Enzymatic transglycosylation – the transfer of the carbohydrate moiety from one heterocyclic base to another – is being actively developed and applied for the synthesis of practically important nucleosides. This reaction is catalyzed by nucleoside phosphorylases (NPs), which are responsible for reversible phosphorolysis of nucleosides to yield the corresponding heterocyclic bases and monosaccharide 1‐phosphates. We found that 7‐methyl‐2′‐deoxyguanosine (7‐Me‐dGuo) is an efficient and novel donor of the 2‐deoxyribose moiety in the enzymatic transglycosylation for the synthesis of purine and pyrimidine 2′‐deoxyribonucleosides in excellent yields. Unlike 7‐methylguanosine, its 2′‐deoxy derivative is dramatically less stable. Fortunately, we have found that 7‐methyl‐2′‐deoxyguanosine hydroiodide may be stored for 24 h in Tris‐HCl buffer (pH 7.5) at room temperature without significant decomposition. In order to optimize the reagent ratio, a series of analytical transglycosylation reactions were conducted at ambient temperature. According to HPLC analysis of the transglycosylation reactions, the product 5‐ethyl‐2′‐deoxyuridine (5‐Et‐dUrd) was obtained in high yield (84–93%) by using a small excess (1.5 and 2.0 equiv.) of 7‐Me‐dGuo over 5‐ethyluracil (5‐Et‐Ura) and 0.5 equiv. of inorganic phosphate. Thymidine is a less effective precursor of α‐d‐2‐deoxyribofuranose 1‐phosphate (dRib‐1p) compared to 7‐Me‐dGuo. We synthesized 2′‐deoxyuridine, 5‐Et‐dUrd, 2′‐deoxyadenosine and 2′‐deoxyinosine on a semi‐preparative scale using the optimized reagent ratio (1.5:1:0.5) in high yields. Unlike other transglycosylation reactions, the synthesis of 2‐chloro‐2′‐deoxyadenosine was performed in a heterogeneous medium because of the poor solubility of the initial 2‐chloro‐6‐aminopurine. Nevertheless, this nucleoside was prepared in good yield. The developed enzymatic procedure for the preparation of 2′‐deoxynucleosides may compete with the known chemical approaches.magnified image

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Biological effects of hormones in both plants and animals are based on high-affinity interaction with cognate receptors resulting in their activation. The signal of cytokinins, classical plant hormones, is perceived in Arabidopsis by three homologous membrane receptors: AHK2, AHK3, and CRE1/AHK4. To study the cytokinin-receptor interaction, we used 25 derivatives of potent cytokinin N-benzyladenine (BA) with substituents in the purine heterocycle and/or in the side chain. The study was focused primarily on individual cytokinin receptors from Arabidopsis. The main in planta assay system was based on Arabidopsis double mutants retaining only one isoform of cytokinin receptors and harboring cytokinin-sensitive reporter gene. Classical cytokinin biotest with Amaranthus seedlings was used as an additional biotest. In parallel, the binding of ligands to individual cytokinin receptors was assessed in the in vitro test system. Quantitative comparison of results of different assays confirmed the partial similarity of ligand-binding properties of receptor isoforms. Substituents at positions 8 and 9 of adenine moiety, elongated linker up to 4 methylene units, and replacement of N by sulfur or oxygen have resulted in the suppression of cytokinin activity of the derivative toward all receptors. Introduction of a halogen into position 2 of adenine moiety, on the contrary, often increased the ligand activity, especially toward AHK3. Features both common and distinctive of cytokinin receptors in Arabidopsis and Amaranthus were revealed, highlighting species specificity of the cytokinin perception apparatus. Correlations between the extent to which a compound binds to a receptor in vitro and its ability to activate the same receptor in planta were evaluated for each AHK protein. Interaction patterns between individual receptors and ligands were rationalized by structure analysis and molecular docking in sensory modules of AHK receptors. The best correlation between docking scores and specific binding was observed for AHK3. In addition, receptor-specific ligands have been discovered with unique properties to predominantly activate or block distinct cytokinin receptors. These ligands are promising for practical application and as molecular tools in the study of the cytokinin perception by plant cells.

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Oligonucleotide–peptide conjugates (OPCs) are a promising class of biologically active compounds with proven potential for improving nucleic acid therapeutics. OPCs are commonly recognized as an efficient instrument to enhance the cellular delivery of therapeutic nucleic acids. In addition to this application field, OPCs have an as yet unexplored potential for the post-SELEX optimization of DNA aptamers. In this paper, we report the preparation of designer thrombin aptamer OPCs with peptide side chains anchored to a particular thymidine residue of the aptamer. The current conjugation strategy utilizes unmodified short peptides and support-bound protected oligonucleotides with activated carboxyl functionality at the T3 thymine nucleobase. The respective modification of the oligonucleotide strand was implemented using N3-derivatized thymidine phosphoramidite. Aptamer OPCs retained the G-quadruplex architecture of the parent DNA structure and showed minor to moderate stabilization. In a series of five OPCs, conjugates bearing T3–Ser–Phe–Asn (SFN) or T3–Tyr–Trp–Asn (YWN) side chains exhibited considerably improved anticoagulant characteristics. Molecular dynamics studies of the aptamer OPC complexes with thrombin revealed the roles of the amino acid nature and sequence in the peptide subunit in modulating the anticoagulant activity.