Introduction

In 1991, an outbreak of food poisoning caused by a species of red algae known as ‘Polycavernosa tsudai’ occurred in Guam, which resulted in killing of three people. Two novel macrolide glycosides, polycavernosides A (2) and B (3), were reported as the causative compounds for the illness . After that, the second fatal food poisoning incidents occurred in the Philippines caused by the ingestion of polycavernoside A (2)-contaminated red algae . Subsequently, polycavernoside analogs such as polycavernoside C (4) were isolated from red algae . In 2015, Navarro et al. isolated polycavernoside D (5) from a marine Okeania sp. cyanobacterium . They suggested that polycavernosides were produced by marine cyanobacteria based on their high content and structural similarity to other cyanobacterial metabolites. In this study, polycavernoside E (1), a new polycavernoside analog, was isolated from a marine Okeania sp. cyanobacterium obtained from Okinawa Prefecture, Japan (Figure 1). This finding provides additional evidence that polycavernosides are secondary metabolites derived from marine Okeania sp. cyanobacteria.

Figure 1: Structures of compounds 1, 2, and 5.

Results and Discussion

The EtOH extract of marine Okeania sp. cyanobacterium (340 g, wet weight) collected from Akuna Beach, Okinawa, Japan, was partitioned between EtOAc and H2O. The EtOAc fraction was further partitioned into 90% aqueous MeOH and hexane. The aqueous MeOH portion was purified by reversed-phase column chromatography (ODS silica gel, MeOH/H2O), automated flash chromatography (hexane/EtOAc), and repeated reversed-phase HPLC to give polycavernoside E (1, 0.5 mg as a colorless oil). The isolation of compound 1 was directed by its characteristic UV absorption around 270 nm.

The molecular formula of 1 was determined to be C44H66O15 based on the HRESIMS data. The NMR data for 1 are summarized in Table 1. The 1H NMR spectrum of compound 1 was similar to those of known polycavernosides but matched none of them, suggesting that 1 was a new analog of polycavernosides . A detailed analysis of the NMR data revealed the planar structure of 1, as shown in Figure 2. COSY and HMQC spectral analyses revealed several partial structures, indicated by the bold bonds in Figure 2. Four HMBC were observed from singlet methyl signals: δH 0.85 (H-28)/δC 19.4 (C-29), δH 0.86 (H-29)/δC 17.8 (C-28), δH 0.94 (H-30)/δC 13.9 (C-31), and δH 0.90 (H-31)/δC 22.2 (C-30). These correlations elucidated the presence of two gem-dimethyl groups. Moreover, three HMBC, δH 4.03 (H-5a’)/δC 106.1 (C-1’), δH 3.61 (H-6’)/δC 83.8 (C-2’), and δH 3.45 (H-7’)/δC 78.5 (C-4’), revealed the presence of a 2,4-di-O-methylpyranose substructure. Furthermore, an HMBC, δH 3.48 (H-6”)/δC 78.7 (C-4”), along with typical chemical shifts and coupling constants from C-1” to C-5” obtained in CD3OD (Table 2), indicated the presence of a 4-O-methylpyranose substructure. The HMBC, δH 3.64 (H-3’)/δC 103.0 (C-1”), indicated that these two sugar structures were connected through a glycosidic bond.

Table 1: NMR data for polycavernoside E (1) in CDCl3.

position δC, typea δHb (J in Hz) COSY selected HMBC 1 171.9, C       2 35.6, CH2 2.29, m 3 1 3 82.0, CH 3.43, m 2   4 38.3, C       5 85.3, CH 3.32, m 6a, 6b   6a 37.7, CH2 1.95, m 5, 6b, 7   6b   1.61, m 5, 6a, 7   7 83.8, CH 3.07, m 6a, 6b, 8a, 8b   8a 42.1, CH2 3.08, m 7, 8b 9 8b   2.00, m 7, 8a   9 206.9, C       10 103.0, C       11 39.7, CH 2.74, m 12a, 12b, 27   12a 33.6, CH2 2.01, m 11, 12b, 13   12b   1.70, m 11, 12a   13 83.5, CH 4.18, dd (11.3, 5.0) 12a, 12b   14 39.8, C       15 78.4, CH 5.17, d (8.2) 16 1 16 127.4, CH 5.55, dd (8.2, 15.0) 15, 17   17 135.4, CH 6.26, m 16, 18   18 130.1, CH 6.09, m 17, 19   19 133.9, CH 6.13, m 18, 20   20 131.2, CH 6.08, m 19, 21   21 134.6, CH 5.67, dt (15.0, 7.3) 20, 22   22 31.8, CH2 2.19, m 21, 23   23 28.1, CH2 1.62, m 22, 24 25 24 17.9, CH2 2.18, m 23, 26 25, 26 25 84.6, C       26 68.6, CH 1.95, t (2.7) 24   27 13.3, CH3 0.99, d (6.8) 11 10 28 17.8, CH3 0.85, s   13, 14, 15, 29 29 19.4, CH3 0.86, s   13, 14, 15, 28 30 22.2, CH3 0.94, s   3, 4, 5, 31 31 13.9, CH3 0.90, s   3, 4, 5, 30 32 OH 4.47, s   9, 10, 11 1’ 106.1, CH 4.27, d (7.7) 2’ 5 2’ 83.8, CH 3.07, m 1’, 3’   3’ 79.9, CH 3.64, m 2’, 4’ 1” 4’ 78.5, CH 3.27, m 3’, 5a’, 5b’   5a’ 63.2, CH2 4.03, dd (11.3, 5.0) 4’, 5b’ 1’ 5b’   3.12, m 4’,5a’   6’ 61.1, CH3 3.61, s   2’ 7’ 58.8, CH3 3.45, s   4’ 1” 103.0, CH 4.87, d (4.5) 2”   2” 71.7, CH 3.53, m 1”, 3”   3” 71.0, CH 3.75, m 2”, 4”   4” 78.7, CH 3.34, m 3”, 5a”, 5b”   5a” 60.1, CH2 4.23, dd (12.2, 3.2) 5b”, 4”   5b”   3.46, m 5a”, 4”   6” 58.1, CH3 3.48, s   4” 7” - OH     8” - OH    

aMeasured at 400 MHz. bMeasured at 100 MHz.

Figure 2: Planar structure of polycavernoside E (1) based on 2D NMR analysis.

The geometries of the two olefins at C-16 and C-20 were determined to be trans based on the large coupling constants, 3JH-16/H-17 15.0 Hz and 3JH-20/H-21 15.0 Hz, respectively. The geometry of the remaining double bond at C-18 was established to be trans by comparing the 13C NMR chemical shifts at C-16 and C-21 between 1 and polycavernoside D (5) (Table S1 in Supporting Information File 1) . In addition, a 4J long-range coupling between δH 1.95 (H-26) and δH 2.18 (H-24) and three HMBC δH 1.62 (H-23)/δC 84.6 (C-25), δH 2.18 (H-24)/δC 84.6 (C-25), and δH 2.18 (H-24)/δC 68.6 (C-26) revealed a terminal alkyne structure. Additionally, COSY correlations shown in Figure 2 revealed the side chain structure of 1 containing a terminal alkyne and a conjugated trans triene (C-15 to C-26).

We then focused on the macrolide structure of 1. Six HMBC, δH 0.94 (H-30)/δC 82.0 (C-3), δH 0.94 (H-30)/δC 38.3 (C-4), δH 0.94 (H-30)/δC 85.3 (C-5), δH 0.90 (H-31)/δC 82.0 (C-3), δH 0.90 (H-31)/δC 38.3 (C-4), and δH 0.90 (H-31)/δC 85.3 (C-5), along with COSY correlations shown in Figure 2, revealed a chain structure from C-2 to C-8. In addition, eight HMBC, δH 5.17 (H-15)/δC 171.9 (C-1), δH 2.29 (H-2)/δC 171.9 (C-1), δH 0.85 (H-28)/δC 83.5 (C-13), δH 0.85 (H-28)/δC 39.8 (C-14), δH 0.85 (H-28)/δC 78.4 (C-15), δH 0.86 (H-29)/δC 83.5 (C-13), δH 0.86 (H-29)/δC 39.8 (C-14), and δH 0.86 (H-29)/δC 78.4 (C-15), and COSY correlations shown in Figure 2, clarified the connection of C-1 to C-8 and C-15 to C-11(-C27) through an ester bond. Furthermore, five HMBC, δH 0.99 (H-27)/δC 103.0 (C-10), δH 4.47 (H-32)/δC 206.9 (C-9), δH 4.47 (H-32)/δC 103.0 (C-10), δH 4.47 (H-32)/δC 39.7 (C-11), and δH 3.08 (H-8a)/δC 206.9 (C-9) connected C-11 and C-8 through a ketone carbonyl carbon at C-9 and hemiacetal carbon at C-10, revealing the 16-membered macrolide structure of 1. The HMBC, δH 4.27 (H-1’)/δC 85.3 (C-5), revealed that the disaccharide moiety was connected to C-5. Finally, considering the molecular formula of 1 and the chemical shifts of known polycavernosides, we established the presence of a THP ring containing C-3 to C-7 and a THF ring containing C-10 to C-13 in the macrolide structure. Consequently, we established the planar structure of 1, as shown in Figure 2.

The relative configuration of compound 1 was determined based on the NMR data obtained in CD3OD and CDCl3 (Table 1 and Table 2). The relative configuration of the THP ring and the disaccharide moiety of 1 was determined by analyzing the proton coupling constants and NOESY correlations (Figure 3). The two coupling constants in CD3OD, 3JH-5/H-6b (11.9 Hz) and 3JH-6b/H-7 (11.9 Hz), indicated that H-5, H-6b, and H-7 were in the axial position. The two NOESY correlations in CD3OD, δH 1.80 (H-6b)/δH 0.91 (H-31) and δH 2.36 (H-2)/δH 0.91 (H-31), revealed that H-6b, C-31, and C-2 were located in the same face of the THP ring as shown in Figure 3. Consequently, the relative configuration of the THP ring was determined to be 3S*,5S*,7S*. For the 2,4-di-O-methylpyranose moiety, the two large coupling constants in CD3OD, 3JH-1’/H-2’ (7.8 Hz) and 3JH-2’/H-3’ (9.0 Hz), indicated that H-1’, H-2’, and H-3’ were in the axial position. The two NOESY correlations in CDCl3, δH 3.45 (H-7’)/δH 4.87 (H-1”) and δH 3.45 (H-7’)/δH 4.23 (H-5a”), revealed that the methoxy group at C-4’ was in the equatorial position and H-4’ was in the axial position. The 2,4-di-O-methylpyranose moiety was identified as 2,4-di-O-methylxylose. For the 4-O-methylpyranose moiety, the two large coupling constants in CD3OD, 3JH-1”/H-2” (7.3 Hz) and 3JH-2”/H-3” (9.1 Hz), indicated that H-1”, H-2”, and H-3” were in the axial position. NOESY correlations in CDCl3, δH 3.75 (H-3”)/δH 3.48 (H-6”), revealed that the methoxy group at C-4” was in the equatorial position and H-4” was in the axial position. The 4-O-methylpyranose moiety was identified as 4-O-methylxylose. The relationship between the relative configuration of the 2,4-di-O-methylxylose moiety and 4-O-methylxylose moiety was identified using three NOESY correlations in CDCl3, δH 3.45 (H-7’)/δH 4.87 (H-1”), δH 3.45 (H-7’)/δH 4.23 (H-5a”), and δH 3.64 (H-3’)/δH 4.87 (H-1”), as shown in Figure 3. Furthermore, the relationship of the relative configuration between the disaccharide moiety and the THP ring was revealed by two NOESY correlations in CDCl3, δH 4.27 (H-1’)/δH 3.32 (H-5) and δH 4.27 (H-1’)/δH 0.94 (H-30), shown in Figure 3. The validity of the relative configurations shown in Figure 3 is further substantiated by the good agreement with the corresponding chemical shifts of polycavernoside D (5), both of which possess the same disaccharide moiety attached to a THP ring (Tables S1 and S2 in Supporting Information File 1) .

Table 2: NMR data for polycavernoside E (1) in CD3OD.

position δC, typea δHb (J in Hz) COSY selected HMBC 1 174.3, C       2 36.5, CH2 2.36, d (7.7) 3 1 3 83.2, CH 3.40, m 2   4 39.4, C       5 86.1, CH 3.40, m 6a, 6b   6a 38.0, CH2 1.91, m 5, 6b, 7   6b   1.80, ddd (11.9, 11.9, 11.9) 5, 6a, 7   7 76.6, CH 3.65, m 6a, 6b, 8a, 8b   8a 42.3, CH2 2.85, m 7, 8b 9 8b   2.37, m 7, 8a   9 207.4, C       10 104.8, C       11 39.7, CH 2.82, m 12a, 12b, 27   12a 34.6, CH2 1.98, m 11, 12b, 13   12b   1.62, m 11, 12a   13 83.8, CH 4.12, dd (11.6, 4.7) 12a, 12b   14 40.6, C       15 80.3, CH 5.10, d (8.1) 16 1 16 128.5, CH 5.61, dd (8.1, 15.1) 15, 17 18 17 136.4, CH 6.21, m 16, 18   18 131.1, CH 6.13, m 17   19 135.0, CH 6.19, m     20 132.4, CH 6.12, m 21   21 135.6, CH 5.72, dt (15.3, 7.1) 20, 22 19 22 32.7, CH2 2.22, m 21, 23