A Catalytic Approach for the Synthesis of Peptide-Oligonucleotides Conjugates in Aqueous Solution or On-Column.

Peptide-oligonucleotide conjugates (POCs) are covalent architectures composed of a DNA or RNA molecules linked to a peptide. These constructs have found widespread applications ranging from hybrid nanomaterials to gene-targeted therapies. Considering the important role of POCs, a new catalytic approach for their preparation is reported here, that could be applied either on solid support in anhydrous media, or post-synthetically in aqueous buffer. Single amino acids, peptides and cell penetrating peptides (CPPs) were conjugated to various oligo(ribo)nucleotides with high conversions and good isolated yields. The applicability of the method was demonstrated on more than 35 examples including an analogue of a commercial therapeutic oligonucleotide. Other conjugation partners, such as deoxycholic acid and biotin were also successfully conjugated to oligonucleotides. To highlight the potential of this catalytic approach, these conditions have been applied to iterative processes, which is of high interest for the development of DNA-Encoded Libraries.

The hydrazine moiety in the synthesis of modified nucleosides and nucleotides.

Synthetic nucleoside mimics are re-emerging as crucial contenders for antiviral and anticancer medications. While, Ribavirin stands out for its unique antiviral properties, predominantly associated with its distinctive triazole heterocycle as a nucleobase, the exploration of alternative nitrogen-based aromatic heterocycles hold great promises for the discovery of novel bioactive nucleoside mimics. Although nucleoside derivatives synthesised from hydrazine-ribose units have been in development for many decades, they have been little evaluated biologically and even less for their antiviral properties. With the aim of taking a closer look at these under-explored derivatives and investigating their synthetic pathways, this concise review provides an overview of the molecular design, the chemical synthesis, and the biological activity, when available, of these nucleoside analogues. Overall, the entire body of work already done motivates further exploration of theses analogues and encourages us of formulating structurally novel nucleoside drug candidates featuring innovative mode of action.

Synthesis and properties of RNA constrained by a 2'-O-disulfide bridge.

We recently reported the properties of RNA hairpins constrained by a dimethylene (DME) disulfide (S-S) linker incorporated between two adjacent nucleosides in the loop and showed that this linker locked the hairpin conformation thus disturbing the duplex/hairpin equilibrium. We have now investigated the influence of the length of the linker and synthesized oligoribonucleotides containing diethylene (DEE) and dipropylene (DPE) S-S bridges. This was achieved via the preparation of building blocks, namely 2'-O-acetylthioethyl (2'-O-AcSE) and 2'-O-acetylthiopropyl (2'-O-AcSP) uridine phosphoramidites, which were successfully incorporated into RNA sequences. Thermal denaturation analysis revealed that the DEE and DPE disulfide bridges destabilize RNA duplexes but do not disrupt the hairpin conformation. Furthermore, our investigation of the duplex/hairpin equilibrium indicated that sequences modified with DME and DEE S-S linkers predominantly lock the hairpin form, whereas the DPE S-S linker provides flexibility. These findings highlight the potential of S-S linkers to study RNA interactions.

Synthesis of Original Cyclic Dinucleotide Analogues Using the Sulfo-click Reaction.

The stimulator of interferon genes (STING) protein plays a crucial role in the activation of the innate immune response. Activation of STING is initiated by cyclic dinucleotides (CDNs) which prompted the community to synthesize structural analogues to enhance their biological properties. We present here the synthesis and biological evaluation of four novel CDN analogues composed of an N-acylsulfonamide linkage. These CDNs were obtained in high overall yields via the sulfo-click reaction as a key step.

Stimuli-Responsive Boronate Formation to Control Nucleic Acid-Based Functional Architectures.

Boronate esters, formed by the reaction of an oligonucleotide bearing a 5'-boronic acid moiety with the 3'-terminal cis-diol of another oligonucleotide, support the assembly of functional nucleic acid architectures. Reversible formation of boronate esters occurs in templated fashion and has been shown to restore the activity of split DNA and RNA enzymes as well as a split fluorescent light-up aptamer. Apart from their suitability for the design and application of split nucleic acid enzymes and aptamers in the field of biosensing, boronate esters may have played an important role in early life as surrogates of the natural phosphodiester bond. Their formation is reversible and thus fulfills an important requirement for biological self-assembly. Here we discuss the general concept of stimuli-dependent boronate formation and its application in biomolecules with implications for future research.

Boron-containing carbonic anhydrases inhibitors.

Over the last decades, the medicinal chemistry of boron-based compounds has been extensively explored, designing valuable small molecule drugs to tackle diseases and conditions, such as cancer, infections, inflammatory and neurological disorders. Notably, boron has proven to also be a valuable element for the development of inhibitors of the metalloenzymes carbonic anhydrases (CAs), a class of drug targets with significant potential in medicinal chemistry. Incorporating boron into carbonic anhydrase inhibitors (CAIs) can modulate the ligand ability to recognize the target and/or influence selectivity towards different CA isoforms, using the tail approach and boron-based tails. The electron-deficient nature of boron and its associated properties have also led to the discovery of novel zinc-binding CAIs, such as boronic acids and the benzoxaboroles, capable of inhibiting the CAs upon a Lewis acid-base mechanism of action. The present manuscript reviews the state-of-the-art of boron-based CAIs. As research in the applications of boron compounds in medicinal chemistry continues, it is anticipated that new boron-based CAIs will soon expand the current array of such compounds. However, further research is imperative to fully unlock the potential of boron-based CAIs and to advance them towards clinical applications.

Probing naphthalene diimide and 3-hydroxypropylphosphate as end-conjugating moieties for improved thrombin binding aptamers: Structural and biological effects.

The limitations associated with the in vivo use of the thrombin binding aptamer (TBA or TBA15) have dramatically stimulated the search of suitable chemically modified analogues in order to discover effective and reversible inhibitors of thrombin activity. In this context, we previously proposed cyclic and pseudo-cyclic TBA analogues with improved stability that proved to be more active than the parent aptamer. Herein, we have investigated a novel library of TBA derivatives carrying naphthalene diimide (NDI) moieties at the 3'- or 5'-end. In a subset of the investigated oligonucleotides, additional 3-hydroxypropylphosphate (HPP) groups were introduced at one or both ends of the TBA sequence. Evaluation of the G-quadruplex thermal stability, serum nuclease resistance and in vitro anticoagulant activity of the new TBA analogues allowed rationalizing the effect of these appendages on the activity of the aptamer on the basis of their relative position. Notably, most of the different TBA analogues tested were more potent thrombin inhibitors than unmodified TBA. Particularly, the analogue carrying an NDI group at the 5'-end and an HPP group at the 3'-end, named N-TBA-p, exhibited enhanced G-quadruplex thermal stability (ΔTm + 14° C) and ca. 10-fold improved nuclease resistance in serum compared to the native aptamer. N-TBA-p also induced prolonged and dose-dependent clotting times, showing a ca. 11-fold higher anticoagulant activity compared to unmodified TBA, as determined by spectroscopic methods. Overall, N-TBA-p proved to be in vitro a more efficient thrombin inhibitor than all the best ones previously investigated in our group. Its interesting features, associated with its easy preparation, make it a very promising candidate for future in vivo studies.

N-Acylsulfonamide: a valuable moiety to design new sulfa drug analogues.

Sulfonamides are the oldest class of antibiotics, discovered more than 80 years ago. They are still used today despite the appearance of drug resistance phenomena that limit their prescription. Since the discovery and use of the first sulfa drugs, many analogues have been synthesized in order to obtain new active molecules able to circumvent bacterial resistance. Structurally similar to sulfonamide, the N-acylsulfonamide group arouses interest in the field of medicinal chemistry due to specific physico-chemical properties. We report here the synthesis and antibacterial/antibiofilm activities of 18 sulfa drug analogues with an N-acylsulfonamide moiety. These derivatives were obtained efficiently by sulfo-click reactions between readily available thioacid and sulfonyl azide synthons.

6-Substituted Triazolyl Benzoxaboroles as Selective Carbonic Anhydrase Inhibitors: In Silico Design, Synthesis, and X-ray Crystallography.

Benzoxaborole is currently a scaffold of great relevance in medicinal chemistry. In 2016, it was reported to be a new and valuable chemotype for designing carbonic anhydrase (CA) inhibitors. Herein, using an in silico design, we report the synthesis and characterization of substituted 6-(1H-1,2,3-triazol-1-yl)benzoxaboroles. 6-Azidobenzoxaborole was described for the first time as a molecular platform to prepare libraries of inhibitors by a copper(I)-catalyzed azide-alkyne cycloaddition via a click chemistry strategy. With inhibition constants below 30 nM, some derivatives, such as compound 20, showed efficacy as selective hCA VII and IX inhibitors. The design hypothesis was validated by crystallographic investigation on the hCA II/20 adduct, which provided explanations over the different inhibition behavior observed against the five evaluated hCA isoforms. Overall, this study identified 20 as a new promising lead compound to develop novel anticancer agents targeting the tumor-associated hCA IX but also potent neuropathic pain relievers targeting hCA VII.

Stabilization of DNA Duplexes and Hairpins by Charge-Transfer Interactions Using DAN:NDI Pairs.

Electron-rich 1,5-dialkoxynaphthalene (DAN) and electron-deficient 1,8,4,5-naphthalenetetracarboxylic diimide (NDI) are known to interact through the formation of charge-transfer complexes. The introduction of DAN and NDI into various DNA duplexes and hairpins was investigated by ultraviolet (UV) melting curve analysis. The positioning of the DAN:NDI pair was found to strongly influence the stability of DNA duplex and hairpins. In particular, while the introduction of one DAN/NDI pair in front of each other in the center of a DNA duplex led to a decrease of the thermal stability (ΔTm - 6 °C), the addition of a second pair restored or even increased the stability. In contrast, the introduction of DAN:NDI pairs at the end of a duplex always induced a strong stabilization (ΔTm up to +20 °C). Finally, a DAN:NDI pair positioned in the loop of a hairpin induced a stronger stabilization than a T4 loop (ΔTm + 10 °C). Based on charge-transfer interactions, the strong stabilizations observed allow the preparation of highly stabilized DNA nanostructures opening the way to numerous applications in nanotechnology.

Boronic Acid Assisted Self-Assembly of Functional RNAs.

Boronate esters formed by reaction of an oligonucleotide carrying a 5'-boronic acid moiety with the 3'-terminal cis-diol of another have been shown previously to assist assembly of fragmented DNAzymes. Here we demonstrate that boronate esters replacing the natural phosphodiester linkage at selected sites of two functional RNAs, the hairpin ribozyme and the Mango aptamer, allow assembly of functional structures. The hairpin ribozyme, a small naturally occurring RNA that supports the reversible cleavage of appropriate RNA substrates, is very sensitive to fragmentation. Splitting the ribozyme at four different sites led to a significant decrease or even loss of cleavage and ligation activity. Ribozymes assembled from fragments capable of boronate ester formation showed restoration of cleavage activity in some but not all cases, dependent on the split site. Ligation proved to be more challenging, no supportive effect of the boronate ester was observed. Split variants of the Mango aptamer also showed a dramatic loss of functionality, which however, was restored when 5'-boronic acid modified fragments were used for assembly. These studies show for the first time that boronate esters as internucleoside linkages can act as surrogates of natural phosphodiesters in functional RNA molecules.

Expanding the 'aplysinospin cascade' through DNA-templated [2+2] photocycloaddition.

Inspired by the unique ability of nucleic acids to template chemical transformations that are otherwise impossible in solution, we embarked on the generalisation of our DNA-templated [2+2] photo-induced homo- and heterodimerization of aplysinopsins. Our process ensures a straightforward access to cyclobutane containing natural products and analogues thereof. Most importantly, this conceptual biomimetic achievement presents interesting arguments to build a biosynthetic scenario.

Supramolecular Recognition of Phosphodiester-Based Donor and Acceptor Oligomers Forming Gels in Water.

Inspired by automated DNA synthesis, electron-rich dialkoxynaphthalene (DAN) donor and electron-deficient naphthalene-tetracarboxylic diimide (NDI) acceptor phosphodiester-linked homohexamers were synthesized by the phosphoramidite method. Two types of hexamers were prepared, one with only one phosphodiester between the aromatics (i.e., DAN or NDI) and a second with two phosphodiesters around a propanediol between the aromatics, leading to the latter more flexible and more hydrophilic hexamers. The folding properties of these homohexamers alone or mixed together, in water only, were studied by UV-visible absorption spectroscopy and atomic force microscopy (AFM). AFM imaging revealed that a 1:1 mixture of hexaDAN and hexaNDI formed fibers by charge transfer donor-acceptor recognition leading to a hydrogel after drying. The organization of the resulting structures is strongly dependent on the nature of the complementary partner, leading to the formation of mono- or multilayer hydrogel networks with different compactness.

A terminal functionalization strategy reveals unusual binding abilities of anti-thrombin anticoagulant aptamers.

Despite their unquestionable properties, oligonucleotide aptamers display some drawbacks that continue to hinder their applications. Several strategies have been undertaken to overcome these weaknesses, using thrombin binding aptamers as proof-of-concept. In particular, the functionalization of a thrombin exosite I binding aptamer (TBA) with aromatic moieties, e.g., naphthalene dimides (N) and dialkoxynaphthalenes (D), attached at the 5' and 3' ends, respectively, proved to be highly promising. To obtain a molecular view of the effects of these modifications on aptamers, we performed a crystallographic analysis of one of these engineered oligonucleotides (TBA-NNp/DDp) in complex with thrombin. Surprisingly, three of the four examined crystallographic structures are ternary complexes in which thrombin binds a TBA-NNp/DDp molecule at exosite II as well as at exosite I, highlighting the ability of this aptamer, differently from unmodified TBA, to also recognize a localized region of exosite II. This novel ability is strictly related to the solvophobic behavior of the terminal modifications. Studies were also performed in solution to examine the properties of TBA-NNp/DDp in a crystal-free environment. The present results throw new light on the importance of appendages inducing a pseudo-cyclic charge-transfer structure in nucleic acid-based ligands to improve the interactions with proteins, thus considerably widening their potentialities.

Facile access to 4'-(N-acylsulfonamide) modified nucleosides and evaluation of their inhibitory activity against SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14.

N-Acylsulfonamides possess an additional carbonyl function compared to their sulfonamide analogues. Due to their unique physico-chemical properties, interest in molecules containing the N-acylsulfonamide moiety and especially nucleoside derivatives is growing in the field of medicinal chemistry. The recent renewal of interest in antiviral drugs derived from nucleosides containing a sulfonamide function has led us to evaluate the therapeutic potential of N-acylsulfonamide analogues. While these compounds are usually obtained by a difficult acylation of sulfonamides, we report here the easy and efficient synthesis of 20 4'-(N-acylsulfonamide) adenosine derivatives via the sulfo-click reaction. The target compounds were obtained from thioacid and sulfonyl azide synthons in excellent yields and were evaluated as potential inhibitors of the SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14.

New Benchmark in DNA-Based Asymmetric Catalysis: Prevalence of Modified DNA/RNA Hybrid Systems.

By harnessing the chirality of the DNA double helix, chemists have been able to obtain new, reliable, selective, and environmentally friendly biohybrid catalytic systems with tailor-made functions. Nonetheless, despite all the advances made throughout the years in the field of DNA-based asymmetric catalysis, many challenges still remain to be faced, in particular when it comes to designing a "universal" catalyst with broad reactivity and unprecedented selectivity. Rational design and rounds of selection have allowed us to approach this goal. We report here the development of a DNA/RNA hybrid catalytic system featuring a covalently attached bipyridine ligand, which exhibits unmatched levels of selectivity throughout the current DNA toolbox and opens new avenues in asymmetric catalysis.

Applications of the Reversible Boronic Acids/Boronate Switch to Nucleic Acids.

Over the last decades, boron and nucleic acids chemistries have gained a lot of attention for biological, medicinal and analytical applications. Our laboratory has a long-standing interest in both chemistries and owing to the ability of boronic acids to react with cis-diol function in aqueous media we developed over the years a variety of applications ranging from molecular recognition and sensing to the development of reversible dynamic systems in which the natural phosphodiester linkage was replaced by a boronate. In this account, we summarize research results from our group from our preliminary studies on molecular recognition of ribonucleosides to the dynamic assembly of functional DNAzymes. In particular, the various parameters influencing the dynamic nature of these reversible covalent bonds able to respond to external stimuli are discussed. Finally, current challenges and opportunities for boron-based nucleic acids are also addressed.

Design and NMR characterization of reversible head-to-tail boronate-linked macrocyclic nucleic acids.

Inspired by the ability of boronic acids to bind with compounds containing diol moieties, we envisioned the formation in solution of boronate ester-based macrocycles by the head-to-tail assembly of a nucleosidic precursor that contains both a boronic acid and the natural 2',3'-diol of ribose. DOSY NMR spectroscopy experiments in water and anhydrous DMF revealed the dynamic assembly of this precursor into dimeric and trimeric macrocycles in a concentration-dependent fashion as well as the reversibility of the self-assembly process. NMR experimental values and quantum mechanics calculations provided further insight into the sugar pucker conformation profile of these macrocycles.

Charge-Transfer Interactions Stabilize G-Quadruplex-Forming Thrombin Binding Aptamers and Can Improve Their Anticoagulant Activity.

In the search for optimized thrombin binding aptamers (TBAs), we herein describe the synthesis of a library of TBA analogues obtained by end-functionalization with the electron-rich 1,5-dialkoxy naphthalene (DAN) and the electron-deficient 1,8,4,5-naphthalenetetra-carboxylic diimide (NDI) moieties. Indeed, when these G-rich oligonucleotides were folded into the peculiar TBA G-quadruplex (G4) structure, effective donor-acceptor charge transfer interactions between the DAN and NDI residues attached to the extremities of the sequence were induced, providing pseudo-cyclic structures. Alternatively, insertion of NDI groups at both extremities produced TBA analogues stabilized by π-π stacking interactions. All the doubly-modified TBAs were characterized by different biophysical techniques and compared with the analogues carrying only the DAN or NDI residue and unmodified TBA. These modified TBAs exhibited higher nuclease resistance, and their G4 structures were markedly stabilized, as evidenced by increased Tm values compared to TBA. These favorable properties were also associated with improved anticoagulant activity for one DAN/NDI-modified TBA, and for one NDI/NDI-modified TBA. Our results indicated that TBA pseudo-cyclic structuring by ad hoc designed end-functionalization represents an efficient approach to improve the aptamer features, while pre-organizing and stabilizing the G4 structure but allowing sufficient flexibility to the aptamer folding, which is necessary for optimal thrombin recognition.

Modified internucleoside linkages for nuclease-resistant oligonucleotides.

In the past few years, several drugs derived from nucleic acids have been approved for commercialization and many more are in clinical trials. The sensitivity of these molecules to nuclease digestion in vivo implies the need to exploit resistant non-natural nucleotides. Among all the possible modifications, the one concerning the internucleoside linkage is of particular interest. Indeed minor changes to the natural phosphodiester may result in major modifications of the physico-chemical properties of nucleic acids. As this linkage is a key element of nucleic acids' chemical structures, its alteration can strongly modulate the plasma stability, binding properties, solubility, cell penetration and ultimately biological activity of nucleic acids. Over the past few decades, many research groups have provided knowledge about non-natural internucleoside linkage properties and participated in building biologically active nucleic acid derivatives. The recent renewing interest in nucleic acids as drugs, demonstrated by the emergence of new antisense, siRNA, aptamer and cyclic dinucleotide molecules, justifies the review of all these studies in order to provide new perspectives in this field. Thus, in this review we aim at providing the reader insights into modified internucleoside linkages that have been described over the years whose impact on annealing properties and resistance to nucleases have been evaluated in order to assess their potential for biological applications. The syntheses of modified nucleotides as well as the protocols developed for their incorporation within oligonucleotides are described. Given the intended biological applications, the modifications described in the literature that have not been tested for their resistance to nucleases are not reported.