A Polyol-Polyol Super-Carbon-Chain Compound Containing Thirty-Six Carbon Stereocenters from the Dinoflagellate Amphidinium gibbosum: Absolute Configuration and Multi-Segment Modification

A super-carbon-chain compound, named gibbosol C, featuring a polyoxygenated C70-linear-carbon-chain backbone encompassing two acyclic polyol chains, was obtained from the South China Sea dinoflagellate Amphidinium gibbosum. Its planar structure was elucidated by extensive NMR investigations, whereas its absolute configurations, featuring the presence of 36 carbon stereocenters and 30 hydroxy groups, were successfully established by comparison of NMR data of the ozonolyzed products with those of gibbosol A, combined with J-based configuration analysis, Kishi’s universal NMR database, and the modified Mosher’s MTPA ester method. Multi-segment modification was revealed as the smart biosynthetic strategy for the dinoflagellate to create remarkable super-carbon-chain compounds with structural diversity.

Polyol-polyol compounds, however, are SCCCs featuring the presence of two polyol chains connected by a central core containing tetrahydropyran rings. So far, few examples have been reported. Ostreol B, isolated from the marine dinoflagellate Ostreopsis cf. ovata, could be classified as a polyol-polyol compound, although it only contains a tetrahydropyran ring as the central

Planar Structure and Ozonolysis of Gibbosol C (1)
The molecular formula of 1 was established as C76H142O32 with six degrees of unsaturation by the positive high resolution-electrospray ionization-mass spectrometry [ (Table 1), four degrees of unsaturation result from four carbon-carbon double bonds. Thus, the molecule must contain two rings. The 13 C NMR data and the DEPT135 experiment of 1 indicated the presence of 76 carbon resonances that can be categorized as seven methyl groups, 25 methylene groups (including an oxymethylene), 41 methine groups (including 5 olefinic and 33 oxymethine groups), and three non-protonated olefinic carbons.   (Table 1), four degrees of unsaturation result from four carbon-carbon double bonds. Thus, the molecule must contain two rings. The 13 C NMR data and the DEPT135 experiment of 1 indicated the presence of 76 carbon resonances that can be categorized as seven methyl groups, 25 methylene groups (including an oxymethylene), 41 methine groups (including 5 olefinic and 33 oxymethine groups), and three non-protonated olefinic carbons. Three substructures, viz., I (from C-1 to C-13, C-71, C-72, C-73, and C-74, in orange), II (from C-14 to C-33, C-75, and C-76, in green), and III (from C-34 to C-70, in pink), were determined by analysis of key 1 H-1 H COSY, H2BC, and HMBC correlations of 1 (Figure 1c).
Due to the heavy overlap of the 1 H and 13 C NMR signals of 1, the relative configurations of its stereogenic carbons could not be determined based on various 1D and 2D NMR data of the intact 1.
Comparison of the planar structure of 1 with that of gibbosol A (Figure 1) revealed that the structures of the C-5-C-11, C-17-C-33, and C-37-C-70 segments in 1 are the same as those of the C-4-C-10, C-18-C-34, and C-36-C-69 segments in gibbosol A, respectively. Based on the same biosynthetic machinery, the absolute configurations of the corresponding segments above should be identical. With detailed NMR data of three ozonolyzed products of gibbosol A (viz., gAa-c, Figure 2a) at hand [25], ozonolysis reaction was carried out for 1 to obtain the corresponding NMR data for comparison. As a result, O 3 /NaBH 4 -mediated cleavage of the carbon-carbon double bonds of 1 afforded three main fragments, viz., 1a, 1b, and 1c (Figure 2b, Tables S1-S3). Both 1b and 1c were obtained as epimeric pairs at C-13 and C-33, respectively.

Relative and Absolute Configurations of Gibbosol C (1)
Because gibbosols A and C were produced by the same marine dinoflagellate, the common biosynthetic origins of the two SCCCs should lead to identical absolute configurations of the corresponding segments in the three main pairs of the ozonolyzed fragments above. Detailed analysis of the NMR data led to the conclusion that the relative configurations of 1a, 1b, and 1c were similar to those of gAa, gAb, and gAc, respectively, except for the insertion of an additional methylene group between C-2 and C-4 in 1a, the presence of an additional 16-OH group in 1b and an additional 36-OH group in 1c, and the absence of the 13,15-diol and 16-Me (Me-73 in gAb) groups in 1b (Figure 2). Coincidentally, all these modifications appear on the starting segments within three ozonolyzed products of gibbosol A [25].
Mar. Drugs 2020, 18, x FOR PEER REVIEW 6 of 13 Comparison of the planar structure of 1 with that of gibbosol A (Figure 1) revealed that the structures of the C-5-C-11, C-17-C-33, and C-37-C-70 segments in 1 are the same as those of the C-4-C-10, C-18-C-34, and C-36-C-69 segments in gibbosol A, respectively. Based on the same biosynthetic machinery, the absolute configurations of the corresponding segments above should be identical.
With detailed NMR data of three ozonolyzed products of gibbosol A (viz., gAa-c, Figure 2a) at hand [25], ozonolysis reaction was carried out for 1 to obtain the corresponding NMR data for comparison. As a result, O3/NaBH4-mediated cleavage of the carbon-carbon double bonds of 1 afforded three main fragments, viz., 1a, 1b, and 1c (Figure 2b, Tables S1-S3). Both 1b and 1c were obtained as epimeric pairs at C-13 and C-33, respectively.

Relative and Absolute Configurations of Gibbosol C (1)
Because gibbosols A and C were produced by the same marine dinoflagellate, the common biosynthetic origins of the two SCCCs should lead to identical absolute configurations of the corresponding segments in the three main pairs of the ozonolyzed fragments above. Detailed analysis of the NMR data led to the conclusion that the relative configurations of 1a, 1b, and 1c were similar to those of gAa, gAb, and gAc, respectively, except for the insertion of an additional methylene group between C-2 and C-4 in 1a, the presence of an additional 16-OH group in 1b and an additional 36-OH group in 1c, and the absence of the 13,15-diol and 16-Me (Me-73 in gAb) groups in 1b ( Figure 2). Coincidentally, all these modifications appear on the starting segments within three ozonolyzed products of gibbosol A [25].
To determine the absolute configurations of C-2, C-7, and C-9 in 1a, the modified Mosher's MTPA method was used. Through a comparison of the sign of ∆δ SR values between 1as/1ar, the absolute configurations of C-2 and C-7 in 1a were determined to be S and R (Figure 3b), respectively [29]. In addition, the absolute configuration of C-9 was assigned as R by the widely separated H 2 -10 signals of 1ar (δ 4.29, 4.18) when compared with those of 1as (δ 4.18, 4.13) [30,31]. Based on this syn relationship between Me-71 and 7-OH, the absolute configuration of C-5 in 1a was established as R. Therefore, the absolute configurations of the stereogenic carbons in 1a were established as 2S,5R,7R,9R (Figure 3b), which are the same as those in gAa. Mar. Drugs 2020, 18, x FOR PEER REVIEW 7 of 13 3 Hz) revealed that both C-72/H-8a and C-10/H-8b were in gauche orientations. Therefore, two alternating conformers were assigned for the C-8−C-9 segment. Based on the results above, the relative configuration between 7-OH/Me-72 was determined as syn (Figure 3a).
To determine the absolute configurations of C-2, C-7, and C-9 in 1a, the modified Mosher's MTPA method was used. Through a comparison of the sign of Δδ SR values between 1as/1ar, the absolute configurations of C-2 and C-7 in 1a were determined to be S and R (Figure 3b), respectively [29]. In addition, the absolute configuration of C-9 was assigned as R by the widely separated H2-10 signals of 1ar (δ 4.29, 4.18) when compared with those of 1as (δ 4.18, 4.13) [30,31]. Based on this syn relationship between Me-71 and 7-OH, the absolute configuration of C-5 in 1a was established as R. Therefore, the absolute configurations of the stereogenic carbons in 1a were established as 2S,5R,7R,9R (Figure 3b), which are the same as those in gAa.
Based on Kishi's universal NMR database, the relative configurations of the C-17−C-25 segment in 1b were assigned as (anti/anti/anti/anti), the same as those of the C-18−C-26 segment in gAb (Figure  ). Therefore, the relationship between 36-OH and 37-OH was concluded to be syn (Figure 4b).
Furthermore, the relative configurations of the C-39−C-42 segment in 1c were assigned as syn/anti/anti (Table S1), the same as those of the C-38−C-41 segment in gAc [25], on the basis of Kishi's universal NMR database [27,28].
In the pathogenesis of atherosclerosis, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) promote the accumulation of macrophages within the intima, leading to formation of the atherosclerotic lesion [34][35][36]. The above two adhesion molecules, particularly VCAM-1, have been considered as potential therapeutic targets for anti-atherogenic drug development [37]. It is important to find promising VCAM-1 inhibitors from natural products. Thus, the effects of gibbosol C (1) on VCAM-1 and ICAM-1 in human umbilical vein endothelial cells were investigated according to previous procedures [25]. At the concentration range of 10.0 and 100.0 µg/mL, gibbosol C (1) showed no obvious activities on VCAM-1 and ICAM-1 expression ( Figure S1), whereas gibbosol A displayed remarkable activation effects on VCAM-1 expression. On the contrary, gibbosol B exhibited marked inhibitory activities on VCAM-1 expression [25].
The co-isolation of gibbosols A-C enabled us to summarize their structural features, which may shed light on the biosynthesis of these polyol-polyol SCCCs. The 13 carbon central cores (C-43-C-55); the C-4-C-10, C-18-C-34, and C-36-C-42 segments of the starting polyol chain; and the whole terminal polyol chain (C-56-C-69) of gibbosol A are quite conserved, whereas other segments of the starting polyol chain are variable. Diverse modification patterns, such as insertion, substitution, oxidation, and reduction, appear on the C-1-C-4, C-11-C-17, or C-34-C-35 segment of the starting polyol chain of gibbosol A. In other words, multi-segment modification on the starting polyol chain is the biosynthetic strategy for the generation of gibbosol C (1).
In addition, glycolate should be the starter unit in the biosynthesis of gibbosols A-C [38]. Though acetate labeling patterns of some polyol-polyene SCCCs, such as amphidinols A [38], 4 [2,39], and 17 [39,40], have been reported, no enzymatic mechanisms involved in the biosynthesis of these SCCCs have been uncovered so far [38][39][40][41]. Of course, the acetate labeling patterns of gibbosols A-C are worthy of further investigation in future.

General Experimental Procedures
HR-ESI-MS was obtained on a Bruker maXis ESI-QTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in the positive-ion mode. LR-ESI-MS was recorded on a Bruker amaZon SL mass spectrometer (Bruker Daltonics, Bremen, Germany) in both the positive-and negative-ion modes. One-and two-dimensional NMR spectra were measured on a Bruker AV-700 MHz NMR spectrometer (Bruker Scientific Technology Co. Ltd., Karlsruhe, Germany). UV spectra were recorded on a UV-2600 UV-Vis spectrophotometer (SHIMADZU, Kyoto, Japan) and optical rotations determined on an MCP 500 modular circular polarimeter (Anton Paar GmbH, Seelze, Germany) with a 1.0 cm cell at 25 • C. Preparative HPLC was performed on a Waters 2535 pump equipped with a YMC C 18 reversed-phase column (250 × 10 mm i.d., 5 µm, Kyoto, Japan) and a 2998 photodiode array detector coupled with a 2424 evaporative light scattering detector (Waters Corporation, Milford, NY, USA). For column chromatography, silica gel (100-200 mesh, Qingdao Mar. Chem. Ind. Co. Ltd., Qingdao, China) and C 18 reversed-phase silica gel (ODS-A-HG 12 nm, 50 µm, YMC, Kyoto, Japan) were employed.

Isolation of the Dinoflagellate and the Large-Scale Culture
The isolation and culture of the marine dinoflagellate Amphidinium gibbosum was described in our previous publication [25].

Isolation of Gibbosol C (1)
The filtrate of the culture medium (1200 L) was loaded onto a macroporous resin column (DIAION, HP-20, 120 cm × 15 cm i.d.), eluted with freshwater to remove sea salt. The loaded sample was successively eluted with 25%, 50%, 75%, and 95% aqueous ethanol. All the eluates were concentrated under reduced pressure to afford the resultant solid (5.5 g), which was separated by a C 18
1as: For 1 H NMR spectroscopic data, see

Conclusions
In summary, a new polyol-polyol SCCC, named gibbosol C, was isolated from the South China Sea dinoflagellate A. gibbosum. Its planar structure and absolute configurations, featuring the presence of 36 carbon stereocenters and 30 hydroxy groups, were successfully established by extensive NMR investigations, ozonolysis of the carbon-carbon double bonds, J-based configuration analysis, Kishi's universal NMR database, the modified Mosher's MTPA ester method, and comparison of the NMR data of the ozonolyzed products with those of gibbosol A. Multi-segment modification seems to be the smart biosynthetic strategy for the dinoflagellate to create remarkable SCCCs with diverse structures. Marine dinoflagellates of the genus Amphidinium harbor novel and complex SCCC biosynthetic routes. New integrated chemical, spectroscopic, and computational approaches or intelligent databases should be developed to cope with the stereochemical complexity of SCCCs. Evidently, specific carbon-carbon bond cleavages are an important means for the determination of the relative and absolute configurations of polyol-polyol SCCCs in the future.