aβ_1-42 (PDB ID: 2BEG) was used as a receptor structure. The docking search space for the receptor was confined to edge strands of β-sheets to reflect the experimental results (Figure 7). The top 10 docking models for each conformer were retrieved to infer binding site information (center_x, center_y, and center_z parameters) for the subsequent docking refinement by Autodock Vina [35]. The final docking model was selected based on the Autodock Vina score.

3.13. Statistical Data Evaluation

All results were given as a mean ± standard error of the mean (SEM). All data were analyzed through GraphPad Prism 9.0 software and compared using One-way ANOVA analysis followed by Bonferroni’s post-hoc comparison. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

4. Conclusions

Succinyl glyco-oxydifficidin (1), a new glycosylated and succinylated member of the difficidin family, was discovered in an intertidal mudflat-derived Bacillus sp. AMD05. Compared its structure with that of oxydifficidin, succinyl glyco-oxydifficidin (1) was modified through glycosylation and succinylation, along with a double-bond migration from C-6 to C-7. Although difficidin was discovered more than 35 years ago in 1986, its absolute configuration had remained undetermined until now. By using combinational tools of spectroscopic analysis, the modified Mosher’s method, and DP4 computational calculation, we elucidated the absolute configuration of succinyl glyco-oxydifficidin (1) for the first time among the difficidin family compounds. Interestingly, succinyl macrolactin O (2) is the only compound in the macrolactin family to bear both glucose and succinyl acid.

Furthermore, succinyl glyco-oxydifficidin (1) inhibited/dissociated Alzheimer-disease-related Aβ aggregation and succinyl macrolactin O (2) inhibited Aβ aggregation, indicating their therapeutic potential to regulate Aβ aggregation. Even though the difficidin and macrolactin families were discovered in the 1980s with anti-microbial bioactivity [11,23], their Aβ-regulating activities were observed for the first time here. Succination and glycosylation may distribute to bioactive modification. Our discovery of the new glycosylated and succinylated macrocyclic lactones with Aβ-regulating activity from marine Bacillus sp. highlights that marine bacteria are prolific sources of natural products diversified by glycosylation, an important biological process for changing the structures and bioactivity of compounds.