Christelle Dupouy

Metallophyte wastes and polymetallic catalysis: a promising combination in green chemistry. The illustrative synthesis of 5′-capped RNA

The compatibility of metallic catalysts derived from plants was studied for the chemical solid-phase synthesis of a high added-value biomolecule: 5′-capped RNA (GpppRNA or 7mGpppRNA) involved in many essential biological processes. These molecules are of great interest for biologists for structural and mechanistic studies of their complexes with RNA capping enzymes. Several polymetallic catalysts were prepared from various metallophytes species growing in mine sites in a context of phytoremediation programs. Their catalytic efficacy was evaluated for the coupling reaction of guanosine diphosphate (GDP) with a preactivated 5′-monophosphate hexathymidylate (pT6) or a pRNA to form 5′-GpppT6 or 5′-GpppRNA respectively. The direct coupling of 7mGDP with pRNA was also investigated. With Zn-enriched catalysts obtained from Zn metallophytes, the capping yields were better than those obtained with conventional catalyst ZnCl2. Interestingly with Ni-enriched plant extracts the yield was higher than with NiCl2, a polymetallic catalysis was certainly involved. With this plant-inspired catalysis, metallophytes wastes become efficient and greener polymetallic catalysts. Finally, environmental benefits and effectiveness can be combined.

Chemical Synthesis of RNA with Base‐Labile 2′‐O‐(Pivaloyloxymethyl)‐Protected Ribonucleoside Phosphoramidites

The efficiency of chemical RNA synthesis has been radically improved by the use of pivaloyloxymethyl (PivOM) groups as 2′‐protection, containing an acetal spacer that minimizes the steric effect of the ester group on the neighboring amidite during the coupling. However, the major benefit of the base‐labile PivOM groups is their simple removal upon standard basic conditions applied to deprotect the RNA and release it from solid supports. Combined with standard acyl groups for nucleobases, cyanoethyl groups for phosphates, and base‐cleavable linkers, PivOM groups make RNA deprotection as simple as DNA deprotection. Thus, no additional deprotection step with tedious desalting workup procedures is required, and RNA synthesis becomes as convenient and efficient as DNA synthesis. Curr. Protoc. Nucleic Acid Chem. 43:3.19.1‐3.19.27.

Direct Synthesis of Partially Modified 2′-O-Pivaloyloxymethyl RNAs by a Base-Labile Protecting Group Strategy and their Potential for Prodrug-Based Gene-Silencing Applications

An original and straightforward synthesis of partially modified 2′‐O‐pivaloyloxymethyl‐substituted (PivOM‐substituted) oligoribonucleotides has been achieved. The aim of this 2′‐enzymolabile modification was to enhance nuclease stability of RNA and transmembrane transport. To make these modified RNAs easily available we developed a base‐labile protecting group strategy with standard protections for nucleobases (acyl) and phosphates (cyanoethyl), a Q‐linker and two different acetalester protection groups for 2′‐OH: propionyloxymethyl (PrOM) and PivOM. Interestingly, orthogonal deprotection conditions based on anhydrous butylamine in THF were found to remove propionyloxymethyl groups selectively, while preserving PivOM groups. Duplex stability, circular dichroism studies and nuclease resistance, as well as the ability to inhibit gene expression of modified 2′‐O‐PivOM RNA, were evaluated.

Stimuli-responsive oligonucleotides in prodrug-based approaches for gene silencing

Oligonucleotides (ONs) have been envisaged for therapeutic applications for more than thirty years. However, their broad use requires overcoming several hurdles such as instability in biological fluids, low cell penetration, limited tissue distribution, and off-target effects. With this aim, many chemical modifications have been introduced into ONs definitively as a means of modifying and better improving their properties as gene silencing agents and some of them have been successful. Moreover, in the search for an alternative way to make efficient ON-based drugs, the general concept of prodrugs was applied to the oligonucleotide field. A prodrug is defined as a compound that undergoes transformations in vivo to yield the parent active drug under different stimuli. The interest in stimuli-responsive ONs for gene silencing functions has been notable in recent years. The ON prodrug strategies usually help to overcome limitations of natural ONs due to their low metabolic stability and poor delivery. Nevertheless, compared to permanent ON modifications, transient modifications in prodrugs offer the opportunity to regulate ON activity as a function of stimuli acting as switches. Generally, the ON prodrug is not active until it is triggered to release an unmodified ON. However, as it will be described in some examples, the opposite effect can be sought. This review examines ON modifications in response to various stimuli. These stimuli may be internal or external to the cell, chemical (glutathione), biochemical (enzymes), or physical (heat, light). For each stimulus, the discussion has been separated into sections corresponding to the site of the modification in the nucleotide: the internucleosidic phosphate, the nucleobase, the sugar or the extremities of ONs. Moreover, the review provides a current and detailed account of stimuli-responsive ONs with the main goal of gene silencing. However, for some stimuli-responsive ONs reported in this review, no application for controlling gene expression has been shown, but a certain potential in this field could be demonstrated. Additionally, other applications in different domains have been mentioned to extend the interest in such molecules.

Lipophilic 2-O-Acetal Ester RNAs: Synthesis, Thermal Duplex Stability, Nuclease Resistance, Cellular Uptake, and siRNA Activity after Spontaneous Naked Delivery

The in vivo application of siRNA depends on its cellular uptake and intracellular release, and this is an unsatisfactorily resolved technical hurdle in medicinal applications. Promising concepts directed towards providing efficient cellular and intracellular delivery include lipophilic chemical modification of siRNA. Here we describe chemistry for the production of modified siRNAs designed to display improved transmembrane transport into human cells while preserving the potency of the RNAi‐based inhibitors. We report the synthesis and the biochemical and biophysical characteristics of 2′‐O‐phenylisobutyryloxymethyl (PiBuOM)‐modified siRNAs and their impact on biological activity. In the case of spontaneous cellular uptake of naked PiBuOM‐modified siRNA, we observed increased target suppression in human cells relative to unmodified or pivaloyloxymethyl (PivOM)‐modified siRNA. We provide evidence of improved spontaneous cellular uptake of naked PiBuOM‐modified siRNA and of substantial target suppression in human cells in serum‐containing medium.