Boerelasin B Structure Elucidation

September 25, 2024

by Mikhail Elyashberg, Leading Researcher, ACD/Labs

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Boerelasin B

Cytochalasans, a unique class of natural products derived from fungal polyketide synthase−nonribosomal peptide synthetase (PKS−NRPS) pathways, are distinguished by a perhydro-isoindolone core fused with a macrocyclic ring. These compounds exhibit intricate structures and a wide range of biological activities. Through chemical investigation of the endophytic fungus *Boeremia exigua* (Didymellaceae), isolated from the leaves of the medicinal plant *Fritillaria monatha* Migo (syn.: *Fritillaria hupehensis* P. K. Hsiao & K. C. Hsia) (Liliaceae), Shi et al. [1] identified four novel compounds, including boerelasin B (1):

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Boerelasin B (1), a white amorphous powder, was determined to have the molecular formula C₂₉H₃₅NO₄ based on HR-ESI-MS. Its structure was determined from 1D and 2D NMR spectra as well as by comparison of the spectroscopic data with those of a known similar structure isolated from the same fungus.

The NMR data obtained (Table 1) were used by us for challenging Structure Elucidator Suite. The data were entered into Structure Elucidator, and a molecular Connectivity Diagram (MCD) was created (Figure 1).

Table 1. Spectroscopic NMR data obtained for boerelasin B.

Figure 1. Molecular connectivity diagram (MCD) of boerelasin B. Hybridizations of carbon atoms are marked by corresponding colors: sp² – violet, sp³ – blue, not sp – light blue. Labels “ob” and “fb” are set by the program to carbon atoms for which neighboring with heteroatom is either obligatory (ob) or forbidden (fb). HMBC connectivities are marked by green arrows, while COSY connectivities – by blue arrows. Ambiguous connectivities are marked by dotted lines.

We see that the MCD contains many ambiguous COSY connectivities, which is explained by the presence of four pairs of identical ¹H chemical shifts (marked by red in Table 1). Hybridization of the carbon at 99.50 ppm colored in light blue is also ambiguous as this atom can be in the states sp² (C=C) or sp³ (C-O). The mentioned ambiguities make the problem challenging for manual solution, but the program has means for automatically resolving this issue. To accelerate structure generation two evident carbonyl bonds and an O-H bond were manually drawn. The presence of a hydroxyl group was postulated as a characteristic strong absorption band was observed at 3300 cm^-1 in the IR spectrum of a similar compound isolated in work [1]. Introducing the O-H bond will prevent the generation of large number of erroneous structures containing an NH₂ group. IR spectra can give quite valuable structural information and should not be ignored by NMR spectroscopists!

Structure generation accompanied with ¹³C NMR chemical shift prediction by neural networks and incremental approaches was initiated, which was completed in 6.5 min with the following results: k = 5,388 → (structure filtering) → 90 → (duplicate removal) → 17. After the preliminary structure ranking in increasing order of deviations between experimental ¹³C chemical shifts and those predicted by neural networks [d_N(¹³C )], chemical shift calculation was performed for the top 15 structures using the HOSE code- based approach, and the file was ranked again. The top eight ranked structures of the output file are shown in Figure 2.

Figure 2. The top eight ranked structures of the output file. ¹³C chemical shift prediction was carried out using the HOSE code-based method, the neural networks, and the incremental approach. Average deviations of ¹³C chemical shifts determined by these methods are denoted as d_A, d_N and d_I correspondingly. Each atom is colored to mark a difference between its experimental and calculated ¹³C chemical shifts. The green color represents a difference between 0 to 3 ppm, yellow was >3 to 15 ppm, red > 15 ppm,.

As shown in Figure 2 the first ranked structure is identical to that which was proposed for boerelasin B by the authors [1]. To additionally check this structure’s validity, the DP4 probabilities of structure correctness were calculated for the four top ranked structures (Figure 3).

Figure 3. The top four ranked structures of the ranked output file. ¹³C chemical shift prediction was carried out using the HOSE code-based method, the neural networks, and the incremental approach. Average deviations of ¹³C chemical shifts determined by these methods are denoted as d_A, d_N and d_I correspondingly. Each atom is colored to mark a difference between its experimental and calculated ¹³C chemical shifts. The green color represents a difference between 0 to 3 ppm, yellow was >3 to 15 ppm. The DP4_A, DP4_N and DP4_I are probabilities of structure correctness calculated by the program.

We see that the DP4_A value calculated from HOSE code-based chemical shift prediction (the most accurate approach among three ones implemented into Structure Elucidator), is equal ~100% for the first ranked structure, and DP4_I = 100% for it. These data confirm correctness of the found solution to the problem.

The structure of boerelasin B with ¹³C chemical shifts assigned to carbon atoms by the program is shown below:

This example shows how Structure Elucidator successfully copes with ambiguity stemming from the presence of four pairs of identical chemical shifts in ¹H spectrum during structure elucidation of complex natural product.

References

1. B.-B. Shi, C. Tian, X. Lv, J. Schinnerl, K. Ye, H. Guo, F. Xu, Y. He, H.-L. Ai, J.-K. Liu. (2023). Boerelasins A−D, Four Unprecedented Cytochalasins from the Endophytic Fungus Boeremia Exigua. J. Org. Chem., 88, 13926−13933.

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