Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells

Abstract

<jats:p>The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS2 method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS1 data confirms the presence of the protonated alkaloids, and the MS2 fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN’s lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue.</jats:p>

Biosynthesis of Cylindrospermopsin in Cyanobacteria: Characterization of CyrJ the Sulfotransferase

“…7-Deoxy-desulfo-CYN has already been extracted and purified from C. raciborskii cell extracts. , We chose to purify this metabolite from the extracellular medium of a culture of Oscillatoria sp. PCC 10702 (Figure S3) because this strain produced a higher level of cylindrospermopsins compared to other strains, including Oscillatoria sp.…”

“…The purified 7-deoxy-desulfo-CYN showed a characteristic mass spectrum obtained by LC–MS using the product ion mode at different collision energies; the [M + H] + ion at m / z 320 gave the following characteristic product ions at m / z 194, 176, and 136 (Figure S4). , Furthermore, we unambiguously assigned the structure of 7-deoxy-desulfo-CYN by enzymatically transforming this compound into 7-deoxy-CYN, for which a standard is commercially available. We also quantified 7-deoxy-desulfo-CYN using this enzymatic transformation (see below).…”

“…These transitions are characteristic of the cylindrospermopsins and correspond to the loss of SO 3 and the uracil moiety (Figures S5–S8). , Figure shows the rate of incorporation of glycine into the cylindrospermopsins. Clearly, the initial incorporation rates as well as the maxima of incorporation followed the order of 7-deoxy-desulfo-CYN > 7-deoxy-CYN > 7- epi -CYN ≈ CYN.…”

“…Following the discovery of CYN, several close analogues (Figure ) have been isolated from cyanobacteria. − 7- epi -Cylindrospermopsin (7- epi -CYN, 2 ) is as toxic as CYN, while controversial data have been published regarding the toxicity of 7-deoxy-cylindrospermopsin (7-deoxy-CYN, 3 ). , 7-Deoxy-desulfo-cylindrospermopsin (7-deoxy-desulfo-CYN, 4 ) was isolated from a C. raciborskii strain, and its toxicity was recently assessed and determined that this compound is much less toxic than CYN . However, as stated above, more work is needed to fully understand the toxicity of CYN and its analogues.…”

Identification of 7-Deoxy-desulfo-argino-cylindrospermopsin, the Missing Piece in Cylindrospermopsin Biosynthesis

“…This fragmentation gives the m/z 194 ion that loses water to give the m/z 176 ion, both observed in the product ion spectra of cylindrospermopsins. 15,24,25 Figure 2B shows a typical precursor ion chromatogram (m/z 194 product ion) obtained from a sample of an extracellular culture medium of Oscillatoria sp. PCC 6506.…”