Norflavicansone Structure Elucidation

July 18, 2024

by Mikhail Elyashberg, Leading Researcher, ACD/Labs

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Norflavicansone

Biodiscovery initiatives in Indonesia have focused on exploring the largely untapped chemical diversity of the country’s abundant lichen flora, aiming to discover new products with notable biological properties. Chemical screening of the Teloschistes flavicans extract identified this species as a candidate for further study, with recent findings published by Ferron and coworkers [1].

Six chlorodepsidones from the thallus of a sample of lichen have been isolated and identified, including three new compounds: norflavicansone, flavicansone, and isocaloploicin.

The structure elucidation of these proton-deficient metabolites was carried out on the basis of a synergistic combination of Computer-Assisted Structure Elucidation (CASE) and Density Functional Theory (DFT) calculations. The CASE expert system ACD/Structure Elucidator Suite was used in this research.

Here we will discuss the structure elucidation of norflavicansone (1) as was described in work [1].

1

Compound 1 was isolated as an amorphous solid with an exact mass of C₁₇H₁₄Cl₂O₅, as derived from the HR-ESI-MS [M−H]⁻ signal detected at m/z 367.0145 (calcd. for C₁₇H₁₃³⁵Cl₂O₅⁻, 367.0146). We see that the molecule is a highly hydrogen deficient one: the ratio of hydrogen atoms to skeletal atoms is 0.6, which makes structure elucidation very challenging. Using chemical knowledge, reference data and biosynthetic considerations, authors suggested the possible structure of norflavicansone but its confirmation by computational methods was necessary.

The 1D and 2D NMR data (COSY, HSQC and HMBC) which were used for structure elucidation are presented in Table 1.

Table 1. Spectroscopic NMR data of compound 1. Weak HMBC peaks are denoted by “w”.

The data presented in Table 1 were entered into Structure Elucidator, and a molecular Connectivity Diagram (MCD) was automatically created by the program (Figure 1). The lengths of the weak HMBC correlations was set as 2-4 chemical bonds.

Figure 1. The Molecular Connectivity Diagram for compound 1. Carbon atoms’ hybridizations are color-coded as violet for sp² and blue for sp³. Standard HMBC connectivities (2-3 bonds length) are marked by green arrows, long-range connectivities (2-4 bonds lengths) are displayed as violet arrows. Labels ob (obligatory) and fb (forbidden) indicate the admissibility of a neighbor heteroatom. One hydrogen, four oxygen, and two chlorine heteroatoms appear in the lower left corner for consistency with the determined molecular formula.

Checking the MCD for consistency showed that 2D NMR data contain contradictions, meaning that there are nonstandard correlations (NSC) present. NSCs are those whose lengths exceed 3 chemical bonds. Therefore, Fuzzy Structure Generation was initiated. The structure generation was completed with the following results: k = 69472 → 185 → 22, t_g = 8 min 50 s, where k is the number of generated structures from which 185 passed spectral and structural filtering and 22 structures being finally saved after removing duplicates. t_g is the processing time.

¹³C chemical shift prediction was performed for all structures of the output file using the three empirical methods of chemical shift calculation supported by ACD/SE: HOSE code based, neural networks and incremental. The structures were then ranked in increasing order of average deviations of experimental chemical shift from calculated values. The four top ranked structures are shown in Figure 2.

Figure 2. The four top-ranked structures of the output file according to d_A values. Atoms are colored to indicate the difference between its experimental and calculated ¹³C NMR chemical shift values. Green color corresponds to a difference between 0 and 3 ppm while yellow indicates a difference between 3 and 15 ppm. Average deviations are provided for the three methods of prediction: d_A, HOSE code; d_N, neural networks; d_I, incremental approach, along with the maximal deviation for each approach.

Only the two first ranked structures can be considered as likely candidates because the third and fourth have larger deviations and have no chemical sense. It is known that methyl groups connected to aromatic rings with oxygen atoms on both sides have chemical shifts between 7 and 12 ppm, hinting that the yellow atoms 10.90 and 18.6 should be swapped. Interconverting these two assignments led to the final solution with decreased deviations:

Figure 3. The two top-ranked candidates from the output file of the CASE analysis of compound 1. DP4_A, DP4_N and DP4_I are probabilities of structure validity calculated by the program.

The first-ranked structure coincided with the hypothesis which was suggested from a manual structure elucidation. Besides having the smallest deviation, its DP4 probability values ranging from 97.68% and 99.99%, strongly suggesting this CASE-proposed structure to be correct. To further strengthen this conclusion (as recommended in [2]), DFT calculations of the ¹³C chemical shifts were performed on the two first candidates at the mPW1PW91/6-311+G(2d,p) level on geometries optimized at the B3LYP/6-31+G(d,p) level. All statistical parameters of DFT-predicted chemical shifts (RMSD, max_dev and r) are clearly in favor of the CASE-preferred structure:

Parameters	Structure 1	Structure 2
RMSD, ppm	2.18	3.09
max_dev, ppm	5.0	7.7
r	0.9991	0.9981

 

Thus, this CASE-DFT analysis reliably established the challenging structure of norflavicansone.

References

1. Ferron, S.; Ismed, F.; Elyashberg, M. E.; Buevich, A. V.; Arifa, N.; Boustie, J.; Uriac, P.; Le Pogam, P.; Le Dévéhat, F. (2024). CASE-DFT Structure Elucidation of Proton-Deficient Chlorodepsidones from the Indonesian Lichen Teloschistes flavicans and Structure Revision of Flavicansone. Nat. Prod. In print. https://doi.org/10.1021/acs.jnatprod.4c00277
2. Buevich, A. V.; Elyashberg, M. E. (2016). Synergistic combination of CASE algorithms and DFT chemical shift predictions: a powerful approach for structure elucidation, verification and revision. J. Nat. Prod., 79(12), 3105–3116. DOI: https://doi.org/10.1021/acs.jnatprod.6b00799

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