Ekaterina V. Efimtseva

An efficient synthesis and physico-chemical properties of 2'-O-D-ribofuranosylnucleosides, minor tRNA components

ABSTRACT A high yield preparation of 9-(2-O-β-D-ribofuranosyl-β-D-ribofuranosyl)adenine, guanine- and the pyrimidine analogs (cytosine, thymine and uracil base moiety) has been achieved, and the conformational properties of the ring systems were investigated using NMR spectroscopy and X-ray.

Oligonucleotides Containing Disaccharide Nucleosides

Disaccharide nucleosides with 2′-O-(D-arabinofuranosyl), 2′-O-(L-arabinofuranosyl), 2′-O-(D-ribopyranosyl), 2′-O-(D-erythrofuranosyl), and 2′-O-(5-azido-5-deoxy-D-ribofuranosyl) substituents were synthesized. These modified nucleosides were incorporated into oligonucleotides (see Table). Single substitution resulted in a ΔTm of +0.5 to −1.4° for DNA/RNA and a ΔTm of −0.8 to −4.7° for DNA/DNA duplexes. These disaccharide nucleosides can be well accommodated in RNA/DNA duplexes, and the presence of a NH2−C(5″) group has a beneficial effect on duplex stability.

Disaccharide Nucleosides and Oligonucleotides on Their Basis. New Tools for the Study of Enzymes of Nucleic Acid Metabolism

The main structural features of an important group of natural compounds, disaccharide nucleosides, are reviewed. The synthesis and properties of modified oligonucleotides on their basis as well as the methods of introduction of reactive aldehyde groups are described. The last part is devoted to the application of these compounds for studies of enzymes of nucleic acid metabolism.

Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups

Oligonucleotides of a novel type containing 2′‐O‐β‐ribofuranosyl‐cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild‐type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter‐containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of protein‐nucleic acid interactions.

Determination of the nucleotide conformation in the productive enzyme-substrate complexes of RNA-depolymerases

© 1997 Federation of European Biochemical Societies.

Synthesis and properties of O-beta-D-ribofuranosyl-(1"-2')-adenosine-5"-O-phosphate and its derivatives.

Abstract The synthesis of O-β-D-ribofuranosyl-(1″-2′)-adenosine-5″-O-phosphate and its suitably protected derivative for oligonucleotide synthesis have been developed.

Effective Anomerisation of 2′‐Deoxyadenosine Derivatives During Disaccharide Nucleoside Synthesis

The formation of a disaccharide nucleoside (11) by O3′‐glycosylation of 5′‐O‐protected 2′‐deoxyadenosine or its N 6‐benzoylated derivative has been observed to be accompanied by anomerisation to the corresponding α‐anomeric product (12). The latter reaction can be explained by instability of the N‐glycosidic bond of purine 2′‐deoxynucleosides in the presence of Lewis acids. An independent study on the anomerisation of partly blocked 2′‐deoxyadenosine has been carried out. Additionally, transglycosylation has been utilized in the synthesis of 3′‐O‐β‐d‐ribofuranosyl‐2′‐deoxyadenosines and its α‐anomer.

Synthesis and Properties of Phosphorylated 3′-O-β-D-Ribofuranosyl-2′-deoxythymidine

Abstract A strategy was developed for the synthesis of 3′-O-β-D-ribofuranosyl 2′-deoxythymidine derivatives using three different protecting groups, which allows the synthesis of a phosphoramidite building block for oligonucleotide synthesis. Likewise the 5′-O- and 5″-O-phosphorylated analogues were synthesized and their conformation was determined using NMR spectroscopy.

Interaction of HIV-1 Reverse Transcriptase and T7 RNA Polymerase with Phosphonate Analogs of NTP and Inorganic Pyrophosphate

We have examined the interaction of human immunodeficiency virus reverse transcriptase (HIV RT) and T7 RNA polymerase (T7 RNAP) with modified nucleoside triphosphates and inorganic pyrophosphate (PPi) analogs containing nonhydrolyzable bisphosphonate groups. We have synthesized a number of derivatives of bisphosphonic acid having different aromatic and nonaromatic side substituents, as well as the NTP derivatives whose incorporation into the growing nucleotide chain during the polymerization reaction results in formation of bisphosphonates as leaving groups. The competitive character of inhibition of both enzymes has been revealed for all the compounds under study, and the inhibition constants have been estimated. One of PPianalogs containing a bulky aromatic substituent is characterized by similar inhibition constants for both T7 RNAP and RT. The universal character of this inhibitor can serve as evidence for a similar structure of the NPT-binding sites in the two polymerases. It has been shown that nonsubstituted methylenebisphosphate is a better leaving group than that containing additional methyl and hydroxyl groups. The NTP analogs are very weak inhibitors of T7 RNAP, whereas HIV RT is more sensitive to this type of compounds. On the basis of the X-ray crystallographic data on the T7 RNAP complex with a template and NTP, we have modeled the binding of some derivatives of bisphosphonic acid in the active center of the enzyme. The peculiarities observed in the model correlate well with the experimental data on inhibition.

Oligodeoxyribonucleosides Containing 1-β-D-Glucopyranosylthymine Synthesis and Substrate Properties

Abstract Regioselective method for 1-β-D-glucopyranosylthymine incorporation into oligonucleotides has been developed and substrate properties of the latters in DNA synthesis and hydrolysis reactions were investigated.