A simple and convenient synthetic protocols based on H-phosphonate chemistry have been developed for the preparation of nucleoside 5′-diphosphates. It consists of oxidation of the silylated H-phosphonate monoesters in pyridine with iodine to produce the corresponding N-pyridiniumphosphonate intermediates, followed by their reactions with orthophosphoric acid used as a nucleophile. The added value of the method is that substrates for the reaction, H-phosphonate monoesters, can be prepared in situ from the corresponding nucleosides and used for the next step without isolation. The procedure is simple and applicable to the preparation of various diphosphates of ribo- and deoxyribonucleosides, and their analogues.

It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (Np_nN’s) and adenosine 5′-phosphoramidate (NH₂-pA) belonging to nucleoside 5′-phosphoramidates (NH₂-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that Np_nN’s induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH₂-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH₂-pA, NH₂-pG) and pyrimidine (NH₂-pU, NH₂-pC) nucleoside 5′-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH₂-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH₂-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5′-phosphoramidates could be considered as new signaling molecules.

The continued advancement of oligonucleotide-based strategies in research and therapeutics relies on expanding the repertoire of chemical modifications to overcome persistent challenges, such as improving cellular uptake and delivery. Addressing these obstacles requires innovative bioconjugation approaches that integrate seamlessly with oligonucleotide modalities. Here, we report the development of a novel phosphotriester trifunctional probe based on the H-phosphonate derivative ammonium (9H-fluoren-9-yl)methyl, introducing significant advancements in synthetic phosphate chemistry. This platform supports robust and versatile chemical transformations, enabling the incorporation of diverse functionalities, such as biotin, fluorescent markers, G4-stabilizing ligands, and azido groups, into oligonucleotide backbones. The resulting multifunctional probes are compatible with different conjugation strategies and phosphorothioate modifications, allowing late-stage functionalization in solution without requiring solid-phase synthesis. We demonstrate the utility of this approach through the synthesis of G4-ligand-conjugated oligonucleotides (GL-Os) designed to target individual G4 structures. However, the strategy's adaptability ensures compatibility with a wide range of oligonucleotide-based applications that benefit from the addition of functional probes. This flexibility broadens accessibility and applicability, facilitating the development of oligonucleotide tools for advanced chemical biology studies, including fluorescence-based imaging and pull-down experiments.

The most common type of modification of a phosphorus center is the replacement of one or two non-bridging atoms of the phosphate group with different heteroatoms (e.g., sulfur, selenium) or groups of atoms (e.g., methyl, borane). Such compounds are referred to as the first generation of nucleotide analogues. We were interested in exploring the possibility of further functionalizing of the heteroatoms present in nucleoside phosphorothioates, phosphoroselenoate or boranephosphonates to create a second generation of nucleotide analogues with hopefully new biological properties. Here we report a preliminary account of our work on introducing additional modifications on the sulfur or selenium heteroatom of nucleotide analogues. Graphical Abstract