Stereochemical determination of NMR chemical shifts in marine terpenoids, antheliol and sangiangol B, using DFT calculations

Abstract Stereochemical determination of the flexible trinor-guaiane sesquiterpenoid, antheliol (1a) and the flexible diterpenoid, sangiangol B (2a), isolated from a marine soft coral, Anthelia sp., was supported by quantum chemical calculations of NMR chemical shifts at DFT levels. The relative configuration of antheliol is now revealed, as 1S*, 4S*, 7S*, 10R* as in 1b, whereas sangiangol B (2c) has complete stereochemistry as 1S*, 7R*, 8R*, 10R*, 11R*, 12S*. Graphical Abstract


Introduction
Structure determination, particularly stereochemical assignment, is often difficult, especially for conformationally flexible natural products, because NOE is sometimes not reliable enough to conclude their 3D chemical structures (Hanif et al. 2019). Moreover, the scarcity and unstable nature of such materials prevent further refinement of their 3D chemical structures through chemical derivatization. To overcome this problem, DFT-based NMR chemical shift calculations constitute a reliable method to circumvent this problem (Lodewyk et al. 2012;Ermanis et al. 2019;Hehre et al. 2019;Marcarino et al. 2020). Some flexible molecules have been revised on the basis of DFT calculations of NMR chemical shifts. Examples include glabramycins B and C (Li 2015), curcusones I and J (Sarotti 2018), cyclohelminthols CP-1, CP-2, CP-3, CP-4 (Inose et al. 2021), to name a few. Moreover, Hehre and co-workers (Hehre et al. 2019) have developed a practical method for DFT-based NMR structure conformation for flexible natural products, comprising five steps: (1) systematic conformational search with the Merck Molecular Force Field (MMFF) molecular mechanic model and removal of duplicate conformers with energies larger than 40 kJ/mol; (2) geometry calculation with the HF/3-21G model and removal of duplicate conformers and those with energies larger than 40 kJ/mol from that of the global minimum; (3) energy calculations with the xB97X-D/6-31G Ã model and removal of conformers with energies larger than 15 kJ/mol from that of the global minimum; (4) geometry calculation with the xB97X-D/6-31G Ã model and removal of conformers with energies larger than 10 kJ/mol from that of the global minimum; and (5) energy calculation with the xB97X-V/6-311 þ G(2df,2p)[6-311G Ã ] model. Finally, chemical shifts for all conformers within 10 kJ/mol of the global minimum were obtained using xB97X-D/6-31G Ã and were empirically corrected. This method has been optimised to give statistical decision d 13 C values expressed as root mean square errors (RMSE) less than 4.0 ppm and has been verified for about 2275 natural products (Hehre et al. 2019). This practical method was also useful for NMR chemical assignment of molecules possessing insufficient hydrogen atoms, such as polybrominated diphenyl ethers (Hanif et al. 2021).
In 2015, we discovered a rare, new trinor-guaiane sesquiterpene, which we name here, antheliol (1a). It was isolated from an Indonesian marine soft coral, Anthelia sp (Hanif et al. 2015). Its structure possesses an octahydroazulene skeleton, which is a flexible natural product. The planar structure of 1a was determined using 1D and 2D NMR, while the stereochemistry was refined with NOE and by comparing observed and calculated optical  rotation values. In 2017, clavuridin B (1c) (Gao et al. 2017) was discovered from the Xisha soft coral, Clavularia viridis, and the structure was confirmed by X-ray crystallography analysis to be same as 1a. Overall comparison of NMR data of the two molecules indicated that antheliol (1a) is a stereoisomer of 1c and the stereochemical revision of 1a, using DFT-based NMR chemical shift calculations, is one of the subjects of this article. Following the revision of antheliol (1a), a complete stereochemical structure of sangiangol B (2a) (Hanif et al. 2020) isolated from a marine soft coral Anthelia sp. is also presented.

Structural revision of antheliol
The original structure of antheliol was reported as in 1a (Figure 1) (Hanif et al. 2015) and it proved to be a stereoisomer of clavuridin B (1c), as determined with X-ray crystallography. Due to the flexible ring, we started an investigation with antheliol 1a and identified two possibilities for its true relative stereochemistry, having different configurations at C7: 1a (antheliol 7R) and 1b (antheliol 7S). After 1a and 1b were subjected to DFT-based NMR calculations using Spartan 0 20, the calculated 13 C NMR chemical shifts of 1a and 1b were obtained together with statistical data for max absolute, mean absolute, RMS errors and DP4 (Figure 2 and Table 1). The structure of 1a gave RMSE 3.29 ppm with DP4 0%, whereas the structure of 1b gave RMSE 1.28 ppm with DP4 100%, indicating that its relative configuration at C7 should be revised as 7S (1b, Figure 1). The relative configuration of antheliol (1b) was then determined as 1S Ã , 4S Ã , 7S Ã , 10R Ã . In addition, the different calculated and experimental 13 C NMR (Gao et al. 2017) chemical shifts for 1c (clavuridin B 7R) gave RMSE 0.91 ppm with DP4 100%, whereas 1d (clavuridin B 7S) gave RMSE 3.60 ppm with DP4 0% indicating that its relative configuration at C7 is truly as 7R. The structure of 1c determined by DFT calculations was in agreement with X-ray crystallography data. Both the results obtained from calculations indicate that the true structure of antheliol is as in 1b.

Complete stereochemical determination of sangiangol B
Structure determination of sangiangol B (2a) was accomplished with extensive NMR, including 1 H, 13 C, 1 H-1 H COSY, HSQC, HMBC, NOE/NOESY, specific optical rotation and molecular modelling as well as HRMS. As a result, five stereocenters were revealed out of six chiral centres. The remaining chirality at C-7 was solved using DFT calculation of NMR chemical shifts. Therefore, two diastereomers of sangiangol B, as in 2b and 2c ( Figure 1) were investigated using Hehre's protocol. After calculation of theoretical 13 C NMR chemical shifts, we found that 2b and 2c differed ( Figure 3 and Table 2). From the statistical analysis, the stereochemistry of sangiangol B is determined as in 2c (Figure 3) with the complete relative configuration: 1S Ã , 7R Ã , 8R Ã , 10R Ã , 11R Ã , 12S Ã .

Conclusion
In conclusion, through computational analysis of 13 C chemical shifts in NMR spectra of antheliol and sangiangol B, we have revised the relative stereochemical assignment at C-7 of antheliol to be 7S Ã and determined the relative configuration of sangiangol B at C-7 to be 7R Ã . The relative configuration of antheliol (1b) is now revealed as 1S Ã , 4S Ã , 7S Ã , 10R Ã , whereas sangiangol B (2c) has complete stereochemistry as 1S Ã , 7R Ã , 8R Ã , 10R Ã , 11R Ã , 12S Ã . This study serves as an additional example in a growing repertoire of work that demonstrates the utility of computational tools to aid structural determination efforts.

Supplementary materials
The following are available online, Figure S1. Experimental 1 H NMR Antheliol (1b