Jordi Robles

A synthetic procedure for the preparation of oligonucleotides without using ammonia and its application for the synthesis of oligonucleotides containing 0-4-alkyl thymidines.

Abstract The preparation of 5′-O-dimethoxytrityl (DMT) and p -nitrophenylethyl (NPEOC, NPE) protected nucleosides linked to 4-(2-hydroxyethyl)-3-nitrobenzoic acid derivatives is described. These products attached to controlled-pore glass supports and together with DMT and NPE-protected nucleoside cyanoethyl phosphoramidites allows for the first time the preparation of short oligonucleotides containing the ammonia sensitive mutagenic bases 0-4-propyl and 0-4-butyl thymidines.

NPE-resin, a new approach to the solid-phase synthesis of protected peptides and oligonucleotides I : Synthesis of the supports and their application to oligonucleotide synthesis.

Abstract The preparation of polymeric supports containing a base labile 2-(2-nitrophenyl) ethyl linkage and the attachment of protected nucleosides is described together with their application to oligonucleotide synthesis.

A single-molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials

An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2‐C‐methyl‐d‐erythritol‐4‐phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single‐molecule force spectroscopy (SMFS), we have probed at the single‐molecule level the interaction of 1‐deoxy‐d‐xylulose 5‐phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde‐3‐phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single‐molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 µM, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2‐step process with dissociation constants of koff = 6.1 × 10−4 ± 7.5 × 10−3 and 1.3 × 10−2 ± 1.0 × 10−2 s−1, and reaction lengths of xβ = 3.98 ± 0.33 and 0.52 ± 0.23 Å. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.—Sisquella, X., de Pourcq, K., Alguacil, J., Robles, J., Sanz, F., Anselmetti, D., Imperial, S., Fernàndez‐Busquets, X. A single‐molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials. FASEB J. 24, 4203–4217 (2010). www.fasebj.org

Synthesis of modified oligonucleotides containing 4-guanidino-2-pyrimidinone nucleobases

Abstract Oligonucleotides incorporating 4-guanidino-2-pyrimidinone nucleobases have been prepared. These nucleobase analogues were designed to mimic the double hydrogen bond donor pattern of protonated cytosines in parallel triple helices. Guanidine-, N-methyl-, N,N-dimethyl-, and N,N′-dimethylguanidine-containing nucleoside H-phosphonates were used for the synthesis of oligonucleotide analogues with minor modifications in standard solid-phase procedures.

Stepwise solid-phase synthesis of nucleopeptide Phac-Ser(p5′CATCAT)-Gly-Asp-OH from adenovirus-2 nucleoprotein

Abstract Nucleopeptide Phac-Ser(p 5′ CATCAT)-Gly-Asp-OH, corresponding to the linking site of adenovirus-2 nucleoprotein, has been synthesised by a stepwise solid-phase methodology. After assembly of the peptide on a solid support, the oligonucleotide chain elongation was carried out at the hydroxyl group of the serine residue by the phosphite triester approach using 5′-phosphoramidite derivatives. The target nucleopeptide was then obtained in good yield after removal of the permanent protecting groups under mild basic conditions.

Homoserine derivatives for the preparation of base-stable nucleopeptide analogues

Covalently linked peptide-oligonucleotide hybrids are good candidates for antisense or anti-gene therapeutics. The use of homoserine as the linking amino acid allows nucleopeptide analogues with a base-stable amino acid-nucleoside phosphate diester linkage to be obtained. Three Nα, O-protected homoserine derivatives (N α-Boc-Hse(DMT)-O− HTEA+ (I),N α-Fmoc-Hse(MMT)-O−Hpyr+ (II) andN α-Phac-Hse(DMT)-O−HTEA+(III) were prepared after transient silylation,N α-acylation, desilylation and protection of the hydroxyl group. The first can be placed at any position in the peptide sequence, while the other two must be placed at theN-terminus to afford nucleopeptides with the N-terminal amine group free or permanently blocked, respectively.

Solid phase synthesis of a model nucleopeptide with a phosphodiester bond between the 5′ end of a trinucleotide and a serine residue

Abstract The model nucleopeptide Boc-Ser(pTCT)-NHcHex has been assmbled on a polystyrene solid support using the phosphite triester approach. Fmoc and cyanoethyl groups have been used to protect the exocyclic amine of cytosine and phosphate groups respectively. Final deprotection has been carried out by treatment with methanolic potassium carbonate.

Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

The internal ribosome entry site (IRES) element located at the 5´untranslated genomic region of various RNA viruses mediates cap-independent initiation of translation. Picornavirus IRES activity is highly dependent on both its structural organization and its interaction with host factors. Small molecules able to interfere with RNA function are valuable candidates for antiviral agents. Here we show that a small molecule based on benzimidazole (IRAB) inhibited foot-and-mouth disease virus (FMDV) IRES-dependent protein synthesis in cells transfected with infectious RNA leading to a decrease of the virus titer, which was higher than that induced by a structurally related benzimidazole derivative. Interestingly, IRAB preferentially inhibited IRES-dependent translation in cell free systems in a dose-dependent manner. RNA structural analysis by SHAPE demonstrated an increased local flexibility of the IRES structure upon incubation with IRAB, which affected 3 stem-loops (SL) of domain 3. Fluorescence binding assays conducted with individual aminopurine-labeled oligoribonucleotides indicated that the SL3A binds IRAB (EC50 18 μM). Taken together, the results derived from SHAPE reactivity and fluorescence binding assays suggested that the target site of IRAB within the FMDV IRES might be a folded RNA structure that involves the entire apical region of domain 3. Our data suggest that the conformational changes induced by this compound on a specific region of the IRES structure which is essential for its activity is, at least in part, responsible for the reduced IRES efficiency observed in cell free lysates and, particularly, in RNA-transfected cells.

Phosphitylation of Primary Carboxamides. Synthesis of Peptide-Oligonucleotide Conjugates with Acylphosphoramidate Linkages

Abstract N-acylphosphoramidates can be obtained from the reaction of phosphitylated primary carboxamides and an alcohol in the presence of an acid catalyst such as tetrazole and subsequent oxidation. The reaction is useful for the preparation of peptide-oligonucleotide conjugates.

SYNTHESIS AND TRIPLE HELIX-FORMING ABILITY OF OLIGONUCLEOTIDES WITH N, N-DIMETHYLAMINOETHYL PHOSPHORAMIDATE LINKAGES

Abstract Two oligonucleotides, partially modified with N,N -dimethylaminoethyl phosphoramidate groups, were obtained by an optimized solid-phase synthesis cycle based on H -phosphonate chemistry. Their use as third strands in parallel triple helices was shown to produce a decrease in stability with respect to all-phosphodiester oligonucleotide complexes, most probably due to unfavourable steric effects. Phosphoramidate-modified oligonucleotides were shown to be notably stable to exonucleases.