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Targeting bromodomain-containing protein 9 (BRD9) represents a promising strategy for the development of new agents endowed with anticancer properties. With this aim, a set of 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based compounds was investigated following a combined approach that relied on in silico studies, chemical synthesis, biophysical and biological evaluation of the most promising items. The protocol was initially based on molecular docking experiments, accounting a library of 1896 potentially synthesizable items tested in silico against the bromodomain of BRD9. A first set of 21 compounds (1-21) was selected and the binding on BDR9 was assessed through AlphaScreen assays. The obtained results disclosed compounds 17 and 20 able to bind BRD9 in the submicromolar range (IC50 = 0.35 ± 0.18 μM and IC50 = 0.14 ± 0.03 μM, respectively) showing a promising selectivity profile when tested against further nine bromodomains. Taking advantage of 3D structure-based pharmacophore models, additional 10 derivatives were selected in silico for the synthetic step and binding assessment, highlighting seven compounds (22, 23, 25, 26, 28, 29, 31) able to selectively bind BRD9 among different bromodomains. The ability of the identified BRD9 binders to cross artificial membranes in vitro was also assessed, revealing a very good passive permeability profile. Preliminary studies were carried out on a panel of healthy and cancer human cell lines to explore the biological behavior of the selected compounds, disclosing a moderate activity and significant selectivity profile towards leukaemia cells. These results highlighted the applicability of the reported multidisciplinary approach for accelerating the selection of promising items and for driving the chemical synthesis of novel selective BRD9 binders. Moreover, the low molecular weight of the reported 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based BRD9 binders suggests the possibility for further exploring the chemical space in order to obtain new analogues with improved potency.

The multiple inhibition of biological targets involved in pro-inflammatory eicosanoid biosynthesis represents an innovative strategy for treating inflammatory disorders in light of higher efficacy and safety. Herein, following a multidisciplinary protocol involving virtual combinatorial screening, chemical synthesis, and in vitro and in vivo validation of the biological activities, we report the identification of 1,2,4-oxadiazole-based eicosanoid biosynthesis multi-target inhibitors. The multidisciplinary scientific approach led to the identification of three 1,2,4-oxadiazole hits (compounds 1, 2 and 5), all endowed with IC50 values in the low micromolar range, acting as 5-lipoxygenase-activating protein (FLAP) antagonists (compounds 1 and 2), and as a multi-target inhibitor (compound 5) of arachidonic acid cascade enzymes, namely cyclooxygenase-1 (COX-1), 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase-1 (mPGES-1). Moreover, our in vivo results demonstrate that compound 5 is able to attenuate leukocyte migration in a model of zymosan-induced peritonitis and to modulate the production of IL-1β and TNF-α. These results are of interest for further expanding the chemical diversity around the 1,2,4-oxadiazole central core, enabling the identification of novel anti-inflammatory agents characterized by a favorable pharmacological profile and considering that moderate interference with multiple targets might have advantages in re-adjusting homeostasis.

[Image: see text] Microsomal prostaglandin E(2) synthase-1 (mPGES-1), the terminal enzyme responsible for the production of inducible prostaglandin E(2), has become an attractive target for the treatment of inflammation and cancer pathologies. Starting from an aminobenzothiazole scaffold, used as an unprecedented chemical core for mPGES-1 inhibition, a Combinatorial Virtual Screening campaign was conducted, using the X-ray crystal structure of human mPGES-1. Two combinatorial libraries (6 × 10(4)) were obtained by decorating the aminobenzothiazole scaffold with all acyl chlorides and boronates available at the Merck database. The scientific multidisciplinary approach included virtual screening workflow, synthesis, and biological evaluation and led to the identification of three novel aminobenzothiazoles 1, 3, and 13 acting as mPGES-1 inhibitors. The three disclosed hits are able to inhibit mPGES-1 in a cell-free system (IC(50) = 1.4 ± 0.2, 0.7 ± 0.1, and 1.7 ± 0.2 μM, respectively), and all are endowed with antitumoral properties against A549 human cancer cell lines at micromolar concentrations (28.5 ± 1.1, 18.1 ± 0.8, and 19.2 ± 1.3 μM, respectively).

Tesi di laurea quinquennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquennale in Chimica e Tecnologie Farmaceutiche

Inverse Virtual Screening (IVS) is a docking based approach aimed to the evaluation of the virtual ability of a single compound to interact with a library of proteins. For the first time, we applied this methodology to a library of synthetic compounds, which proved to be inactive towards the target they were initially designed for. Trifluoromethyl-benzenesulfonamides 3-21 were repositioned by means of IVS identifying new lead compounds (14-16, 19 and 20) for the inhibition of erbB4 in the low micromolar range. Among these, compound 20 exhibited an interesting value of IC50 on MCF7 cell lines, thus validating IVS in lead repurposing.

Tesi di laurea quinquiennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquiennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquiennale in Chimica e Tecnologie Farmaceutiche

[Image: see text] JMJD3 is a member of the KDM6 subfamily and catalyzes the demethylation of lysine 27 on histone H3 (H3K27). This protein was identified as a useful tool in understanding the role of epigenetics in inflammatory conditions and in cancer as well. Guided by a virtual fragment screening approach, we identified the benzoxazole scaffold as a new hit suitable for the development of tighter JMJD3 inhibitors. Compounds were synthesized by a microwave-assisted one-pot reaction under catalyst and solvent-free conditions. Among these, compound 8 presented the highest inhibitory activity (IC(50) = 1.22 ± 0.22 μM) in accordance with molecular modeling calculations. Moreover, 8 induced the cycle arrest in S-phase on A375 melanoma cells.

The quinoline‐5,8 dicarboxylic acid scaffold has been identified by a fragment‐based approach as new potential lead compound for the development of JMJD3 inhibitors. Among them, 3‐(2,4‐dimethoxypyrimidin‐5‐yl)quinoline‐5,8‐dicarboxylic acid (compound 3) shows low micromolar inhibitory activity against Jumonji domain‐containing protein 3 (JMJD3). The experimental evaluation of inhibitory activity against seven related isoforms of JMJD3 highlighted an unprecedented selectivity toward the biological target of interest.

Tesi di laurea quinquiennale in Chimica e Tecnologie Farmaceutiche

tesi di laurea quinquiennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquiennale in Chimica e Tecnologie Farmaceutiche

Microsomal Prostaglandin E syntase 1 (mPGES-1) has been recognized as a novel, promising target for anti-inflammatory and anticancer drugs development being involved in a number of pathologic conditions including arthritis, cancer and Alzheimer's disease. MPGES-1 is the terminal enzyme in the prostaglandin (PG) biosynthesis pathway and catalyzes the conversion of prostaglandin H2 (PGH2) to prostaglandin E2(PGE2). It is an inducible enzyme and its expression is increased in response to pro-inflammatory stimuli. This feature makes mPGES-1 extremely interesting for drugs development because its inhibition is connected to the suppression of inducible PGE2 responsible for inflammatory and tumor pathologies that should reduce the typical side effects of the COX-2 inhibitors commercially available. Thanks to the resolution human mPGES-1 X-ray structure, and starting from our previous results, we report the structure based drug design of new inhibitors against mPGES-1 through a combinatorial approach. We have selected two different 'privileged structures':a) 2-aminobenzothiazoles and b) 1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one scaffolds, which canbe easily manipulated to provide a number of highly functionalized potential ligands. Starting from the analysis of the synthetic procedures , in fact, we have combined (Enumerate phase) and evaluated (Virtual Screening Workflow) all the commercially available synthons in silico before the chemical synthesis, obtaining in this way a large libraries (millions of compounds) that have been prepared and screened against mPGES-1 (dedicated software: CombiGlide, Schrödinger LLC.). The possible inhibitors have been selected by a qualitative computational filter, based on the accordance with specific key interactions with the receptor counterparts, predicted free energy of binding and ligand efficiency, leading to the identification of a focused set of compounds selected for the subsequent phases of chemical synthesis and biological evaluation (cell free and cell-based assays).Following this workflow, we have identified a set of new mPGES-1 inhibitors, all endowed with low micromolar activity. Moreover, the ongoing lead optimization phases will be step-by-step supported by the prediction of the pharmacokinetic properties (dedicated software: QikProp, Schrödinger LLC.), essential to discard the compounds with unfavorable predicted ADME properties that fall outside the normal range of known drugs, eliminating unnecessary testing on compounds that will ultimately fail.

We have applied the inverse virtual screening protocol to a small library of 19 synthetic compounds that showed a weak activity against the enzyme JMJD3. Our analysis suggested the enzyme ErbB4 as a new putative target of the investigated compounds. This macromolecule is a receptor tyrosine-protein kinase, member of the epidermal growth factor receptor subfamily and identified as potential target for cancer therapy. Experimental in vitro assays show that 5 compounds present inhibitory activity against ErbB4 in the low micromolar range. We have also investigated the binding of the identified lead compounds toward the highly structural related isoform ErbB2. The experimental evidences highlight a selectivity towards ErbB4. Moreover, one of the selected compounds shows antiproliferative activity against carcinoma (HCT) and breast (MCF7) cancer cells in low micromolar range

The yellow-pigmented, non-motile, Gram-negative, strictly aerobic, rod-shaped bacterial strain VI.18T was isolated from the Mediterranean sponge Axinella verrucosa collected off the coast near Sdot Yam, Israel. Results from 16S rRNA gene sequence analysis indicated that the isolate belonged to the family Flammeovirgaceae . The highest nucleotide similarity (91.4 %) occurred with Aureibacter tunicatorum A5Q-118T. The predominant cellular fatty acids of strain VI.18T were iso-C15 : 0 (56.0 %), iso-C17 : 1ω9c (22.8 %) and C16 : 0 (7.4 %) and its major respiratory quinone was MK-7. The DNA G+C content was 47.5 mol%. The strain could readily be distinguished from its phylogenetically closest relatives by phenotypic, physiological and chemotaxonomic properties. On the basis of the data from the present polyphasic study, we propose a novel genus and species within the family Flammeovirgaceae , with the name Fulvitalea axinellae gen. nov., sp. nov. Strain VI.18T ( = ATCC BAA-2395T = LMG 26722T) is the type strain of Fulvitalea axinellae.

A novel aerobic bacterium, designated strain PIII.02T, was isolated from a Mediterranean sponge (Axinella polypoides) collected off the Israeli coast near Sdot Yam. The non-motile cells were Gram-staining-negative, oxidase-positive and catalase-positive. The orange pigment of colonies growing on marine agar was neither diffusible nor flexirubin-like. Strain PIII.02T grew at 15–35 °C, at pH 6.0–9.0, with 2.0–7.0 % (w/v) NaCl, and with 1.0-8.0 % (w/v) sea salts. The predominant fatty acids were iso-C15 : 0, iso-C16 : 1 H, iso-C16 : 0, C16 : 0, anteiso-C15 : 0 and C16 : 1ω7c. The major respiratory quinone was MK-7. The genomic DNA G+C content of the novel strain was 38.1 mol%. Results from 16S rRNA gene sequence analysis indicated that strain PIII.02T was distantly related to established members of the phylum Bacteroidetes . The established species found to be most closely related to the novel strain was Persicobacter diffluens NCIMB 1402T (87.6 % 16S rRNA gene sequence similarity). Based on the phenotypic and chemotaxonomic data and the results of the phylogenetic analyses, strain PIII.02T represents a novel species of a new genus, for which the name Luteivirga sdotyamensis gen. nov., sp. nov. is proposed. The type strain is PIII.02T ( = ATCC BAA-2393T = LMG 26723T).

Two bacterial strains, VI.14 and VIII.04T, were isolated from the Mediterranean sponge Axinella verrucosa collected off the Israeli coast near Sdot Yam. The non-motile, aerobic, Gram-negative isolates were oxidase-negative and catalase-positive, and formed golden-brown colonies on marine agar 2216. The pigment was neither diffusible nor flexirubin-like. Strain VIII.04T grew at 15–37 °C, at pH 6.0–9.0, in the presence of 20–50 g NaCl l−1 and 20–80 g sea salts l−1, The spectrum was narrower for strain VI.14, with growth at pH 7.0–8.0. and in the presence of 30–50 g NaCl l−1 and 30–70 g sea salts l−1. The predominant fatty acid (>50 %) in both strains was iso-C15 : 0, and the major respiratory quinone was MK-6. The DNA G+C content was 30.7 and 31.1 mol% for VIII.04T and VI.14, respectively. Results from 16S rRNA sequence similarity and phylogenetic analyses indicated that both strains are closely related to members of the family Flavobacteriaceae within the phylum Bacteroidetes , with as much as 91.7 % 16S rRNA sequence similarity. On the basis of data from the polyphasic analysis, we suggest that the strains represent a novel species in a new genus within the family Flavobacteriaceae , for which the name Aureivirga marina gen. nov., sp. nov. is proposed. Strain VIII.04T ( = ATCC BAA-2394T = LMG 26721T) is the type strain of Aureivirga marina.

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

The structure of a new phosphoglycolipid from the halophilic Gram-negative bacteria Halomonas elongata ATCC 33173(T), Halomonas eurihalina ATCC 49336(T), Halomonas almeriensis CECT 7050(T), strain Sharm (AM238662), Halomonas halophila DSM 4770(T), and Halomonas salina ATCC 49509(T) was elucidated by NMR and mass spectroscopy studies. In all of the species examined, the polar lipid composition consisted of 1,2-diacylglycero-3-phosphorylethanolamine, 1,2-diacylglycero-3-phosphoryl-glycerol, bisphosphatidyl glycerol, and the new phosphoglycolipid PGL1. The structure of PGL1 was established to be (2-(alpha-D-glucopyranosyloxy)-3-hydroxy-propyl)-phosphatidyl diacylglycerol. C16:0;C18:1 and C16:0;C19:cyclopropane are the most abundant acyl chains linked to the phosphatidylglycerol moiety of each isolated PGL1. All of the species presenting the lipid PGL1 belong to Halomonas rRNA group 1, suggesting that the new phosphoglycolipid could be a chemotaxonomic marker of this phylogenetic group.

An alkalitolerant and halotolerant bacterium, designated strain Sharm was isolated from a salt lake inside Ras Muhammad. The morphological, physiological and genetic characteristics were compared with those of related species of the genus Halomonas. The isolate grew optimally at pH 7.0, 5-15% NaCl at 35 degrees C. The cells were Gram-negative rods, facultative anaerobes. They accumulated glycine-betaine, as a major osmolyte, and ectoine and glutamate as minor components. The strain Sharm(T) biosynthetised alpha-glucosidase. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and a novel phosphoglycolipid as major components. Ubiquinone with nine repetitive unities (Q9) was the only quinone found and, nC16:0 and C19:0 with cyclopropane were the main cellular fatty acids, accounting for 87.3% of total fatty acids. The G + C content of the genomic DNA was 64.7 mol %. The 16S rRNA sequence analysis indicated that strain Sharm was a member of the genus Halomonas. The closest relatives of the strain Sharm were Halomonas elongata and Halomonas eurihalina. However, DNA-DNA hybridisation results clearly indicated that strain Sham was a distinct species of Halomonas. On the basis of the evidence, we propose to assign strain Sharm as a new species of the genus Halomonas, H. sinaiensis sp. nov, with strain Sharm(T) as the type strain (DSM 18067(T); ATCC BAA-1308(T)).

A convenient synthesis of beta-galactosyl derivatives of antiviral and anticancer nucleosides, which utilizes the purified beta-galactosidase activity from the hapatopancreas of Aplysia fasciata, is reported. All reactions were extremely stereo- and regioselective, since only anomerically pure 5'-O-beta-galactosyl conjugates were formed. 5'-O-beta-Galactosyl-5-fluorouridine was synthesized with a 60% yield and 5'-O-beta-galactosyl-3-azido- 3'-deoxythymidine, the derivative of the anti-HIV drug, was obtained in 43% yield.

Glycobiology and related disciplines have received an enormous interest in recent years as they shed new light on the functional roles of carbohydrates in biological events leading to the understanding of mechanisms of important pathologies and to the development of new therapeutics. Although carbohydrate can be isolated from natural sources, the synthetic strategy plays its own role allowing access to larger quantities of structurally defined material and entry to analogs of naturally occurring structures. Nowadays, the bottleneck in this field is represented by some limitations of the potential of carbohydrate containing molecules because their complex structures make classical chemical synthesis very difficult. The synthesis of oligosaccharides have to face with three main snags: (i) reactivity of the leaving group on the monosaccharide acting as donor; (ii) regioselectivity towards a single hydroxyl group on the acceptor molecule; (iii) stereoselectivity in forming pure anomers of the new glycosidic product. The protecting-group manipulations that are needed for the stereocontrol of the products are demanding procedures thus yields and selectivity are lowered hindering the efficient production of oligosaccharides needed for biological testing. A particular benefit of the renewed interest for sugar chemistry has been the attention of scientific community to the biological methodologies in the production of oligosaccharides, an area which emerged in recent decades. In the carbohydrate field different enzymes were used for diverse purposes but enzymatic strategies for high-yield and stereospecific construction of glycosidic bonds are based on the action of two types of enzymes: glycosyl hydrolases (endo- and exo-glycosidases) and glycosyltransferases. Glycosyl hydrolases in the cells are responsible for the cleavage of glycosidic linkages; the exo-glycosidase are involved for glycan processing during in vivo glycoprotein synthesis. The glycosyltransferases are instead responsible in vivo for the synthesis of most cell-surface glycoconjugates. This review deals with the application of different types of glycosyl hydrolases. Elegant examples concerning the use of genetically modified representatives of glycosyl hydrolases (glycosynthases and thioglycoligases) will be also reported; some recent advances on the use of glycosyltransferases are also included. In compiling this review we were aware of the huge amount of excellent material published in the recent years on this topic, thus we will limit the covering of literature to the last decade. Attention will be devoted to the regioselectivity towards pyranosidic templates and to the yield of reactions. The molecular diversity obtained by the use of enzymes from different biological sources and the biological importance of the compounds synthesized will be focused as well. This review will put in evidence that glycosyl hydrolases and glycosyltransferases for the synthesis of the glycosidic linkages will play a relevant role in the next future as on the bench bio-catalysts for the chemists involved in the synthesis of oligosaccharides of biological interest.

The marine ecosystem can be considered a rather unexplored source of biological material (e.g. natural substances with therapeutic activity) and can also be a surprising source of enzymes carrying new and interesting catalytic activities to be applied in biocatalysis. The use of glycosyl hydrolases from marine environments dates back to the end of the 1960s and was mainly focused on the development of sensitive and reliable hydrolytic methods for the analysis of sugar chains. As a result not all the benefits of a particular enzymatic activity have been investigated, especially regarding the transglycosylation potential of these enzymes for the synthesis of glycosidic bonds. In this review, the potential of marine sources will be demonstrated reporting on the few examples found in literature for the synthesis and hydrolysis of biologically relevant oligosaccharides catalyzed by glycosyl hydrolases of marine origin. Particular emphasis is given to the synthesis of glycosidic bonds, which is easy by the use of glycosyl hydrolases. Further aspects considered in this review are applications of these biocatalysts for vegetal waste treatment in recovering useful materials, for structural identification and for preparation of target materials from new purified polysaccharides, for the synthesis or modification of food-related compounds and for glycobiology related studies.

A halophilic, alkalitolerant bacterium, strain 20AGT, was isolated from an algal mat collected from a sulfurous spring located in Santa Maria Incaldana (Mondragone, Campania Region, southern Italy). The isolate is Gram-positive, ferments several carbohydrates and has motile, rod-shaped cells that do not sporulate. The isolate grows at pH 6·5–9·5 and in 5–20 % NaCl. On the basis of 16S rRNA gene sequence similarity, the strain was shown to belong to the genus Oceanobacillus; strain 20AGT showed 96·6 % 16S rRNA gene sequence similarity to the type strain of Oceanobacillus iheyensis, DSM 14371T, and 99·5 % similarity to Oceanobacillus oncorhynchi NCIMB 14022T. Levels of DNA–DNA relatedness between strain 20AGT and O. iheyensis DSM 14371T and O. oncorhynchi NCIMB 14022T were respectively 29·4 and 59·0 %. The G+C content of the DNA of strain 20AGT was 40·1 mol%. The predominant respiratory quinone was MK-7, phosphatidylglycerol and phosphatidylcholine were the predominant polar lipids and minor phospholipids were also detected. ai-C14 : 0, ai-C15 : 0 and i-C15 : 0 were the major fatty acids. Strain 20AGT accumulated osmolytes and produced exopolysaccharide. On the basis of phenotypic characteristics, phylogenetic data and DNA–DNA relatedness data, isolate 20AGT should be designated as the type strain of a subspecies of Oceanobacillus oncorhynchi, for which the name Oceanobacillus oncorhynchi subsp incaldanensis subsp. nov. is proposed. The type strain is 20AGT (=DSM 16557T=ATCC BAA-954T).

The purification and characterisation of the alpha-glucosidase from the marine mollusc Aplysia fasciata are reported. Overall substrate specificity of the pure enzyme for both hydrolytic and transglycosylation reactions was studied. Remarkable characteristics of this enzyme are indicated by the results of the interesting survey of transglycosylation reactions reported: pyridoxine glucosylation, synthesis of chromophoric (pNP) di- and trisaccharides, glucosylation of cellobiose and sucrose. For these last two acceptors both the yields of reactions and the concentrations of products are comparable to those obtained using glycosyl transferases; in addition, synthesis of pyridoxine and chromophoric glycosides were still possible using a 1:1 ratio maltose:acceptor which is a very interesting characteristic from a synthetic point of view (effortless purification, productivity of each reaction batch, etc.).

A beta-D-mannosidase was purified to homogeneity from visceral mass extract of Aplysia fasciata a mollusc belonging to the order Anaspidea. The purified enzyme is a homodimer with a subunit mass of 130 kDa. Temperature and pH optima of this enzyme were 45 degrees C and 4.5, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. The K(M) and V(max) values for p-nitrophenyl beta-D-mannopyranoside were determined to be 2.4 mM and 50.3 micromol min(-1)mg(-1), respectively. The catalytic efficiency of this beta-mannosidase (11,519 min(-1)) was significantly higher than those reported for beta-mannosidases from other sources. It was verified that this is an exo-acting glycosyl hydrolase with transglycosidase activity. When the enzyme was incubated in the presence of p-nitrophenyl beta-D-mannopyranoside, self-transfer of the mannosyl group was observed, and a 10-15% yield of a beta-1-4 disaccharide was obtained. When the reaction was performed in the presence of o-nitrophenyl alpha-D-2-deoxy-N-acetyl glucopyranoside in 3:1 molar ratio with respect to the p-nitrophenyl beta-D-mannopyranoside, two regioisomers (85:15, 12% yield) due to the beta-mannosylation of the heteroacceptor in 4 and in 6 positions were formed.

A Gram-negative, aerobic, motile and rod-shaped haloalkaliphilic bacterial strain 5AGT (DSM 15293 and ATCC BAA-966) was isolated from water with algal mat of a mineral pool in Malvizza site (Campania-Italy) and was subjected to a polyphasic study. The isolate grew at temperature of 10.0-43.0 degrees C with an optimum at 37.0 degrees C. Strain 5AGT grew optimally in the presence of 10% NaCl and grew also in the absence of salt. The isolate grew in the pH range 7.0-10.0 with an optimum at pH 9.0. It accumulated glycine-betaine, ectoine, and glutamate, as osmoprotectants. Strain 5AGT was also characterized chemotaxonomically by having ubiquinone-8 (Q8) as the predominant isoprenoid quinone, phosphoethanolamine (PEA), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG), as major polar lipids and aiC16:0 and C18:1cis as the major fatty acids. The DNA G+C content was 63.7mol%. Phylogenetic analyses based on 16S rRNA gene sequence showed that the isolate belonged to the genus Halomonas. The DNA-DNA hybridization of the type strain 5AGT with the most related Halomonas campisalis showed a re-association value of 35.0%. On the basis of phenotypic properties and phylogeny, strain 5AGT should be placed in the genus Halomonas as a member of a novel species for which we propose the name Halomonas campaniensis sp. nov.

The use of crude extract of the hepatopancreas of Aplysia fasciata, a large mollusc belonging to the order Anaspidea containing a beta-galactosidase activity, was reported for the synthesis of different galactosides. Good yields with polar acceptors and the uncommon beta-1-3 selectivity in the transgalactosylation reactions with most of the acceptors were observed. A beta-1-2 selectivity in the hydrolytic conditions was also observed and discussed.

Carbohydrates serve as structural components and en- ergy sources of cells. More interestingly, however, these biomolecules are involved in a variety of molecular recognition processes in intercellular communication and signal transduction such as cell adhesion, differ- entiation, development and regulation. For these rea- sons, great interest has arisen in carbohydrate-based pharmaceuticals and on the development of techniques for the analysis and synthesis of oligosaccharides. In this respect, enzymes involved in carbohydrates hydrolysis and modification are increasingly being utilised for the bioconversion of sugars, for the synthesis of oligosac- charides with potential application, and for the charac- terisation of carbohydrate compounds of unknown structure. In this review, the enzymology and the applications of three glycosyl hydrolases from the archaeon Sulfolobus solfataricus are described. In particular, we focus on the enzymological properties of a b-glycosidase, an a-xylo- sidase, and an a-fucosidase; their exploitation in oligo- saccharides synthesis will be also described.

The analysis of the complete genome of the thermoacidophilic Archaeon Sulfolobus solfataricus revealed two open reading frames (ORF), named SSO11867 and SSO3060, interrupted by a -1 frameshift and encoding for the N- and the C-terminal fragments, respectively, of an alpha-l-fucosidase. We report here that these ORFs are actively transcribed in vivo, and we confirm the presence of the -1 frameshift between them at the cDNA level, explaining why we could not find alpha-fucosidase activity in S. solfataricus extracts. Detailed analysis of the region of overlap between the two ORFs revealed the presence of the consensus sequence for a programmed -1 frameshifting. Two specific mutations, mimicking this regulative frameshifting event, allow the expression, in Escherichia coli, of a fully active thermophilic and thermostable alpha-l-fucosidase (EC ) with micromolar substrate specificity and showing transfucosylating activity. The analysis of the fucosylated products of this enzyme allows, for the first time, assigning a retaining reaction mechanism to family 29 of glycosyl hydrolases. The presence of an alpha-fucosidase putatively regulated by programmed -1 frameshifting is intriguing both with respect to the regulation of gene expression and, in post-genomic era, for the definition of gene function in Archaea.

The taxomony of strain M8, isolated from algal mat formed at the origin of a sulfurous spring in Rifieto (Savignano Irpino, Campania, Italy), was investigated in a polyphasic approach. The morphological, physiological and genetic characteristics were compared with of Planococcus and Planomicrobium species. The isolate grew optimally at pH 9.0, 1.8 M NaCl at 37 degrees C. The cells were Gram-positive cocci that form pairs, tetrads and aggregates of several cells. The isolate was aerobic/microaerophilic and accumulated glycine-betaine, as a major osmolyte, with minor components glutamate and an unknown compound. M8 was able to hydrolyse X-Glc (5-bromo-4-chloro-3-indoyl beta-d-glucopyranoside). The polar lipid profile consisted of phosphatidylglycerol and diphosphatidylglycerol as major components, and phosphocholine as a minor compound. MK8 was the only quinone found and the fatty acid composition was dominated by branched acids, mainly aiC15:0. The G+C content of DNA was 47.9% and its phylogenetic position was established by 16S rRNA gene sequencing as a member of the genus Planococcus. The DNA/DNA similarity of M8 to the type species Planococcus citreus was less than 55%. For this reason and for physiological and chemotaxonomic features, it is proposed to create a new species Planococcus rifietensis sp. nov.

The stereochemistry of janolusimide, a lipophilic tripeptide marine toxin, has been fully elucidated by stereoselective synthesis of the lactam component (5S)-3,3′-dimethyl-5-isopropylpyrrolidin-2,4-dione. The peptide was then synthesized in 13 steps and in 0.8% total yield.

The synthesis of (17 R )-17-methylincisterol, a highly degraded marine steroid, has been achieved starting from vitamin D 2 in 8 steps and in 13% overall yield. Its O-demethyl analog, which is an intermediate in the synthetic sequence, is possibly the true naturally occurring molecule.

The enantioselective synthesis of methyl (R)-mandelate and methyl (R)-o-chloromandelate was investigated using an NADH-dependent carbonyl reductase from Thermus thermophilus (TtADH) and, separately, archaeal glucose dehydrogenase and Bacillus stearothermophilus alcohol dehydrogenase (BsADH) for NADH regeneration. Optimal reaction times and substrate concentrations in the absence and presence of organic solvents were determined. The enantiofacial selectivity of TtADH was shown to be inversely proportional to the hydrophobicity of the short-chain linear alcohols employed as co-substrates of the bacillar ADH. The bioreduction of methyl benzoylformate yielded the (R)-alcohol with a 77 % yield (ee = 96 %) using glucose dehydrogenase and glucose, and 81 % yield (ee = 94 %) applying BsADH and ethanol. The bioreduction of methyl o-chlorobenzoylformate yielded the halogenated (R)-alcohol with 95 % and 92 % ee, and 62 % and 78 % yield using glucose dehydrogenase and BsADH, respectively.

The stereochemistry of janolusimide, a lipophilic tripeptide marine toxin, has been fully elucidated by stereoselective synthesis of the lactam component (5S)-3,3′-dimethyl-5-isopropylpyrrolidin-2,4-dione. The peptide was then synthesized in 13 steps and in 0.8% total yield.

Strain 7SM30T , an aerobic marine, Gram-negative, heterotrophic and yellow- to orange-pigmented bacterium isolated from seawater from Castellón, Spain, was characterized using a polyphasic approach. Analysis of the 16S rRNA gene sequence showed that the isolate represented a novel lineage within the family Flavobacteriaceae. The most closely related genera were Pseudozobellia, Zobellia and Kriegella. Cells of strain 7SM30T were non-motile rods that required sea salts for growth, used a wide variety of carbohydrates as sole carbon and energy sources and, unlike species of the genera Pseudozobellia and Zobellia, did not possess flexirubin-type pigment or hydrolyse agar. Strain 7SM30T contained MK6 as the sole respiratory quinone. Phosphatidylethanolamine (PE) was the only identifiable polar lipid, although other lipids were also detected. The predominant cellular fatty acids were saturated C15 and monounsaturated C15. The DNA G+C content was around 40 mol%. On the basis of extensive phenotypic and phylogenetic comparative analysis, it is concluded that the new strain represents a novel genus and species, for which the name Euzebyella saccharophila gen. nov., sp. nov., is proposed. The type strain of the type species is 7SM30T (=CECT 7477T=KCTC 22655T).

Glycosylation is considered to be an important reaction for the chemical modification of compounds with useful biological activities. Glycoside hydrolases are biotechnologically attractive enzymes which can be used in synthetic reactions for assembling glycosidic linkages with absolute stereoselectivity at an anomeric centre. Most of these enzymes are commercially available but there is great interest in the search for new biocatalysts with original catalytic characteristics. The marine environment has shown to be a very interesting source for new glycosyl hydrolases for both hydrolytic and synthetic aspects. In particular, Aplysia fasciata a marine herbivorous mollusc has been shown to be a potent producer of a library of glycoside hydrolases applied to the synthesis of glycosidic bonds. The impressive assortment of glycosidases in marine organisms clearly indicates that the potential biodiversity of these enzymes is still largely unexplored and that potential applications of biocatalysts from the sea will increase in the near future.

Archaea , like Eukarya and Bacteria , are able to N glycosylate select protein targets. However, in contrast to relatively advanced understanding of the eukaryal N glycosylation process and the information being amassed on the bacterial process, little is known of this posttranslational modification in Archaea . Toward remedying this situation, the present report continues ongoing efforts to identify components involved in the N glycosylation of the Haloferax volcanii S-layer glycoprotein. By combining gene deletion together with mass spectrometry, AglE, originally identified as a homologue of murine Dpm1, was shown to play a role in the addition of the 190-Da sugar subunit of the novel pentasaccharide decorating the S-layer glycoprotein. Topological analysis of an AglE-based chimeric reporter assigns AglE as an integral membrane protein, with its N terminus and putative active site facing the cytoplasm. These finding, therefore, contribute to the developing picture of the N glycosylation pathway in Archaea .

Arabinan polysaccharide side-chains are present in both Mycobacterium tuberculosis and Corynebacterium glutamicum in the heteropolysaccharide arabinogalactan (AG), and in M. tuberculosis in the lipoglycan lipoarabinomannan (LAM). This study shows by quantitative sugar and glycosyl linkage analysis that C. glutamicum possesses a much smaller LAM version, Cg-LAM, characterized by single t-Araf residues linked to the alpha(1--6)-linked mannan backbone. MALDI-TOF MS showed an average molecular mass of 13,800-15 400 Da for Cg-LAM. The biosynthetic origin of Araf residues found in the extracytoplasmic arabinan domain of AG and LAM is well known to be provided by decaprenyl-monophosphoryl-D-arabinose (DPA). However, the characterization of LAM in a C. glutamicum : : ubiA mutant devoid of prenyltransferase activity and devoid of DPA-dependent arabinan deposition into AG revealed partial formation of LAM, albeit with a slightly altered molecular mass. These data suggest that in addition to DPA utilization as an Araf donor, alternative pathways exist in Corynebacterianeae for Araf delivery, possibly via an unknown sugar nucleotide.