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. 2020 Aug 20;25(17):3786.
doi: 10.3390/molecules25173786.

Discovery of Unusual Cyanobacterial Tryptophan-Containing Anabaenopeptins by MS/MS-Based Molecular Networking

Affiliations

Discovery of Unusual Cyanobacterial Tryptophan-Containing Anabaenopeptins by MS/MS-Based Molecular Networking

Subhasish Saha et al. Molecules. .

Abstract

Heterocytous cyanobacteria are among the most prolific sources of bioactive secondary metabolites, including anabaenopeptins (APTs). A terrestrial filamentous Brasilonema sp. CT11 collected in Costa Rica bamboo forest as a black mat, was studied using a multidisciplinary approach: genome mining and HPLC-HRMS/MS coupled with bioinformatic analyses. Herein, we report the nearly complete genome consisting of 8.79 Mbp with a GC content of 42.4%. Moreover, we report on three novel tryptophan-containing APTs; anabaenopeptin 788 (1), anabaenopeptin 802 (2), and anabaenopeptin 816 (3). Furthermore, the structure of two homologues, i.e., anabaenopeptin 802 (2a) and anabaenopeptin 802 (2b), was determined by spectroscopic analysis (NMR and MS). Both compounds were shown to exert weak to moderate antiproliferative activity against HeLa cell lines. This study also provides the unique and diverse potential of biosynthetic gene clusters and an assessment of the predicted chemical space yet to be discovered from this genus.

Keywords: Brasilonema; anabaenopeptins; antiproliferative activity; hexapeptides; molecular networking; tryptophan-containing peptides.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A). The molecular network with anabaenopeptins (APT) cluster obtained from the organic extract of Brasilonema sp. CT11. Nodes are labeled with parent m/z values and edges with the mass difference. Node size is indicative of the ion count; edge thickness is relative to cosine score. (B). Four APTs detected using LC-HRMS/MS following general structure X1-CO-[Lys2-X3-X4-MeX5-X6], wherein the X1 and X3-6 are non-proteinogenic or proteinogenic amino acids.
Figure 2
Figure 2
Anabaenopeptin synthetase gene cluster arrangement of Brasilonema CT11 yielding four anabaenopeptins 1, 2a, 2b, and 3. (A) Gene map of the apt biosynthetic gene cluster. (B) Proposed biosynthesis of anabaenopeptins in Brasilonema sp. CT11. A—adenylation domain; C—condensation domain; E—epimerization domain; MT—methyltransferase domain; PCP—peptidyl-carrier protein; TE—thioesterase domain.
Figure 3
Figure 3
HR-MS/MS product ion spectra of protonated anabaenopeptins from Brasilonema CT11; (a) compound 1, (b) compound 2a/2b, and (c) compound 3.
Figure 4
Figure 4
Most significant correlations provided by COSY and HMBC 2D spectra of compounds 2a and 2b.
Figure 5
Figure 5
Cell viability was assessed by MTT assay at 48 h exposure time. Cell viability was calculated as the percentage of viable cells in 2a and 2b compounds-treated cells relative to control at 20, 10, and 5 µM concentration. Data are presented as mean ± SD; n = 3. * p ≤ 0.05. STS stands for Staurosporine, used as positive control at 1 µM concentration.

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