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.
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.
Ionophores are an important class of synthetic molecules which mimic natural ion channels or carriers. Here we report the aggregation behavior in pseudo-physiological environment of three Cyclic Phosphate-Linked Oligosaccharides (CyPLOS) derivatives, synthetic ion transporters based on cyclic, phosphate-linked disaccharide skeleton differing for the nature of the tails (tetraethylene-TEG glycol and/or n-undecyl chains) attached to the C-2 and C-3 of the constitutive monosaccharides. Their aggregation behavior has been studied by a combined use of dynamic light scattering (DLS), electron paramagnetic resonance spectroscopy (EPR) and Small Angle Neutron Scattering (SANS). DLS measurements were performed to reveal the formation and size distribution of the CyPLOS aggregates. EPR measurements, by using 5-doxyl stearic acid (5-DSA) as spin-probe, showed that the aggregates are mainly due to the formation of double layers and allowed to analyze the local fluidity. Finally, SANS measurements allowed estimating the layer thickness of the double layers. Our results indicate that the three CyPLOS analogs show self-aggregation properties that depend on the different nature of the inserted tails.
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.
The ion transport properties of a new family of synthetic ionophores based on cyclic phosphate-linked oligosaccharide (CyPLOS) macrocycles are described.
Novel hybrid oligonucleotides carrying the G-quadruplex-forming d(5'TGGGAG3') sequence, conjugated with mono- or disaccharides at the 3' or 5'-end through phosphodiester bonds, have been synthesized as potential anti-HIV agents, via a fully automated, online phosphoramidite-based solid-phase strategy. CD-monitored thermal denaturation studies on the resulting quadruplexes indicated the insertion of a single monosaccharide at the 3'-end as the optimal modification, conferring improved stability to the quadruplex complex. In addition, the 3'-conjugation with glucose or mannose converted the anti-HIV inactive unmodified oligomer into active compounds. On the contrary, the 5'-tethering with these monosaccharides, as well as the conjugation, either at the 5' or 3'-end, with sucrose, were in all cases detrimental to quadruplex stability and did not improve the biological activity. On the basis of the assumption that the kinetically and thermodynamically favored formation of the quadruplex complex is a prerequisite for efficient antiviral activity, a novel bis-conjugated oligonucleotide was designed. This combined a mannose residue at the 3'-phosphate end with bulky aromatic tert-butyldiphenylsilyl (TBDPS) group at the 5'-end, previously shown to markedly favor the formation of quadruplex complexes. The 5',3'-bis-conjugated 6-mer, for which a detailed biophysical characterization has been carried out, resulted in 3-fold greater antiviral activity against HIV-1 than the sole 3'-glyco-conjugated oligonucleotide.
We here describe the synthesis of a series of novel bicyclic ribonucleoside derivatives, with 18-crown-6 ether moieties attached via their ribose 2- and 3- positions, as first examples of crown ether ring-fused nucleosides, to be evaluated as antiviral and/or antitumoral agents.
A versatile approach to develop libraries of diverse 5',3'-bis-conjugated oligonucleotides (ODNs) is here described. The usage of ad hoc derivatized solid supports, to which the first nucleoside unit is attached through a phosphate linkage, opens easy synthetic access to a large variety of hybrid bis-conjugated oligomers. The G-quadruplex forming d((5')TGGGAG(3')) sequence, as a potential anti-HIV agent, has been here used as a model system.
The intense search for oligonucleotides (ODNs) endowed with pharmacological activities has led, in the past decade, to the identification of tens of candidate drugs, now being evaluated in preclinical or clinical trials. Based on G-rich DNA sequences, several aptamers, adopting G-quadruplex structures with different topologies, have been selected as potent in vitro antiviral and/or antitumoral agents. In order to develop novel therapeutically relevant G-quadruplex-based aptamers, we have investigated - as a model compound - the (5')d(TGGGAG)(3') sequence, known to be anti-HIV-1 active if 5'-modified with bulky aromatic residues. A set of 5'-conjugated analogues has been analyzed by integrated CD, DSC and molecular modelling studies, allowing a detailed biophysical characterization of the resulting G-quadruplexes. Following the assumption that the kinetically and thermodynamically favoured formation of the quadruplex complexes is a pre-requisite for their efficient antiviral activity, novel hybrid ODNs, carrying diverse terminal modifications, were prepared via a fully automated, on-line phosphoramidite-based strategy and evaluated for anti-HIV activity.
Novel amphiphilic cyclic disaccharide analogues, in which the saccharide units are connected through stable phosphodiester linkages (CyPLOS, Cyclic Phosphate-Linked OligoSaccharides) and decorated with long lipophilic tentacles at the 2- and 3-OH moieties, have been synthesized. Their propensity to self-aggregation has been investigated by means of 1H and 31P NMR experiments, making it possible to determine for these macrocycles critical aggregation concentration values in the millimolar range.