Controlling the G-Quadruplex Topology: Toward the Formation of a Lipid Thrombin Binding Aptamer Prodrug.

Important efforts have been devoted toward the development of modified oligonucleotides capable of controlling the secondary structures of the G-quadruplex (G4). Herein, we introduce a photocleavable, lipidated construct of the well-known Thrombin Binding Aptamer (TBA) whose conformation can be dual-controlled by light and/or the ionic strength of the aqueous solution. This novel lipid-modified TBA oligonucleotide spontaneously self-assembles and switches from the conventional antiparallel aptameric fold at low ionic strength to the parallel, inactive conformation of the TBA oligonucleotide strands under physiologically relevant conditions. The latter parallel conformation can be readily and chemoselectively switched back to the antiparallel native aptamer conformation upon light irradiation. Our lipidated construct constitutes an original prodrug of the original TBA with properties that are prone to improving the pharmacodynamic profile of the unmodified TBA.

Bioconjugated Oligonucleotides: Recent Developments and Therapeutic Applications.

Oligonucleotide-based agents have the potential to treat or cure almost any disease, and are one of the key therapeutic drug classes of the future. Bioconjugated oligonucleotides, a subset of this class, are emerging from basic research and being successfully translated to the clinic. In this Review, we first briefly describe two approaches for inhibiting specific genes using oligonucleotides-antisense DNA (ASO) and RNA interference (RNAi)-followed by a discussion on delivery to cells. We then summarize and analyze recent developments in bioconjugated oligonucleotides including those possessing GalNAc, cell penetrating peptides, α-tocopherol, aptamers, antibodies, cholesterol, squalene, fatty acids, or nucleolipids. These novel conjugates provide a means to enhance tissue targeting, cell internalization, endosomal escape, target binding specificity, resistance to nucleases, and more. We next describe those bioconjugated oligonucleotides approved for patient use or in clinical trials. Finally, we summarize the state of the field, describe current limitations, and discuss future prospects. Bioconjugation chemistry is at the centerpiece of this therapeutic oligonucleotide revolution, and significant opportunities exist for development of new modification chemistries, for mechanistic studies at the chemical-biology interface, and for translating such agents to the clinic.

Synthesis and evaluation of apoptosis induction of thienopyrimidine compounds on KRAS and BRAF mutated colorectal cancer cell lines.

Monoclonal antibodies (MoAb) and tyrosine kinase inhibitors (TKI) targeting the EGFR (Epidermal Growth Factor Receptor) pathways are currently used in colorectal cancer treatment. Despite the improvement of median overall survival, resistance is observed notably due to KRAS and BRAF gene mutations. We synthesized four series of thienopyrimidines whose scaffold is structurally close to TKI used in clinical practice. We evaluated apoptosis induced by these compounds using flow cytometry on KRAS and BRAF mutated cell lines. Our results confirm that the mutated cell lines (HCT116 and HT29) are more resistant to apoptosis than the non-mutated cell line (Hela). Interestingly, among the 13 compounds tested, three of them (5b, 6b and 6d) and gefitinib exhibited a noteworthy pro-apoptotic effect, especially on mutated cell lines with an IC(50) value between 70 and 110µM. These three compounds seem particularly attractive for the development of novel treatments for colorectal cancer patients harboring EGFR pathway mutations.

Hybrid lipid oligonucleotide conjugates: synthesis, self-assemblies and biomedical applications.

Hybrid lipid oligonucleotide conjugates are finding more and more biotechnological applications. This short critical review highlights their synthesis, supramolecular organization as well as their applications in the field of biotechnology (111 references).

An analytical study of lipid-oligonucleotide aggregation properties.

Lipid-oligonucleotides (LON) attract great interest as supramolecular scaffolds to improve the intracellular delivery of nucleic acids. Analytical characterization of LON assemblies is critical to formulation development, understanding in-vivo performance, as well as quality control. For this study, we selected LONs featuring different modifications on both oligonucleotide (with or without a G4 prone sequence) and lipid (mono or bis-alkyl chain covalently attached to the oligonucleotide sequence). Size exclusion chromatography (SEC) and, for the first time, capillary electrophoresis (CE) were investigated to study LON supramolecular self-assemblies. Results were correlated to those obtained with conventional physico-chemical characterization techniques i.e. gel electrophoresis, dynamic light scattering, and circular dichroism. In SEC, a separation between LON monomers and micelles was achieved in 5min on a TSK-gel G3000PW column at 70°C with 100% water, as mobile phase. CE conditions were optimized using a fused-silica capillary length of 10.0cm effective length at 15°C. Different background electrolytes were tested by varying the nature and the concentration of salts added. A sodium tetraborate buffer with 75mM NaCl appeared suitable to promote LON assembly. CE offers benefits to LON micelle analysis in terms of speed of analysis, high resolution, and low quantity of sample injected. Moreover, CE provides an appropriate tool to assess the impact of media of biological relevance on LON self-assembly. In this work, the key role of lipophilic tails and the formation of tetramolecular G-quadruplexes on the stability of LON micelles was confirmed.

Intramolecular Michael Additions in Uridine Derivatives: Isolation of the Labile 5'O-C6 Cyclonucleoside.

Uridine derivatives undergo a diastereospecific intramolecular hetero Michael addition onto uracil C6 to give cyclo-adducts. In contrast to the potent amine and thiol nucleophiles at the 5' position of ribose, which readily give the N- and S-cyclonucleosides in good yields, the cyclization reaction from the "natural" 5'-hydroxyl is tedious and has so far been overlooked most probably because of the thermodynamic instability of the O-cyclo-adduct. Here, we show that the O-cyclonucleoside 1 can be isolated, although in low isolated yields, in acidic conditions following an original mechanism. Whether such cyclization reactions occur from biologically relevant pyrimidine-based nucleosides is an open question of interest. Given the structures of thymidine-based antiviral drugs, our results suggest a new hypothetical mode of action for these drugs.

Sensitive liposomes encoded with oligonucleotide amphiphiles: a biocompatible switch.

DNA-tagged liposomes made of DOPC specifically bind to a fluorescently labelled complementary ss-DNA with virtually no influence from the lipid bilayer despite the absence of a linker; depending on an external stimulus, either physical (temperature) or chemical (competitive complementary ON sequences), the liposomes switch between an on and off fluorescent state depending on the location of the probe either at the surface or in the bulk.

Controlling G-quadruplex formation via lipid modification of oligonucleotide sequences.

G-quadruplexes (G4) represent attractive supramolecular scaffolds. In this communication, we show that the lipid modification of a G4 prone oligonucleotide sequence drastically increases the probability of forming tetramolecular parallel G4s with unprecedented conformational control over other unspecific oligomers or folds.

Lipid oligonucleotide conjugates as responsive nanomaterials for drug delivery.

We report Lipid OligoNucleotide conjugates (LONs) bearing either two or three hydrophobic chains. LONs self-assemble into micellar aggregates, which provide a suitable reservoir for hydrophobic drugs such as paclitaxel. Our results demonstrate that the composition of the LONs both in terms of the lipid and the oligonucleotide sequence impacts their ability to host lipophilic molecules. Interestingly, binding of the complementary oligonucleotide selectively induces the release of part of the drug payload of the aggregates. These LON based micelles, which efficiently host hydrophobic drugs, represent an original stimuli-responsive drug delivery system.

Synthesis and study of antiproliferative activity of novel thienopyrimidines on glioblastoma cells.

The receptor tyrosine kinases (for example EGFR, PDGFR, VEGFR) are a transmembrane protein family which plays a crucial role in tumor growth, survival, metastasis dissemination and angiogenesis. During the past 10 years, many tyrosine kinase inhibitors (TKIs) have been approved for cancer treatment (imatinib, gefitinib, erlotinib, sunitinib, sorafenib). These compounds generally possess a pyrrolo- or pyrimido- pyrimidine scaffold or approaching molecular structure. We synthesized 10 thienopyrimidine compounds (including 5 newly synthesized) whose scaffold is very similar to the agents cited above. The cytotoxicity of these agents was evaluated using a MTT assay and a flow cytometry technique on glioblastoma cell lines. Two compounds showed a similar cytotoxicity to the standard anti-EGFR gefitinib (IC50: gefitinib=51.9 microM, 6b=61.8 microM, 6c=41.2 microM), suggesting a blockade of the EGFR pathway by binding to the TK receptor.

Nucleoside, nucleotide and oligonucleotide based amphiphiles: a successful marriage of nucleic acids with lipids.

Amphiphilic molecules based on nucleosides, nucleotides and oligonucleotides are finding more and more biotechnological applications. This Perspective highlights their synthesis, supramolecular organization as well as their applications in the field of biotechnology.

One-step synthesis of O-benzyl hydroxamates from unactivated aliphatic and aromatic esters.

We have developed a simple and high yielding one-step method for the synthesis of hydroxamate derivatives directly from a broad range of unactivated esters and the anion of O-benzyl-hydroxylamine generated in situ. The reaction takes place in minutes at -78 degrees C. Very importantly, the method was successfully employed with enolizable esters, including chiral alpha-amino acid esters and peptides, with no trace of racemization/epimerization at the alpha carbon detected.

A deep cavitand provides a structured environment for the Menschutkin reaction.

We report that a synthetic, vase-shaped host molecule forms complexes with quinuclidine and accelerates the Menschutkin reaction in the nanoenvironment that the host provides. Kinetic simulation is used to determine rate constants for reactions of the complex, and these are compared to control reactions in the absence of the host. The efficacy of the host in effecting this supramolecular acceleration is dependent on the structures of the alkylating agent and its leaving group.

A functionalized, deep cavitand catalyzes the aminolysis of a choline derivative.

The aminolysis of choline p-nitrophenyl carbonate is catalyzed with turnover by a deep cavitand bearing an introverted pyridone function. The synergy of action between the recognition of the guest in the binding pocket and the catalytic activity brought to bear by the pyridone is responsible for the high substrate specificity observed.

Reinvestigation of the noncatalyzed coupling of aryllithium with haloarene: a novel aromatic nucleophilic substitution pathway.

Noncatalyzed coupling reactions of aryllithiums and haloarenes proceed not only through the well-known aryne route but also, in some cases, through a novel addition-elimination pathway. Indeed, ortho-chloro- and ortho-bromomethoxyarenes lead selectively to the corresponding ortho-biaryls through a chelation-driven aromatic nucleophilic substitution pathway. Contrary to common belief, such noncatalyzed coupling reactions often proceed with high regioselectivity and high yield. These results underline the potency of such simple reactions and open up a straightforward access to a wide range of biaryl structures; this also appears particularly useful for large-scale and biaryl building-block syntheses, as only cheap and readily available substrates are involved.

NaNO2-mediated transformation of aliphatic secondary nitroalkanes into ketones or oximes under neutral, aqueous conditions: how the nitro derivative catalyzes its own transformation.

The nitrosation of secondary nitro derivatives into ketones or oximes depending on the nitro substituents has been reinvestigated. The reaction efficiently takes place under neutral conditions, thus allowing acid-sensitive substrates to be converted in very good yields. The generation of nitrosating species under such mild conditions is unprecedented. Mechanistic investigations strongly suggest that they result from the nucleophilic attack of the nitrite anion on the aci-nitro(nate) form of the secondary nitroalkane. The latter acts in turn as an autocatalyst for its own transformation by means of the nitrosating species generated in situ from it.