Dmitry A. Stetsenko

New approach to solid phase synthesis of polyamide nucleic acids analogues (PNA) and PNA-DNA conjugates

Abstract New version of sequential DNA and polyamide solid phase synthesis is applied to the preparation of PNA and PNA-DNA conjugates bearing free 3′-end. N-Mmt protected uracil and thymine PNA monomers are obtained in high yields. Novel support derived from TSK Gel Toyopearl ® and efficient coupling procedure with 2,4,6-triisopropylbenzenesulfonyloxy-l-benzotriazole (TPSOBt) are also developed. Preliminary results of biological testing of prepared compounds are reported.

DNA enzymes as potential therapeutics: towards clinical application of 10-23 DNAzymes

Introduction: Ongoing studies on the inhibition of gene expression at the mRNA level have identified several types of specific inhibitors such as antisense oligonucleotides, small interfering RNA, ribozymes and DNAzymes (Dz). After its discovery in 1997, the 10-23 Dz (which can cleave RNA efficiently and site-specifically, has flexible design, is independent from cell mechanisms, does not require expensive chemical modifications for effective use in vivo) has been employed to downregulate a range of therapeutically important genes. Recently, 10-23 Dzs have taken their first steps into clinical trials. Areas covered: This review focuses predominantly on Dz applications as potential antiviral, antibacterial, anti-cancer and anti-inflammatory agents as well as for the treatment of cardiovascular disease and diseases of CNS, summarizing results of their clinical trials up to the present day. Expert opinion: In comparison with antisense oligonucleotides and small interfering RNAs, Dzs do not usually show off-target effects due to their high specificity and lack of immunogenicity in vivo. As more results of clinical trials carried out so far are gradually becoming available, Dzs may turn out to be safe and well-tolerated therapeutics in humans. Therefore, there is a good chance that we may witness a deoxyribozyme drug reaching the clinic in the near future.

A New Phosphoramidite Reagent for the Incorporation of Diazaphenoxazinone Nucleoside With Enhanced Base-Pairing Properties into Oligodeoxynucleotides

Abstract New phosphoramidite reagent 7 suitable for incorporation of dC analogue was synthesized. ODNs containing diazaphenoxaziione residues in defined positions were prepared. The stability of duplexes formed was increased up to 3-5°C per modified base. Preliminary results of molecular biological testing were reported. This work was partly supported by RFBR grant 96-04-49209, by grant HHMI#75195-544201, LLNL P99U, and grant DOE agreement No. B307902 mod. 04. The authors would like to thank Dr. Tatyana L. Azhikina for valuable help in DNA sequencing and Dr. Natalya P. Potapova for NMR spectra recording.

Delivery of therapeutic RNA-cleaving oligodeoxyribonucleotides (deoxyribozymes): from cell culture studies to clinical trials

ABSTRACT Introduction. Development of efficient in vivo delivery systems remains a major challenge en route to clinical application of antisense technology, including RNA-cleaving molecules such as deoxyribozymes (DNAzymes). The mechanisms of oligonucleotide uptake and trafficking are clearly dependent on cell type and the type of oligonucleotide analogue. It appears likely that each particular disease target would pose its own specific requirements for a delivery method. Areas covered. In this review we will discuss the available options for DNAzyme delivery in vitro and in vivo, outline various exogenous and endogenous strategies that have been, or are still being, developed and ascertain their applicability with emphasis on those methods that are currently being used in clinical trials. Expert opinion. The available information suggests that a practical system for in vivo delivery has to be biodegradable, as to minimize concerns over long-term toxicity, it should not accumulate in the organism. Extracellular vesicles may offer the most organic way for drug delivery especially as they can be fused with artificial liposomes to produce hybrid nanoparticles. Chemical modification of DNAzymes holds great potential to apply oligonucleotide analogs that would not only be resistant to nuclease digestion, but also able to penetrate cells without external delivery agents.

Oligonucleotide Functionalization by a Novel Alkyne-Modified Nonnucleosidic Reagent Obtained by Versatile Building Block Chemistry

A convenient synthetic strategy has been designed to prepare an alkyne-modified synthon for automated DNA synthesis. It is based on the key O-DMTr-protected 4-(2-hydroxyethyl)morpholin-2,3-dione and building blocks obtained by its functionalization by various aliphatic amines. A respective nonnucleosidic phosphoramidite monomer containing a terminal alkyne in the side-chain was synthesized, and corresponding oligothymidylates incorporating the modification in various positions were prepared. The presence of the alkyne group was confirmed by Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) between the functionalized oligonucleotide and an azide derivative of 7-nitro-2,1,3-benzoxadiazole.

Synthesis and DNA Duplex Stabilities of Oligonucleotides Containing C-5-(3-Methoxypropynyl)-2′-deoxyuridine Residues

Abstract 5′-Dimethoxytrityl-5-(3-methoxypropynyl)-2′-deoxyuridine phosphoroamidite was synthesized with the use of commercial 3- methoxypropyne. Oligonucleotides (ODNs) containing 5-(3- ethoxypropynyl)- 2′-deoxyuridine in different positions were prepared. The stabilities of the duplexes formed by these ODNs with the complementary templates are increased in comparison with the unmodified counterparts. On average modified residue incorporated, the Tm is raised by 1°C.

New oligodeoxyribonucleotide derivatives bearing internucleotide N-tosyl phosphoramidate groups: Synthesis and complementary binding to DNA and RNA

N-Sulfonyl phosphoramidate derivatives of oligodeoxyribonucleotides containing N-tosyl phosphoramidate groups are first reported. The synthesis is based on Staudinger reaction between tosyl azide and 3′,5′-dinucleoside β-cyanoethyl phosphite comprising the immobilized oligonucleotide, which is obtained by the phosphoramidite coupling during the solid-phase oligonucleotide synthesis. The N-tosyl phosphoramidate group was stable under conditions of the oligonucleotide synthesis, in particular, upon acidic detritylation followed by the removal of protective groups and cleavage from the polymer support by the treatment with concentrated aqueous ammonia at 55°C. The stability of DNA and RNA duplexes of the model oligonucleotides containing N-tosyl phosphoramidate groups was only slightly lower than that of native DNA:DNA and DNA:RNA duplexes, respectively.

Pre-steady state kinetics of DNA binding and abasic site hydrolysis by tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3′-end-blocking modifications, possesses DNA and RNA 3′-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3′-end of DNA have been used to prevent 3′-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3′-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3′-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3′-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.

Removal of acid-labile protecting or anchoring groups in the presence of polyfluorinated alcohol: Application to solid-phase peptide synthesis

We describe herein a new method for cleaving from resin and removing acid-labile protecting groups in solid-phase peptide synthesis in the presence of a polyfluorinated alcohol (either trifluoroethanol, TFE, or hexafluoroisopropanol, HFIP). It was shown that 0.1 M HCl in hexafluoroisopropanol or trifluoroethanol removes the acid-labile protecting groups commonly used in Fmoc SPPS for the protection of amino acid side-chains, such as t-butyl ester and ether, Boc, trityl, and Pbf groups including the most acid-resistant p-hydroxymethylphenoxyacetyl group (HMPA), p-benzyloxy benzyl ester (Wang resin), Rink amide, and peptide amide linker (PAL). The addition of 5–10% of a hydrogen-bonding solvent was shown to considerably retard or even fully inhibit the reaction. However, nonhydrogen-bonding solvents, such as dichloromethane, do not slow down the reaction.

A new simple and convenient method for preparation of oligonucleotides containing a pyrene or a cholesterol moiety

A new simple and convenient approach to the synthesis of oligonucleotides containing pyrene or cholesterol moieties is described. The approach does not require a special phosphoramidite reagent as it is based on Staudinger reaction between an azidoalkyl derivative of either pyrene or cholesterol and a polymer-supported internucleoside 2-cyanoethyl phosphite, which is formed during phosphoramidite condensation.