Chemical and Biological Study of Novel Aplysiatoxin Derivatives from the Marine Cyanobacterium Lyngbya sp.

Since 1970s, aplysiatoxins (ATXs), a class of biologically active dermatoxins, were identified from the marine mollusk Stylocheilus longicauda, whilst further research indicated that ATXs were originally metabolized by cyanobacteria. So far, there have been 45 aplysiatoxin derivatives discovered from marine cyanobacteria with various geographies. Recently, we isolated two neo-debromoaplysiatoxins, neo-debromoaplysiatoxin G (1) and neo-debromoaplysiatoxin H (2) from the cyanobacterium Lyngbya sp. collected from the South China Sea. The freeze-dried cyanobacterium was extracted with liquid–liquid extraction of organic solvents, and then was subjected to multiple chromatographies to yield neo-debromoaplysiatoxin G (1) (3.6 mg) and neo-debromoaplysiatoxin H (2) (4.3 mg). They were elucidated with spectroscopic methods. Moreover, the brine shrimp toxicity of the aplysiatoxin derivatives representing differential structural classifications indicated that the debromoaplysiatoxin was the most toxic compound (half inhibitory concentration (IC50) value = 0.34 ± 0.036 µM). While neo-aplysiatoxins (neo-ATXs) did not exhibit apparent brine shrimp toxicity, but showed potent blocking action against potassium channel Kv1.5, likewise, compounds 1 and 2 with IC50 values of 1.79 ± 0.22 µM and 1.46 ± 0.14 µM, respectively. Therefore, much of the current knowledge suggests the ATXs with different structure modifications may modulate multiple cellular signaling processes in animal systems leading to the harmful effects on public health.


Morphological and Molecular Identification of Cyanobacterium
The cyanobacterium strain used in this study were collected from Harbor of Hainan Sanya, China, Named as cyanobacterium HN. Colonies of cyanobacterium HN appeared as dark red, brown, or black tufts ranging from 15 to 25 cm in length and grew attached to sea rock and surface of the sea.
Filament width, cell width, and cell length of cyanobacterium HN were measured on the compound light microscope (Zeiss, Germany) with a 20× objective and 10× ocular lens with a calibrated optical micrometer. Filaments were long, of indeterminate length, 55-65 μm wide, formed by a uniseriate row of discoid cells encased in a firm, colorless, hyaline sheath which, when old, became yellowed and distinctly lamellated. Cells were discoid, 6-8 μm long, 30-40 μm broad, with rounded end cells without calyptra. Cell contents were finely granular without prominent granular inclusions ( Figure S1.1A).
Cyanobacterium HN held the highest 16S rRNA gene similarity with Lyngbya sp. CENA128 T with the value of 99%, revealing that cyanobacterium HN might belong to Lyngbya sp. The phylogenetic trees based on the 16S rRNA gene sequences, reconstructed with the Bayesian MCMC methods, showed that cyanobacterium HN fell into the clade comprising Lyngbya species and formed a stable clade with Lyngbya sp. CENA128 T ( Figure S1.1B). According to these results, cyanobacterium HN belonged to Lyngbya sp.

Brine Shrimp Cytotoxicity Assay
Commercially available Artemia salina (A. salina) or brine shrimp cysts were purchased and cultivated in 3.2% of saline water. Before cultivation, the saline was aerated, and then cysts were kept at room temperature for 24 h. For toxicity screening, hatched larvae were collected and introduced in saline water. Add saline water and equivalent larvae in per well of 96 wells to make test culture plate. Aplysiatoxins with 0.1 μM, 1 μM, 10 μM, 30 μM was added to test culture plate, while DMSO and dichloromethane were added as blank control test and positive control separately. After 24 h in 25 °C, the percent of survival of A. salina was calculated.

Methods for NMR Calculation of Neo-Debromoaplysiatoxin G (1)
Monte Carlo conformational searches were carried out by means of the Spartan's 10 software (Spartan Software, San Francisco, CA, USA) using Merck Molecular Force Field (MMFF). The conformers with Boltzmann-population of over 1% were chosen for NMR calculations, and then the conformers were initially optimized at B3LYP/6-31g (d, p) level in gas. Meanwhile, gaugeindependent atomic orbital (GIAO) calculations of 1 H and 13 C NMR chemical shifts were accomplished by density functional theory (DFT) at the mPWLPW91-SCRF (methanol)/6−311+g (d, p) level with the PCM solvent continuum model in Gaussian 09 software (Gaussian, Wallingford, CT, USA). The calculated NMR data of the lowest energy conformers for isomer 1, isomer 2 were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy. The 1 H and 13 C NMR chemical shifts for TMS were calculated by the same protocol and used as reference. The experimental and calculated data were analyzed by the improved probability DP4 + method for isomeric compounds. A significant higher DP4 + probability score suggested the correctness of its configuration.                        (Table S3.1).