Formation of Trideuteromethylated Artifacts of Pyrrole-Containing Natural Products.

Pyrrole-containing natural products form a large group of structurally diverse compounds that occur in both terrestrial and marine organisms. In the present study the formation of trideuteromethylated artifacts of pyrrole-containing natural products was investigated, focusing on the discorhabdins. Three deuterated discorhabdins, 1, 3, and 5, were identified to be isolation procedure artifacts caused by the presence of DMSO-d6 during NMR sample preparation and handling. Three additional semisynthetic derivatives, 7-9, were made during the investigation of the mechanism of formation, which was shown to be driven by trideuteromethyl radicals in the presence of water, methanol, TFA, and traces of iron in the deuterated solvent. Generation of trideuteromethylated artifacts was also confirmed for other classes of pyrrole-containing metabolites, namely, makaluvamines, tambjamines, and dibromotryptamines, which had also been dissolved in DMSO-d6 during the structure elucidation process. Semisynthetic discorhabdins were assessed for antiproliferative activity against a panel of human tumor cell lines, and 14-trideuteromethyldiscorhabdin L (3) averaged low micromolar potency.

Isolation and Structure Elucidation of Novel Mycosporine-like Amino Acids from the Two Intertidal Red Macroalgae Bostrychia scorpioides and Catenella caespitosa.

This study presents a phytochemical survey of two common intertidal red algal species, Bostrychia scorpioides and Catenella caespitosa, regarding their MAA (mycosporine-like amino acid) composition, which are known as biogenic sunscreen compounds. Six novel MAAs from Bostrychia scorpioides named bostrychines and two novel MAAs from Catenella caespitosa named catenellines were isolated using a protocol which included silica gel column chromatography, flash chromatography on reversed phase material and semipreparative HPLC (High-Performance Liquid Chromatography). The structure of the novel MAAs was elucidated using NMR (Nuclear Magnetic Resonance) and HR-MS (High-Resolution Mass Spectrometry), and their absolute configuration was confirmed by ECD (Electronic Circular Dichroism). All isolated MAAs possess a cyclohexenimine scaffold, and the metabolites from B. scorpioides are related to the known MAAs bostrychines A-F, which contain glutamine, glutamic acid and/or threonine in their side chains. The new MAAs from C. caespitosa contain taurine, an amino sulfonic acid that is also present in another MAA isolated from this species, namely, catenelline. Previous and new data confirm that intertidal red algae are chemically rich in MAAs, which explains their high tolerance against biologically harmful ultraviolet radiation.

An Investigation of Structure-Activity Relationships and Cell Death Mechanisms of the Marine Alkaloids Discorhabdins in Merkel Cell Carcinoma Cells.

A library of naturally occurring and semi-synthetic discorhabdins was assessed for their effects on Merkel cell carcinoma (MCC) cell viability. The set included five new natural products and semi-synthetic compounds whose structures were elucidated with NMR, HRMS, and ECD techniques. Several discorhabdins averaged sub-micromolar potency against the MCC cell lines tested and most of the active compounds showed selectivity towards virus-positive MCC cell lines. An investigation of structure-activity relationships resulted in an expanded understanding of the crucial structural features of the discorhabdin scaffold. Mechanistic cell death assays suggested that discorhabdins, unlike many other MCC-active small molecules, do not induce apoptosis, as shown by the lack of caspase activation, annexin V staining, and response to caspase inhibition. Similarly, discorhabdin treatment failed to increase MCC intracellular calcium and ROS levels. In contrast, the rapid loss of cellular reducing potential and mitochondrial membrane potential suggested that discorhabdins induce mitochondrial dysfunction leading to non-apoptotic cell death.

Analysis of the Mycosporine-Like Amino Acid (MAA) Pattern of the Salt Marsh Red Alga Bostrychia scorpioides.

This study presents the validation of a high-performance liquid chromatography diode array detector (HPLC-DAD) method for the determination of different mycosporine-like amino acids (MAAs) in the red alga Bostrychia scorpioides. The investigated MAAs, named bostrychines, have only been found in this specific species so far. The developed HPLC-DAD method was successfully applied for the quantification of the major MAAs in Bostrychia scorpioides extracts, collected from four different countries in Europe showing only minor differences between the investigated samples. In the past, several Bostrychia spp. have been reported to include cryptic species, and in some cases such as B. calliptera, B. simpliciuscula, and B. moritziana, the polyphyly was supported by differences in their MAA composition. The uniformity in the MAA composition of the investigated B. scorpioides samples is in agreement with the reported monophyly of this Bostrychia sp.

Cytotoxic Compounds of Two Demosponges (Aplysina aerophoba and Spongia sp.) from the Aegean Sea.

The class of demosponges is the biggest and most diverse of all described sponge species and it is reported to produce a plethora of chemically different metabolites with interesting biological activities. The focus of the present study was to investigate the chemical composition of two Mediterranean demosponges, targeting their brominated compounds and prenylated hydroquinones, compounds with interesting cytotoxic and anti-microbial properties. In order to gain a deeper insight into the chemical diversity of their metabolites and their activities, 20 pure secondary metabolites including new natural products were isolated from two different species (Aplysina aerophoba and Spongia sp.) using various chromatographic techniques. Their structures were confirmed by NMR and HRMS, revealing molecules with various chemical scaffolds, mainly prenylated hydroquinones from Spongia sp. and halogenated compounds from Aplysina aerophoba, including 5 novel natural products. The isolated compounds were investigated for their cytotoxic properties using 9 different cell lines, and especially one compound, 2,6-dibromo-4-hydroxy-4-methoxycarbonylmethylcyclohexa-2,5-dien-1-one showed good activities in all tested models.

Low temporal dynamics of mycosporine-like amino acids in benthic cyanobacteria from an alpine lake.

Cyanobacteria are one of the oldest organisms on Earth and they originated at a time when damaging ultraviolet (UV) C radiation still reached the surface. Their long evolution led to several adaptations to avoid deleterious effects caused by exposure to solar UV radiation. Synthesis of sunscreen substances, such as mycosporine-like amino acids (MAAs), allows them to photosynthesise with reduced risk of cell damage. The interplay of solar UV radiation and MAAs is well documented for cyanobacteria in the plankton realm, but little is known for those in the benthic realm, particularly of clear alpine lakes.Here, we assessed the temporal dynamics of MAAs in the benthic algal community of one clear alpine lake dominated by cyanobacteria during the ice-free season and along a depth gradient using state-of-the-art analytical methods (high-performance liquid chromatography, nuclear magnetic resonance, liquid chromatography-mass spectrometry). We differentiated between the epilithic cyanobacterial community and the overlying loosely attached filamentous cyanobacteria, as we expected they will have an important shielding/shading effect on the former. We hypothesised that in contrast to the case of phytoplankton, benthic cyanobacteria will show less pronounced temporal changes in MAAs concentration in response to changes in solar UV exposure.Three UV-absorbing substances were present in both types of communities, whereby all were unknown. The chemical structure of the dominant unknown substance (maximum absorption at 334 nm) resulted in the identification of a novel MAA that we named aplysiapalythine-D for its similarity to the previously described aplysiapalythine-C.Chlorophyll-a-specific MAA concentrations for epilithic and filamentous cyanobacteria showed a significant decrease with depth, although only traces were found in the former community. The temporal dynamics in MAA concentrations of filamentous cyanobacteria showed no significant variations during the ice-free season.Our result on the low temporal MAA dynamics agrees with the reduced growth rates of benthic cyanobacteria reported for cold ecosystems. The permanent presence of this community, which is adapted to the high UV levels characteristic of clear alpine lakes, probably represents the most important primary producers of these ecosystems.

Chemotaxonomic Study of Bostrychia spp. (Ceramiales, Rhodophyta) Based on Their Mycosporine-Like Amino Acid Content.

This study presents a chemotaxonomic investigation of the genus Bostrychia through the quantitation of the major mycosporine-like amino acids (MAAs). The presence of some cryptic species had been suggested in the B. moritziana/B. radicans complex and MAA-profiling in respective samples revealed different chemotypes within this species complex. Another possibly polyphyletic species is Bostrychia simpliciuscula; previous molecular phylogenetic analyses showed four genetic lineages within this species, one of which was recently distinguished as a new species. Phytochemical profiling of those samples used for DNA analyses revealed four different chemotypes, corresponding to the above four lineages and it supports the re-circumscription of the other three B. simpliciuscula lineages. Therefore, mycosporine-like amino acids are considered as suitable chemotaxonomic markers for the reassessment of the classification of B. simpliciuscula. The determination of the MAA patterns in these algae was possible after developing and validating a suitable high-performance liquid chromatography-diode array detector (HPLC-DAD) method.

Mycosporine-like amino acids, brominated and sulphated phenols: Suitable chemotaxonomic markers for the reassessment of classification of Bostrychia calliptera (Ceramiales, Rhodophyta).

Red algae (Rhodophyta) are primarily found in marine habitats around the world and they have been a prolific source of structurally diverse natural products. Among them the genus Bostrychia (Ceramiales, Rhodomelaceae) consists of approximately 40 taxonomically accepted species, and some, e. g., B. tenella, B. radicans, B. moritziana, B. simpliciuscula and B. intricate, include cryptic species. DNA sequence data show three genetic lineages within Bostrychia calliptera, another polyphyletic species requiring further taxonomic investigation. In an attempt to examine whether there are differences in the metabolite pattern that support the re-circumscription of the species and to uncover biogeographic patterns, phytochemical profiling of those samples used for DNA sequencing was conducted. This investigation revealed clearly three different chemotypes, corresponding to the lineages of the published molecular analysis results. The first lineage had a distinct and recognizably different phytochemical profile in contrast to the second and the third lineage which shared some similarities. Still, variations in the pattern of their phenolic compounds allowed a clear discrimination between the second and third lineage, too. The most important marker substances were isolated and their structures elucidated resulting in the characterization of four undescribed phenols. The isolated substances from B. calliptera are considered as suitable chemotaxonomic markers within this polyphyletic group.

Development and Validation of an HPLC Method for the Quantitative Analysis of Bromophenolic Compounds in the Red Alga Vertebrata lanosa.

Bromophenols are a class of compounds occurring in red algae that are thought to play a role in chemical protection; however, their exact function is still not fully known. In order to investigate their occurrence, pure standards of seven bromophenols were isolated from a methanolic extract of the epiphytic red alga Vertebrata lanosa collected in Brittany, France. The structures of all compounds were determined by NMR and MS. Among the isolated substances, one new natural product, namely, 2-amino-5-(3-(2,3-dibromo-4,5-dihydroxybenzyl)ureido)pentanoic acid was identified. An HPLC method for the separation of all isolated substances was developed using a Phenomenex C8(2) Luna column and a mobile phase comprising 0.05% trifluoroacetic acid in water and acetonitrile. Method validation showed that the applied procedure is selective, linear (R2 0.999), precise (intra-day ≤ 6.28%, inter-day ≤ 5.21%), and accurate (with maximum displacement values of 4.93% for the high spikes, 4.80% for the medium spikes, and 4.30% for the low spikes). For all standards limits of detection (LOD) were lower than 0.04 µg/mL and limits of quantification (LOQ) lower than 0.12 µg/mL. Subsequently, the method was applied to determine the bromophenol content in Vertebrata lanosa samples from varying sampling sites and collection years showing values between 0.678 and 0.005 mg/g dry weight for different bromophenols with significant variations between the sampling years. Bioactivity of seven isolated bromophenols was tested in agar diffusion tests against Staphylococcus aureus and Escherichia coli bacteria. Three compounds showed a small zone of inhibition against both test organisms at a concentration of 100 µg/mL.

Bostrychines A-F, Six Novel Mycosporine-Like Amino-Acids and a Novel Betaine from the Red Alga Bostrychia scorpioides.

Various red algae have repeatedly been reported to produce a variety of UV-absorbing mycosporine-like amino acids (MAAs), compounds that are well-known as natural sun-screens, as well as a plethora of betaines, metabolites which contribute to the osmotic balance under salt stress. Among other Rhodophyta, Bostrychia scorpioides, which is thriving as epiphyte on salt marsh plants in Europe and hence experiences extreme environmental conditions such as desiccation, UV-stress and osmotic stress, has barely been investigated for its secondary metabolites. In the present study, seven mycosporine like-amino acids and two betaines were isolated from Bostrychia scorpioides using various chromatographic techniques. Their structures were confirmed by Nuclear Magnetic Resonance (NMR) spectroscopy and High Resolution Mass Spectrometry (HRMS). Six MAAs and one betaine were chemically characterized as new natural products.

Absolute Configuration of Mycosporine-Like Amino Acids, Their Wound Healing Properties and In Vitro Anti-Aging Effects.

Mycosporine-like amino acids (MAAs) are water-soluble metabolites, reported to exhibit strong UV-absorbing properties. They have been found in a wide range of marine organisms, especially those that are exposed to extreme levels of sunlight, to protect them against solar radiation. In the present study, the absolute configuration of 14 mycosporine-like-amino acids was determined by combining the results of electronic circular dichroism (ECD) experiments and that of advanced Marfey's method using LC-MS. The crystal structure of a shinorine hydrate was determined from single crystal X-ray diffraction data and its absolute configuration was established from anomalous-dispersion effects. Furthermore, the anti-aging and wound-healing properties of these metabolites were evaluated in three different assays namely the inhibition of collagenase, inhibition of advanced glycation end products (AGEs) and wound healing assay (scratch assay).

Chemical profiling of mycosporine-like amino acids in twenty-three red algal species.

Rhodophyta produce a variety of chemically different mycosporine-like amino acids (MAAs), compounds that are known as some of the strongest ultraviolet (UV) absorbing molecules in nature. Accordingly, they primarily act as photoprotectants against harmful levels of solar ultraviolet radiation in the UV-A and UV-B range. In order to get a deeper understanding of the chemical diversity of MAAs in red algae, pure standards of eleven mycosporine-like amino acids were isolated from three different species (Agarophyton chilense, Pyropia plicata and Champia novae-zelandiae) using various chromatographic methods. Their structures were confirmed by nuclear magnetic resonance and mass spectrometry. Four out of the eleven MAAs are reported for the first time in algae. In addition, a new high-performance liquid chromatography method was developed for the separation of all isolated MAAs and successfully applied for the analysis of twenty-three red algal species of marine origin. All of them contained MAAs, the most abundant compounds were shinorine, palythine, asterina-330 and porphyra-334. For some samples, the direct assignment of MAAs based on their UV spectra was not possible; therefore, the target analytes were enriched by a simple concentration step, followed by liquid chromatography-mass spectrometry analysis of the extracts. This approach enabled a deeper insight into the MAA pattern of red algae, indicating that not only the four dominant ones are synthesized but also many others, which were often described as unknown compounds in previous studies.

Isolation and Structure Elucidation by LC-DAD-MS and LC-DAD-SPE-NMR of Cyclopeptide Alkaloids from the Roots of Ziziphus oxyphylla and Evaluation of Their Antiplasmodial Activity.

Nine cyclopeptide alkaloids (1-9), of which five (compounds 2, 3, 5, 8, and 9) are described herein for the first time, were isolated from roots of Ziziphus oxyphylla by means of conventional separation methods as well as semipreparative HPLC with DAD and ESIMS detection and LC-DAD-SPE-NMR. Structure elucidation was done by spectroscopic means. Nummularine-R (1), a previously known constituent from this species, was isolated along with its new derivatives O-desmethylnummularine-R (2) and O-desmethylnummularine-R N-oxide (3). In addition, the known compounds hemsine-A (4) and ramosine-A (6), as well as hemsine-A N-oxide (5), were isolated. Moreover, oxyphylline-C (7), a known constituent of Z. oxyphylla stems, was obtained, and two new compounds were identified, oxyphyllines-E (8) and -F (9). Just like oxyphylline-C, oxyphyllines-E and -F belong to the relatively rare class of neutral cyclopeptide alkaloids. The antiplasmodial activity and cytotoxicity of compounds 1, 2, 4, 6, and 9 were evaluated, and the most promising activity was found for O-desmethylnummularine-R (2), which exhibited an IC50 value of 3.2 ± 2.6 µM against Plasmodium falciparum K1, whereas an IC50 value of >64.0 µM was evident for its cytotoxicity against MRC-5 cells.

2-Aminoisobutyric acid in Co(II) and Co(II)/Ln(III) chemistry: homometallic and heterometallic clusters.

The synthesis and magnetic properties of 13 new homo- and heterometallic Co(II) complexes containing the artificial amino acid 2-amino-isobutyric acid, aibH, are reported: [Co(II)(4)(aib)(3)(aibH)(3)(NO(3))](NO(3))(4)·2.8CH(3)OH·0.2H(2)O (1·2.8CH(3)OH·0.2H(2)O), {Na(2)[Co(II)(2)(aib)(2)(N(3))(4)(CH(3)OH)(4)]}(n) (2), [Co(II)(6)La(III)(aib)(6)(OH)(3)(NO(3))(2)(H(2)O)(4)(CH(3)CN)(2)]·0.5[La(NO(3))(6)]·0.75(ClO(4))·1.75(NO(3))·3.2CH(3)CN·5.9H(2)O (3·3.2CH(3)CN·5.9H(2)O), [Co(II)(6)Pr(III)(aib)(6)(OH)(3)(NO(3))(3)(CH(3)CN)(6)]·[Pr(NO(3))(5)]·0.41[Pr(NO(3))(3)(ClO(4))(0.5)(H(2)O)(1.5)]·0.59[Co(NO(3))(3)(H(2)O)]·0.2(ClO(4))·0.25H(2)O (4·0.25H(2)O), [Co(II)(6)Nd(III)(aib)(6)(OH)(3)(NO(3))(2.8)(CH(3)OH)(4.7)(H(2)O)(1.5)]·2.7(ClO(4))·0.5(NO(3))·2.26CH(3)OH·0.24H(2)O (5·2.26CH(3)OH·0.24H(2)O), [Co(II)(6)Sm(III)(aib)(6)(OH)(3)(NO(3))(3)(CH(3)CN)(6)]·[Sm(NO(3))(5)]·0.44[Sm(NO(3))(3)(ClO(4))(0.5)(H(2)O)(1.5)]·0.56[Co(NO(3))(3)(H(2)O)]·0.22(ClO(4))·0.3H(2)O (6·0.3H(2)O), [Co(II)(6)Eu(III)(aib)(6)(OH)(3)(NO(3))(3)(CH(3)OH)(4.87)(H(2)O)(1.13)](ClO(4))(2.5)(NO(3))(0.5)·2.43CH(3)OH·0.92H(2)O (7·2.43CH(3)OH·0.92H(2)O), [Co(II)(6)Gd(III)(aib)(6)(OH)(3)(NO(3))(2.9)(CH(3)OH)(4.9)(H(2)O)(1.2)]·2.6(ClO(4))·0.5(NO(3))·2.58CH(3)OH·0.47H(2)O (8·2.58CH(3)OH·0.47H(2)O), [Co(II)(6)Tb(III)(aib)(6)(OH)(3)(NO(3))(3)(CH(3)CN)(6)]·[Tb(NO(3))(5)]·0.034[Tb(NO(3))(3)(ClO(4))(0.5)(H(2)O)(0.5)]·0.656[Co(NO(3))(3)(H(2)O)]·0.343(ClO(4))·0.3H(2)O (9·0.3H(2)O), [Co(II)(6)Dy(III)(aib)(6)(OH)(3)(NO(3))(2.9)(CH(3)OH)(4.92)(H(2)O)(1.18)](ClO(4))(2.6)(NO(3))(0.5)·2.5CH(3)OH·0.5H(2)O (10·2.5CH(3)OH·0.5H(2)O), [Co(II)(6)Ho(III)(aib)(6)(OH)(3)(NO(3))(3)(CH(3)CN)(6)]·0.27[Ho(NO(3))(3)(ClO(4))(0.35)(H(2)O)(0.15)]·0.656[Co(NO(3))(3)(H(2)O)]·0.171(ClO(4)) (11), [Co(II)(6)Er(III)(aib)(6)(OH)(4)(NO(3))(2)(CH(3)CN)(2.5)(H(2)O)(3.5)](ClO(4))(3)·CH(3)CN·0.75H(2)O (12·CH(3)CN·0.75H(2)O), and [Co(II)(6)Tm(III)(aib)(6)(OH)(3)(NO(3))(3)(H(2)O)(6)]·1.48(ClO(4))·1.52(NO(3))·3H(2)O (13·3H(2)O). Complex 1 describes a distorted tetrahedral metallic cluster, while complex 2 can be considered to be a 2-D coordination polymer. Complexes 3-13 can all be regarded as metallo-cryptand encapsulated lanthanides in which the central lanthanide ion is captivated within a [Co(II)(6)] trigonal prism. dc and ac magnetic susceptibility studies have been carried out in the 2-300 K range for complexes 1, 3, 5, 7, 8, 10, 12, and 13, revealing the possibility of single molecule magnetism behavior for complex 10.

Unique trigonal prism encapsulated Ln complexes: a [Co(II)6Eu] and a [Co(II)6Dy] cage.

The use of 2-amino-isobutyric acid in Co/Ln chemistry has led to the isolation of two unique [Co(II)(6)Ln(III)] 3d-4f metallic cages in which the Ln(III) centre (Ln = Eu, Dy) is encapsulated within a Co(II)(6) trigonal prism.