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There is an urgent need to discover new antimalarials, due to the spread of chloroquine resistance and the limited number of available drugs. A number of quinones have been shown to be effective antimalarials; the observed effects are most likely related to the most prominent chemical feature of these kind of molecules, that is their ability to undergo redox cycling. Antiplasmodial activity of quinone structures of marine origin has been reported. Examples are xestoquinone and halenaquinone (Laurent et al., 2006, Bioorg. Med. Chem., 14:4477-82), their derivative orlaquinone (Longeon et al., 2010, Bioorg. Med. Chem., 18:6006-11), and ketoadociaquinones (Shmitz et al., 1988, J. Org. Chem., 53:3922-25; Cao et al., 2005, Bioorg. Med. Chem., 13:999-1003), isolated from Xestospongia sponges, and thiaplakortones (Davis et al., 2013, J. Org. Chem., 78: 9608-13), isolated from Plakortis lita. It has been also evidenced that the presence of a dioxothiazine moiety enhances the antiplasmodial activity (Figure 1). Within our search for bioactive compounds from Mediterranean ascidians, we have previously isolated a large group of thiazinoquinones (Menna et al., 2013, Eur. J. Org. Chem,, 16:3241-46; Aiello et al., 2005, Eur. J. Org. Chem., 23:5024-30; Aiello et al., 2005, J. Med. Chem., 48:3410-16; Aiello et al., 2003, Eur. J. Org. Chem., 5: 98-900). Some of these compounds have been used as lead structures for the synthesis of simplified analogues for pharmacological screening (Aiello et al., 2010, Bioorg. Med. Chem., 18: 719-27). Based on the structural analogy with the reported antimalarial quinone structures, the synthetic derivatives prepared using the natural thiazinoquinones aplidinones A and B as model structures (1-12, Figure 2) have been tested in vitro against D10 (chloroquine-sensitive) and W2 (chloroquine-resistant) strains of P. falciparum. The synthetic derivatives have shown a significant antiplasmodial activity and many structural requirements, critical for their activity, have been evidenced. Conformational (MD and MM) and electronic (tautomeric form, pka values, percentage of neutral/ionized forms at different pH values) analysis have been performed by computational means. The propensity for electron acceptance of the active compounds has been explored by LUMO orbital and anionic radical analysis. A correlation between electrochemistry (???direct??? and in the presence of Fe(III)-Heme) of the studied compounds and their antiplasmodial effects has been evidenced by using solid-state electrochemical assays (Dome??nech-Carbo?? et al., 2013, Anal. Chem., 85: 4014-21).

Chemical investigation of the Mediterranean ascidian A.conicum resulted in the isolation of two new meroterpenes, the conithiaquinones A (1) and B (2), in addition to two previously reported chromenols (3 and 4) and conicaquinones (5 and 6). The structures of conithiaquinones A and B were determined by interpretation of spectroscopic data, and regiochemical and stereochemical assignments were achieved with the aid of computational methods, including the recently developed DP4 NMR spectral prediction method. Both conithiaquinones A and B showed significant effects on the growth and viability of cells, with 1 showing interesting cytotoxicity against human breast cancer cells.

The sulphate group is a common feature of echinoderm metabolites, like sulphated saponins with triterpenoid or steroidal aglycones and has also been encountered in sponges’ metabolites, in the form of sulphated sterols and phenol sulphates. Only a few examples of sulfated alkanes/alkenes have been reported from marine sources and, recently, they have been shown to be commonly present in remarkably amounts in Mediterranean tunicates.1 Most of these compounds, often having quite simple structures, are cytotoxic or antimicrobial. More complex polysubstituted alkyl sulfates have also been isolated from ascidians; a remarkable example is turbinamide, a bioactive metabolite isolated few years ago in our laboratory from Sidnyum turbinatum.2 This molecule demonstrated a strong and selective cytotoxic effect against neuronal rather than immune system cells.3 An extensive investigation we have carried out on several species of Mediterranean ascidians evidenced that complex sulphated polyketides are quite widespread in these organisms. We have indeed isolated two new polysubstituted alkyl sulphates from two different Mediterranean ascidians (Sidnyum elegans and Ciona edwardsii) whose structures are referable to that of turbinammide. The new compounds also exhibited a strong cytotoxic activity against C6 glioma cells; their structural analogy with turbinamide allowed also evaluating the influence of slight structural modifications on the activity.

The sulphate group is a common feature of echinoderm metabolites, like sulfated saponins with triterpenoid or steroidal aglycones and has also been encountered in sponges’ metabolites, in the form of sulphated sterols and phenol sulphates. Only a few examples of sulfated alkanes/alkenes have been reported from marine sources and, recently, they have been shown to be commonly present in remarkably amounts in Mediterranean tunicates. Most of these compounds, often having quite simple structures, are cytotoxic or antimicrobial. More complex polysubstituted alkyl sulfates have also been isolated from ascidians; a remarkable example is turbinamide, a bioactive metabolite isolated few years ago in our laboratory from Sidnyum turbinatum. This molecule demonstrated a strong and selective cytotoxic effect against neuronal rather than immune system cells. An extensive investigation we have carried out on several species of Mediterranean ascidians evidenced that complex sulphated polyketides are quite widespread in these organisms. We have indeed isolated two new compounds (1 and 2) very closely related to turbinammide from another collection of S. turbinatum; as well as two new molecules from two different Mediterranean ascidians (Sidnyum elegans and Ciona edwardsii) whose structures are referable to that of turbinammide as well.

Complex polysubstituted alkyl sulfates have been isolated from ascidians; a remarkable example is turbinamide, a bioactive metabolite isolated in our laboratory from Sidnyum turbinatum. This molecule demonstrated a strong and selective cytotoxic effect against neuronal rather than immune system cells. We have indeed isolated two new sulphated compounds (1 and 2) from another collection of S. turbinatum; as well as two new molecules (3 and 4) from two different Mediterranean ascidians (Sidnyum elegans and Ciona edwardsii). Their structural analogy with turbinamide allowed evaluating the influence of slight structural modifications on the activity.

Glycolipid analysis of the Caribbean sponge Amphimedon compressa showed it to contain two novel glycosphingolipids, amphiceramide A and B , possessing an unusual Δ6-phytosphingosine. The saccharide chain of amphiceramide A is composed of a β-glucose residue glycosylated at position 6 by an N-acetyl-β-glucosamine, and has never been found before in a natural product. The saccharide chain of amphiceramide B consists of an allolactose [Gal(1β→6)Glc] residue β-linked to the ceramide and is found here for the first time in a natural glycosphingolipid. In addition, the sponge contains a new molecular species of acetamidoglucosyl ceramide , and known glucosyl ceramide (halicerebroside A) and melibiosyl ceramide (amphimelibioside C).

Tyrosine-derived halometabolites frequently occur in marine organisms and are known to play basic functions related to the survival of the living creatures producing them.1 Bromine is by far the halogen most frequently found in these metabolites; for example, bromotyrosine derivatives are constantly detected among the secondary metabolites of the Demospongiae; thus, they have been used for taxonomic purposes.2-4 Iodinated halometabolites, biosynthetically related to tyrosine, are much less widespread in the marine environment, since they have been isolated just in a few algae, microorganisms and marine invertebrates;5 however, recent research indicates this number will increase in the future. Many of the isolated and identified iodine-containing metabolites exhibited biological activities.6 A chemical investigation of secondary metabolites from the Mediterranean ascidia Ciona edwardsii has been performed and a new iodinated derivative, named iodocionin (1) has been isolated and chemically characterized. Structure of 1 has been elucidated through extensive spectroscopic analysis (1D- and 2D-NMR, and MS). Tests in vitro have been performed by Abteilung für Angewandte Molekularbiologie, Institut für Physiologische Chemie, in order to evaluate the capability of compound 1 to affect cells viability; these studies demonstrated that iodocionin exhibits acute toxicity against lymphoma cells with an IC50 of 7.75 μg/ml.

The widespread use of coupling constant information for stereochemical assignment in pyranosides is based on the conformationally rigid six-membered ring they contain. For most pyranosides, one of the two possible chair conformations is largely predominant at room temperature, and this allows an easy discrimination between the large axial-axial couplings and the small axial-equatorial and equatorial-equatorial couplings. This is not the case for furanosides, where the flexibility of the five-membered ring (leading to the so called pseudorotation) causes large variations in coupling constants between conformers, which prevent such an easy correlation between coupling constant values and relative configuration of adjacent carbon atoms. As a consequence, stereochemical elucidation of furanosides is usually based on NOESY/ROESY correlations and/or chemical degradation. As a matter of fact, some correlation between vicinal couplings and relative configuration of furanosides is present in the literature. As early as in 1963, in a paper reporting the proton NMR spectra of all methyl pentofuranosides, it was clearly recognized that a "small" vicinal coupling in a furanoside implies that the coupled protons are trans oriented. However, this principle has been used in the subsequent structure elucidation work only sporadically, and, even then, in a way that resembles much more the empirical comparison with a model compound than the application of a general rule. The reason for this is probably that the threshold below which a coupling constant can be considered "small" has never been defined, making the practical application of this principle difficult. To clarify this point, we undertook an in-depth computational study of coupling constants of furanosides, and showed that in furanosides a vicinal coupling constant < 2.0 Hz (for H-1/H-2 or H-3/H-4) or < 3.5 Hz (for H-2/H-3) can be considered a proof of the trans orientation of the relevant protons.

The new diglycosylceramide terpioside has been isolated from the marine sponge Terpios sp. Terpioside is a diglycosylated glycosphingolipid which is the first example of a natural glycosphingolipid having an L-fucofuranose unit. The structure of terpioside was elucidated by extensive spectroscopic analysis, whereas chemical degradation was used to establish the nature of the alkyl chains and the absolute configuration of the sugars and of the ceramide stereogenic centers.

Reinvestigation of the glycosphingolipid composition of the marine sponge Spheciospongia vesparia revealed the presence of vesparioside B (2a), a new furanose-rich hexaglycosylated glycosphingolipid which is the most complex glycosphingolipid isolated from a marine sponge to date. The structure of the new compound was elucidated using extensive 2D NMR studies and chemical degradation. Particularly useful for structure elucidation of vesparioside B was a quantum mechanical computational study, showing that in furanosides a vicinal coupling constant < 2.0 Hz (for H-1/H-2 or H-3/H-4) or < 3.5 Hz (for H-2/H-3) is a proof of the trans orientation of the relevant protons. This general rule, combined with ROE data, allowed us to elucidate the relative stereochemistry (including anomeric configuration) of the three furanose five-membered rings.

Corrugoside (1a), a new immunostimulatory triglycosilated a-galactoglycosphingolipid, was isolated from the marine sponge Axinella corrugata, and its structure determined by spectral analysis and chemical degradation. Compound 1a activated murine NKT cells in vitro, with a potency of about 2 logs lower than that of aGalCer. Four stereoisomeric glycosphingolipids (2a–2d) were also obtained, b-glucosylceramides bearing unusual endoperoxide and allylic hydroperoxide functionalities on the sphinganine chain. They were shown to be photooxidation artifacts of the known glycosphingolipids 3, also present in the sponge. A possible role of compound 3 as a singlet oxygen scavenger to protect the organism from oxidative damage is proposed.

Chemical analyses of Plakortis simplex sponge extract provided a number of structurally unique secondary metabolites, including the antimalarial polyketide plakortin, the IL-10 inducing simplexide and other unusual glycolipids, and hopanoids (1-8). The hypothesis that they could be products of symbionts was explored by a multi-step strategy: i) cell separation of the fresh sponge tissue in combination with chromatographic and spectroscopic analysis; ii) PCR experiments to prove the presence of key enzymes of the biosynthesis of glycolipids and polyketides; iii) cultivation and screening of GSL-producing bacteria; iv) screening of metagenomic DNA of P. simplex in search of a PKS compatible with the structure of plakortin.

In the last ten years, glycolipids for marine organisms (sponges in particular) have been shown to be an exceptionally rich source of chemical diversity. These compounds often show peculiar biological activities and, for example, agelasphin, an -galactosylceramide first isolated form the sponge Agelas mauritiana, is the reference ligand for the CD1d protein, an antigen presenting molecule which binds and presents glycolipids to T lymphocytes. The most recent contributions in this field from our group will be presented. Simplexide (1), glycoside of a very-long-chain secondary alcohol, has been isolated sever years ago from the Caribbean sponges Plakortis simplex. We have now completed the total synthesis of simplexide, and shown that it is a potent stimulus for endogenous IL-10 production by Peripheral Blood Mononuclear Cells (PBMC), and more generally induces a unique profile of cytokine release. Vesparioside B (2) is a new glycosphingolipid isolated from the sponge Spheciospongia vesparia, characterized by a complex hexasaccharide chain with three sugars in the furanose form. Its structure elucidation led to establish a new method to correlate the relative configuration of the ring carbons to vicinal proton-proton coupling constant values in the five-membered furanose sugar. The same method was then used for the structure elucidation of another complex glycosphingolipid from a Terpios sponge.

The marine sponge Spheciospongia vesparia produces vesparioside (1a), a diglycosylated glycosphingolipid which is the first example of a natural diglycosylceramide with a pentose sugar residue. The structure of vesparioside was mostly determined by extensive spectroscopic analysis but determination of the nature of the alkyl chains and elucidation of the absolute stereochemistry of the sugars and of theceramide required chemical degradation. In this respect, an improved and simplified procedure for the microscale chemical degradation of glycosphingolipids was employed here for the first time.

The scientific interest on the immunological properties of glycosphingolipids (GSLs) is recently increased on account of the role that they could play as therapeutic agents. An interesting aspect of these properties is the immunomudulating activity reported for a number of GSls. Agelasphins, galactosylceramides isolated from marine sponge Agelas mauritiana, exhibited immunostimulating activity, which was suggested to be related to the interesting in vivo antitumoral properties of these compounds through an activation of the immune system. In our continuing studies on glycolipids from marine sponges we have examined the extracts of four species of the genus Agelas and we have isolated a whole new class of GSLs, characterized by an alpha-galactosyl as the sugar directly linked to the ceramide. All the GSLs isolated were tested for their ability to stimulate T-cell proliferation, and the obtained results suggest that the immunostimulating activity is affected by a specific structural feature of the GSLs, namely glycosylation of the inner sugar at position 2. In fact, all the immunostimulating compounds possess a free 2-OH on the sugar directly linked to the ceramide moiety, while this activity disappears if this position is glycosylated either by an alpha-galactosyl or by an alpha-glucosyl. It remains to be understood whether glycosylated alpha-Gal-GSLs are not active because the binding to the T-cell receptor is prevented by the steric hindrance of the additional sugar, or because the free galactose 2-OH group is directly involved in the binding to the receptor. In an attempt to clarify this point, we have been performing a synthetic program turned to the preparation of a number of analogs modified at the key position 2'. To date, we have developed efficient methodologies to synthesize alpha-Gal-GSLs modified at position 2'. In this communication we will report the total stereoselective synthesys of three new agelasphin-analogues shown below and the results of a study to evaluate their immunomodulating activities.

It is now well established that sponges of the genus Agelas and Axinella produce α-galactoglycosphingolipids (α-Gal-GSLs, e.g. 1–3), unique glycosphingolipids which are different from common β-glucosyl- or β-galactosyl ceramides from higher animals and plants in that they show an α-galactose as the first sugar of the carbohydrate chain. Natural α-Gal-GSLs possess interesting immunomodulating activities. They are potent ligands of the CD1d antigen presenting protein, and are capable to specifically activate natural killer T cells (N KT cells) in vivo. KRN7000 (4), a synthetic analogue the simplest α-Gal GSLs agelasphin (1) is under clinical trial as a novel anticancer agent, acting through stimulation of the immune system. As a part of our ongoing search of glycolipids from sponges, we discovered a number of more complex alpha-Gal-GSLs, which were tested on murine T-cell populations. The obtained data suggested that the immunostimulating activity is greatly reduced by glycosylation of the galactose 2-OH group, although some subsequent results obtained by other researchers seemed to be in contrast with these findings. This inconsistency was solved by a recent study, demonstrating that alpha-Gal-GSLs glycosylated at 2' or 3' positions are not active themselves, but are processed by the antigen presenting cells (APCs) with removal of the sugars linked to the galactose. In this scenario, we felt that the availability of analogs of alpha-Gal-GSLs, modified at the key position 2' in a way that cannot be affected by APC processing (e.g. 5), would be important to obtain clear-cut results. Therefore, we carried out the stereoselective total synthesis of the alpha-Gal-GSL analogs 5, and evaluated the influence of the structural modification on their immunostimulating activity. The synthetic procedures, the results of the assays, and their implications on the structural requirements for the immunostimulating activity will be discussed.Meanwhile, the analysis of natural alpha-Gal-GSLs from Agelas and Axinella sponges is far from being concluded, and these species are continuing to provide new glycosphingolipids with unprecedented glycosylation patterns. The latest results of this studies, which also contributed to clarify the structure-activity relationship of alpha-Gal-GSLs will be discussed.

Reinvestigation of the glycosphingolipid composition of the marine sponge Agelas clathrodes revealed the presence of a new tetraglycosylated R-galactoglycosphingolipid (1a), containing an unusual L-rhamnose unit in the sugar head. The structure of the new compound was elucidated using extensive 2D NMR studies. Because of the strong overlapping of the signals of the sugar protons in the 1H spectrum, 13C-coupled and 13C-decoupled phase-sensitive HMQC spectra were used to study the multiplicity of the overlapping signals. In addition, the absolute configuration of sugars was determined using a simple and efficient, yet underutilized CD method.

I glicosfingolipidi sono componenti delle membrane delle cellule eucariotiche, costituiti da una parte idrofila di natura saccaridica legata ad una parte lipofila, il ceramide. Studi recenti hanno portato all'isolamento e alla determinazione strutturale di una nuova classe di glicosfingolipidi (alfa-Gal-GSL), caratterizzati dalla presenza di un alfa-galattoside come primo zucchero della catena oligosaccaridica. Il più semplice alfa-Gal-GSL naturale, l'agelasfina, ha mostrato una interessante attività sul sistema immunitario, essendo capace di stimolare la proliferazione dei linfociti ed una notevole attività antitumorale. Il suo analogo sintetico, il KRN7000, è attualmente nella fase II della sperimentazione clinica come agente antitumorale. I primi tentativi per stabilire i requisiti strutturali necessari per l'attività immunostimolante hanno prodotto dati discordanti. I nostri studi sugli alfa-Gal-GSL naturali sottoposti a test in vitro condotti su linfociti T prelevati da linfonodi di topo suggerivano una chiara relazione struttura-attività: erano immunostimolanti gli alfa-Gal GSL che presentano il gruppo ossidrilico in posizione 2 del primo zucchero legato al ceramide non impegnato in legami glicosidici; al contrario, l'alfa-glucosil-(1→2)-alfa-galattosil ceramide e l' alfa-galattosil-(1→2)-alfa-galattosil ceramide risultavano essere inattivi. Opposti erano i risultati di test condotti su popolazioni linfocitarie miste da milza di topo, effettuati da un gruppo di ricercatori giapponesi. Studi recenti hanno risolto questa incongruenza, dimostrando che gli alfa-galattosil-GSL glicosilati alla posizione 2 o 3 del galattosio non sono attivi come tali, ma solo dopo la rimozione in vivo dello zucchero addizionale da parte di un alfa-glicosidasi lisosomiale. Non è ancora chiaro se gli alfa-Gal-GSL glicosilati non sono attivi a causa dell' ingombro sterico da parte dello zucchero in posizione 2 che impedisce il legame con il recettore, oppure se il gruppo ossidrilico come tale del galattosio è direttamente coinvolto nel legame. Per ottenere maggiori informazioni su questo punto, abbiamo iniziato un progetto sintetico volto alla preparazione di analoghi dei glicolipidi, modificati nella posizione 2 del galattosio, ma che non potessero subire modificazioni da parte dei sistemi enzimatici delle cellule del sistema immunitario. La comunicazione descrive la sintesi totale stereoselettiva del 2-deossigalattosil ceramide e quella, recentemente completata, del 2-O-metilgalattosil ceramide, ed i risultati preliminari dei saggi effettuati per delineare il profilo farmacologico dei composti sintetizzati.