A novel synthetic strategy for monosubstituted cyclodextrin derivatives.

A first solid phase approach to obtain monosubstituted CD conjugates to different labels has been developed. A new solid support has been designed to get a variety of C-6 monofunctionalized CDs (α, ß, MeßCD and HPßCD) covalently linked through a phosphodiester bridge to different labels, in highly pure form and under very mild detachment conditions.

Synthesis of 2'-O-guanidinopropyl-modified nucleoside phosphoramidites and their incorporation into siRNAs targeting hepatitis B virus.

Synthetic RNAi activators have shown considerable potential for therapeutic application to silencing of pathology-causing genes. Typically these exogenous RNAi activators comprise duplex RNA of approximately 21 bp with 2 nt overhangs at the 3' ends. To improve efficacy of siRNAs, chemical modification at the 2'-OH group of ribose has been employed. Enhanced stability, gene silencing and attenuated immunostimulation have been demonstrated using this approach. Although promising, efficient and controlled delivery of highly negatively charged nucleic acid gene silencers remains problematic. To assess the potential utility of introducing positively charged groups at the 2' position, our investigations aimed at assessing efficacy of novel siRNAs containing 2'-O-guanidinopropyl (GP) moieties. We describe the formation of all four GP-modified nucleosides using the synthesis sequence of Michael addition with acrylonitrile followed by Raney-Ni reduction and guanidinylation. These precursors were used successfully to generate antihepatitis B virus (HBV) siRNAs. Testing in a cell culture model of viral replication demonstrated that the GP modifications improved silencing. Moreover, thermodynamic stability was not affected by the GP moieties and their introduction into each position of the seed region of the siRNA guide strand did not alter the silencing efficacy of the intended HBV target. These results demonstrate that modification of siRNAs with GP groups confers properties that may be useful for advancing therapeutic application of synthetic RNAi activators.

Discovery of novel anti-HIV active G-quadruplex-forming oligonucleotides.

A series of d((5')TGGGAG(3')) sequences, 5'-conjugated with a variety of aromatic groups through phosphodiester linkages, were synthesized, showing CD spectra diagnostic of parallel-stranded, tetramolecular G-quadruplex structures. When tested for anti-HIV-1 and HIV-2 activity, potent inhibition of HIV-1 infection in CEM cell cultures was found, associated with high selectivity index values. Surface Plasmon Resonance assays revealed specific binding to HIV-1 gp120 and gp41.

Fluoride-cleavable, fluorescently labelled reversible terminators: synthesis and use in primer extension.

Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3'-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3'-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach.

Fluorescent labeling of (oligo)nucleotides by a new fluoride cleavable linker capable of versatile attachment modes.

The development of a fluoride cleavable linker 1 for reversibly labeling (oligo)nucleotides is described here. The linker allows different ways of chemical attachment of a reporter molecule, for example, click chemistry or amide formation. The versatile attachment modes of labels are demonstrated by derivatizations with pyrene and fluorescein. Besides the synthesis of the new linker, we also show the derivatization of iodobenzene as a model compound and a nucleoside to demonstrate the applicability. Further, cleavability studies in solution and on a solid-supported oligonucleotide are shown. The linker can be applied in the synthesis of reversible terminators, useful for new DNA sequencing technologies like cyclic reversibly terminating (CRT) sequencing.

CyPLOS: a new family of synthetic ionophores.

The ion transport properties of a new family of synthetic ionophores based on cyclic phosphate-linked oligosaccharide (CyPLOS) macrocycles are described.

A versatile synthetic approach for the development of libraries of 5', 3'-bis-conjugated oligonucleotides.

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.

Synthesis of four colors fluorescently labelled 3'-O-blocked nucleotides with fluoride cleavable blocking group and linker for array based Sequencing-by-Synthesis applications.

Reversible terminators having a fluoride cleavable 3'-O-blocking group are presented. Each nucleotide triphosphate is labelled by a fluorescent dye cleavable by the same reagent. We present here their synthesis, cleavage experiments and polymerase incorporation tests for a possible use in a process of Sequencing-by-Synthesis.

Crown ether ring-fused nucleosides: synthesis and conformational properties.

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.

Synthesis, biophysical characterization, and anti-HIV activity of glyco-conjugated G-quadruplex-forming oligonucleotides.

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.

5'-Modified G-quadruplex forming oligonucleotides endowed with anti-HIV activity: synthesis and biophysical properties.

Oligodeoxyribonucleotides of sequence d(5'TGGGAG3') carrying bulky aromatic groups at the 5' end were found to exhibit potent anti-HIV activity [Hotoda, H., et al. (1998) J. Med. Chem. 41, 3655-3663 and references therein]. Structure-activity relationship investigations indicated that G-quadruplex formation, as well as the presence of large aromatic substituents at the 5'-end, were both essential for their antiviral activity. In this work, we synthesized some representative examples of the anti-HIV active Hotoda's 6-mers and analyzed the resulting G-quadruplexes by CD, DSC, and molecular modeling studies, in comparison with the unmodified oligonucleotide. In the case of the sequence carrying the 3,4-dibenzyloxybenzyl (DBB) group, identified as the best candidate for further drug optimization, we developed an alternative protocol to synthesize the 5'-DBB-thymidine phosphoramidite building block in higher yields. The thermodynamic and kinetic parameters for the association/dissociation processes of the 5'-conjugated quadruplexes, determined with respect to the unmodified one, were discussed in light of the molecular modeling studies. The aromatic groups at the 5' position of d(5'TGGGAG3') dramatically enhance both the equilibrium and the rate of formation of the quadruplex complexes. The overall stability of the investigated quadruplexes was found to correlate with the reported IC50 values, thus furnishing quantitative evidence for the hypothesis that the G-quadruplex structures are the ultimate active species, effectively responsible for the biological activity.

Solid-phase synthesis of cyclic PNA and PNA-DNA chimeras.

[structure: see text] A new and versatile on-line automated solid-phase approach to obtain cyclic PNA (I and III) and cyclic PNA-DNA chimeras (II) in highly pure form has been developed. Starting from a Tentagel matrix functionalized with a 3-chloro-4-hydroxyphenylacetic linker, the synthesis of representative, new cyclic molecules by standard peptide and phosphoramidite-based chemistry has been achieved.

Cyclic phosphate-linked oligosaccharides: synthesis and conformational behavior of novel cyclic oligosaccharide analogues.

CyPLOS (cyclic phosphate-linked oligosaccharides), that is, novel cyclic oligosaccharide surrogates, consisting of two, three, and four phenyl-beta-D-glucopyranoside units, 4,6-linked through stable phosphodiester bonds, were prepared by a straightforward and efficient solid-phase protocol. The assembly of the linear precursors was achieved by standard phosphoramidite chemistry on an automated DNA synthesizer, using a suitably protected 4-phosphoramidite derivative of D-glucose as the building block. For the crucial cyclization step a phosphotriester methodology was exploited, followed by a mild basic treatment releasing the desired cyclic molecules in solution in a highly pure form. The cyclic dimer and trimer were also independently prepared by classical solution synthesis, basically following the same approach. The solution structural preferences of the cyclic dimer and trimer, obtained by detailed NMR analysis, are also reported.

An efficient and versatile solid-phase synthesis of 5'- and 3'-conjugated oligonucleotides.

[reaction: see text] An easy and efficient solid-phase strategy to obtain 5'- and 3'-oligonucleotide conjugates in highly pure form has been developed. Ad hoc derivatized solid supports, to which the first nucleoside unit can be attached through a phosphate linkage, have been exploited both in a pre- and post-DNA assembly conjugation approach. A number of 5'- or 3'- oligonucleotide conjugates, incorporating a variety of labels covalently linked through a phosphodiester or a phosphoramidate bond, have been synthesized and characterized.

Glycomimetics as decorating motifs for oligonucleotides: solid-phase synthesis, stability, and hybridization properties of carbopeptoid-oligonucleotide conjugates.

The online solid-phase synthesis of oligonucleotides conjugated at the 3' end with [1-6]-linked oligosaccharide mimics having the O-glycosidic linkages replaced by amide bonds is here described. The assembly of the carbohydrate domain has been carried out by exploiting classical solid phase peptide synthetic protocols, starting from solid supports functionalized with 1-azido sugars, in association with suitably protected 1-azido uronic acids of glucose and lactose, chosen as model addition monomers. After the insertion of a flexible linker, elongation of the oligodeoxyribonucleotide (ODN) chain was performed by standard automated phosphoramidite protocols. 3'-Glycoconjugated 18-mers exhibited an increased enzymatic stability with respect to the same unmodified ODN sequence. UV thermal denaturation experiments showed that the presence of the oligosaccharide tail at the 3' end of the oligonucleotides did not negatively interfere with their duplex formation abilities.

An efficient solid phase synthesis of 5'-phosphodiester and phosphoramidate monoester nucleoside analogues.

An easy and efficient strategy to obtain libraries of 5'-phosphodiester and 5'-phosphoramidate monoester nucleoside analogues in a highly pure form has been developed, starting from a new nucleoside based solid support. The nucleoside scaffold has been anchored through a 5'-phosphodiester linkage to Tentagel HL resin, functionalized with a 3-chloro-4-hydroxyphenylacetic linker. The solid phase synthesis of small libraries of 5'-phosphodiester and 5'-phosphoramidate monoester thymidine analogues is also reported.