June 22, 2022
by Mikhail Elyashberg, Leading Researcher, ACD/Labs
Previously unknown bacterial species are known to be found in deep sea habitats. The extreme environments found there have been shown to genetically segregate organisms as they adapt to them. Part of these evolutionary adaptations are new biosynthetic pathways giving rise to novel structures. The group of M. Jaspars has been working with samples of Dermacoccus abyssi MT 1.1T, a piezotolerant Actinomycete. It was collected from a depth of 10,898 m in 1998 and isolated in 2006 from a Mariana Trench sediment. A series of new phenazines were isolated and their structures elucidated . One of these compounds was dermacozine O (1).
Dermacozine O (1) was isolated as an ink-blue amorphous powder. The LC-HR-ESI-MSn spectrum gave an m/z of 398.1125 for [M + H]+, consistent with a molecular formula of C23H15O4N3 (calculated m/z 398.1135, D = −2.5 ppm), giving 18 degrees of unsaturation. As the ratio of skeletal atoms to hydrogens is equal to 2 the compound is highly hydrogen deficient and, consequently, its structure should be difficult to elucidate. The structure of dermacozine O was determined by the authors of  using 1D and 2D NMR spectra. We employed these data (see Table 1) for challenging the ACD/Structure Elucidator (ACD/SE).
Table 1. NMR spectroscopic data of compound 1. Weak HMBC correlations are distinguished by letter w.
|C/X Label||C||C Calc (HOSE)||XHn||H||H (M)||COSY||H to C HMBC|
|C 2||126.6||128.4||CH||7.87||u||7.78||C 12, C 13|
|C 3||131.3||132.5||CH||7.78||u||7.87, 7.97||C 1, C 5|
|C 4||120.3||117.21||CH||7.97||u||7.78||C 2, C 12|
|C 9||134.5||130.12||CH||7.21||d||C 7, C 8, C 11|
|C 10||129.7||134.16||CH||7.24||d||C 6, C 8, C 11|
|C 20||131.3||131.14||CH||7.3||u||7.47||C 18, C 22, C 21|
|C 21||128.2||128.41||CH||7.47||u||7.30, 7.41||C 20, C 19|
|C 22||127.9||128.61||CH||7.41||u||7.47||C 20|
|C 23||45.9||38.33||CH3||3.67||s||C 5, C 6|
|N 1||NH||11.27||s||C 7, C 18|
These data were entered into ACD/SE and the program created a Molecular Connectivity Diagram (MCD). The slightly edited MCD is presented in Figure 1.
Figure 1. Molecular connectivity diagram. Hybridizations of carbon atoms are marked by corresponding colors: sp2 – violet, sp3 – blue, not sp (sp2 or sp3) – light blue. Labels “ob” and “fb” are set to carbon atoms for which neighboring with heteroatom is either obligatory (ob) or forbidden (fb).
MCD overview. Only one carbon atom C 100.40 is colored as light blue, which means that, at the given chemical atom composition, the atom may be included into one of the following three fragments:
The carbon atom at 167.20 ppm is most probably a carbonyl, therefore the bond C=O was manually defined. Carbons at 150.6, 163.10 and 163.6 ppm can be assigned as those connected to a heteroatom (O or N) by ordinary or double bonds. These carbons are marked by a label “ob”.
Checking this MCD for contradictions indicated that the data is consistent, and no revisions are necessary. Strict Structure Generation (SSG) was initiated, accompanied by 13C chemical shift prediction, using the incremental approach and neural networks in combination with spectral filtering. The following results were obtained: k = 650,736 → (spectral filtering) → 187 → (removal duplicates) → 93, tg = 6 m 32 s.
Then 13C chemical shift prediction was performed using the HOSE code-based algorithm, and the output file was ranked in ascending order of average deviations dA(13C) of the experimental chemical shifts from the calculated ones. The six top ranked structures of the output file are shown in Figure 2.
Figure 2. The six top ranked structures of the output file.
Figure 2 shows that structure #1 which has the lowest deviations calculated by all three methods coincides with structure 1 suggested by the authors . The difference between deviations obtained for structures #1 and #2 is so large that the validity of the structure cannot be questioned. In addition, the competing structures seem too exotic for natural products. The structure of dermacozine O with the automatically assigned 13C chemical shifts is shown below:
Thus, the structure of a highly hydrogen deficient molecule was successfully elucidated by ACD/SE in ca. 7 min with only minor and self-evident edits of the MCD.