Lorenzo De Napoli

Hydrolysis of oleuropein by recombinant β-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus immobilised on chitosan matrix

The recombinant beta-glycosidase (EcS beta gly) from Sulfolobus solfataricus was immobilised on chitosan to perform the enzymatic hydrolysis of commercial oleuropein (heterosidic ester of elenolic acid and 3,4-dihydroxy-phenylethanol (hydroxytyrosol)) at two temperatures (60 and 70 degrees C). Interestingly, on the basis of the reasonable assumption that the enzyme hydrolyses only the sugar linkage, the biotransformation produces unstable aglycone species formed by oleuropein hydrolysis that, differently from some commercially available beta-glucosidases tested, give rise to the formation of hydroxytyrosol, at the operative temperatures of the bioreactor. The results of the biotransformation at 70 degrees C showed that the main products are hydroxytyrosol, and glucose, being the oleuropein aglycone present in low amount at the end of reaction. Both in single step approach or in recycle approach the amounts of glucose and oleuropein aglycone were lightly dependent from flow rate. The amount of hydroxytyrosol, increased on decreasing the flow rate of bioreactor in recycle approach, following a non-linear trend and obtaining the highest value at a flow rate of 15 ml h-1 while in the single step approach the 3,4-dihydroxy-phenylethanol was at its maximum at higher flow rate (16 ml h-1). For the hydrolysis of the oleuropein by bioreactor at 60 degrees C we used lower molar ratio oleuropein/enzyme only by the single step approach. In these conditions it is possible to obtain high amounts of only two products (glucose and hydroxytyrosol) in short time (2 h). The stability of the bioreactor at the operative temperatures showed a t1/2 of 30 days at 70 degrees C and a t1/2 of 56 days at 60 degrees C.

Solid phase synthesis of oligosaccharides

Abstract An efficient and stereocontrolled solid-phase chemical synthesis of oligosaccarides using a new type of insoluble support (a copolymer of polyethylene glycol and polystyrene) is described. Several Lewis acids were tested as activators of the trichloroacetimidate 12 , used as glycosyl donor, in the dimer 13 formation.

Design, Synthesis and Characterisation of Guanosine-Based Amphiphiles

A small library of sugar-modified guanosine derivatives has been prepared, starting from a common intermediate, fully protected on the nucleobase. Insertion of myristoyl chains and of diverse hydrophilic groups, such as an oligoethylene glycol, an amino acid or a disaccharide chain, connected through in vivo reversible ester linkages, or of a charged functional group provided different examples of amphiphilic guanosine analogues, named G1-G7 herein. All of the sugar-modified derivatives were positive in the potassium picrate test, showing an ability to form G-tetrads. CD spectra demonstrated that, as dilute solutions in CHCl(3), distinctive G-quadruplex systems may be formed, with spatial organisations dependent upon the structural modifications. Two compounds, G1 and G2, proved to be good low-molecular-weight organogelators in polar organic solvents, such as methanol, ethanol and acetonitrile. Ion transportation experiments through phospholipid bilayers were carried out to evaluate their ability to mediate H(+) transportation, with G5 showing the highest activity within the investigated series. Moreover, G3 and G5 exhibited a significant cytotoxic profile against human MCF-7 cancer cells in in vitro bioassays.

Nucleolipid nanovectors as molecular carriers for potential applications in drug delivery

Novel thymidine- or uridine-based nucleolipids, containing one hydrophilic oligo(ethylene glycol) chain and one or two oleic acid residues (called ToThy, HoThy and DoHu), have been synthesized with the aim to develop bio-compatible nanocarriers for drug delivery and/or produce pro-drugs. Microstructural characterization of their aggregates has been determined in pure water and in pseudo-physiological conditions through DLS and SANS experiments. In all cases stable vesicles, with mean hydrodynamic radii ranging between 120 nm and 250 nm have been revealed. Biological validation of the nucleolipidic nanocarriers was ensured by evaluation of their toxicological profiles, performed by administration of the nanoaggregates to a panel of different cell lines. ToThy exhibited a weak cytotoxicity and, at high concentration, some ability to interfere with cell viability and/or proliferation. In contrast, DoHu and HoThy exhibited no toxicological relevance, behaving similarly to POPC-based liposomes, widely used for systemic drug delivery. Taken together, these results show nucleolipid-based nanocarriers as finely tunable, multi-functional self-assembling materials of interest for the in vivo transport of biomolecules or drugs.

Solid phase synthesis of cyclic oligodeoxyribonucleotides.

Abstract The polymer supported synthesis of cyclic oligomers of deoxycytidilic acid is described, by a method based on an oligonucleotide-solid phase linkage through the amino group of the base.

A new ferrocenemethyl-thymidine nucleoside: Synthesis, incorporation into oligonucleotides and optical spectroscopic studies on the resulting single strand, duplex and triplex structures

Abstract A new thymidine analogue, bearing a ferrocenemethyl residue at the N-3 position of the base, was synthesized in high yields via Mitsunobu reaction of ferrocenemethanol with sugar protected thymidine, converted into the corresponding 3′-phosphoramidite and incorporated into oligonucleotides. Duplex and triplex formation experiments, evaluated by UV and CD spectroscopy, showed a dramatic decrease of the affinity towards complementary single strands, while for triplexes, the introduction of a ferrocene residue in the third strand resulted in higher melting temperatures, associated with a reduced content of triplex structure.

Design, Synthesis and Characterisation of a Fluorescently Labelled CyPLOS Ionophore

A novel fluorescently labelled synthetic ionophore, based on a cyclic phosphate-linked disaccharide (CyPLOS) backbone and decorated with four tetraethylene glycol tails carrying dansyl units, has been synthesised in 12 steps in 26% overall yield. The key intermediate in the synthetic strategy is a novel glucoside building block, serving through its 2- and 3-hydroxy groups as the anchor point for flexible tetraethylene glycol tentacles with reactive azido moieties at their ends. To test the versatility of this glucoside scaffold, it was preliminarily functionalised with a set of diverse probes--as fluorescent, redox-active or hydrophobic tags--either by reduction of the azides followed by condensation with activated carboxylic acid derivatives, or by a direct coupling with a terminal alkyne in a Cu(I)-promoted 1,3-dipolar cycloaddition. Tagging of the monomeric building block with dansyl residues allowed us to prepare a fluorescent, amphiphilic macrocycle, which was investigated for its propensity to self-aggregate in CDCl(3)--studied by means of concentration-dependent (31)P NMR spectroscopy experiments--and in aqueous solution, in which combined dynamic light scattering (DLS) and small-angle neutron scattering (SANS) measurements provided a detailed physico-chemical analysis of the self-assembled systems, mainly organised in the form of large vesicles. Its ion-transport properties through phospholipid bilayers, determined by HPTS fluorescence assays, showed this compound to be more active than the previously synthesised CyPLOS congeners. Solvent-dependent fluorescence changes for the labelled ionophore in liposome suspension established that the dansyl moieties are dispersed in environments with polarity intermediate between those of CH(2)Cl(2) and propan-2-ol, suggesting that the CyPLOS tentacles infiltrate the mid-polar region of the membranes.

Solid phase synthesis of oligonucleotides tethered to oligo-glucose phosphate tails

Abstract Oligonucleotides conjugated at both 3′ and 5′-ends with glucose residues, 4,6-linked through a phosphodiester bridge, have been synthesized by sequential addition of a 6- O -DMT-glucose-4-phosphoramidite building block following a standard automated ODN assembly procedure. Two 3′,5′-bis-glycoconjugated 18-mers, designed for antisense experiments, have been prepared and their hybridization properties with a complementary DNA fragment evaluated by UV thermal analysis.

New C-23 modified of silybin and 2,3-dehydrosilybin: Synthesis and preliminary evaluation of antioxidant properties

Silybin is the major flavonolignan of silymarin and it displays a plethora of biological effects, generally ascribed to its antioxidant properties. Herein we shall describe an efficient synthetic strategy to obtain a variety of new and more water-soluble silybin and 2,3-dehydrosilybin (DHS) derivatives in which the 23-hydroxyl group was converted to a sulfate, phosphodiester, or amine group, using a solution-phase approach. Furthermore a new and efficient method for the preparation of DHS from silybin was developed and optimised. The antioxidant properties of the new compounds were evaluated in a cellular model in vivo and they displayed an antioxidant activity comparable to or higher than silybin and DHS, being able to prevent H(2)O(2)-induced generation of intracellular reactive oxygen species (ROS). Most of the derivatives also displayed a better hydrophilicity while retaining the biological activities of silybin and they might broaden the in vivo applications of this class of natural compounds.

Use of controlled pore glass in solid phase oligosaccharide synthesis. Application to the semiautomated synthesis of a glyconucleotide conjugate

Abstract Three polymeric supports (polystyrene, Tentagel and controlled pore glass) have been tested for solid phase synthesis of oligosaccharides based on the trichloroacetimidate methodology. Controlled pore glass has been found to yield satisfactory results with TMSOTf as the glycosylation promoter. An application to the on-line preparation of a glyconucleotide is also reported.