Novel nucleic acid analogs with a chimeric phosphinate/phosphate backbone; synthesis and biophysical properties (original) (raw)
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Synthesis and biophysical studies of nucleic acid-binding oligomers
2016
In general, this thesis describes the design, synthesis, oligomerization and biophysical studies of novel PNA monomers. Our initial aim was to develop an efficient and inexpensive route for the synthesis of a series of novel alkyne PNA monomers bearing thymine, cytosine, adenine and guanine nucleobases suitable for Fmoc solid phase PNA synthesis strategy. These novel monomers allow functionalising the PNA sequences with alkyne functions at their Nterminus during solid phase synthesis. These novel monomers can be exploited in the click reaction applications such as a click ligation of PNA and conjugation of PNA with different substrates such as nucleic acids and proteins. As an application for ligation PNA sequences, the alkyne thymine PNA monomer was incorporated successfully into the target PNA oligomers during oligomerization. Mimicking the click (CuAAC) reaction linkers of the ligation of PNA oligomers, three novel 1,2,3-triazole functionalised building blocks were designed and p...
Tetrahedron Letters, 1999
Two oligonucleotidcs, partially modified with N,N-dimethylaminoethyl phosphoramidate groups, were obtained by an optimized solid-phase synthesis cycle based on H-phosphonate chemistry. Their use as third strands in parallel triple helices was shown to produce a decrease in stability with respect to all-phosphodiester otigonucleotide complexes, most probably due to unfavourable steric cftizcts. Phosphoramidate-modified oligonucleotides were shown to be notably stable to exonucleases.
Organic Letters, 2000
H-Phosphonate monomers of 2′-O-(2-methoxyethyl) ribonucleosides have been synthesized. Oxidation of oligonucleotide H-phosphonates has been optimized to allow the synthesis of oligonucleotides containing either 2′-deoxy or 2′-O-(2-methoxyethyl) ribonucleoside residues combined with three different phosphate modifications in the backbone, i.e., phosphodiester (PO), phosphorothioate (PS), and phosphoramidate (PN). Phosphodiester linkages were introduced by oxidation with a cocktail of 0.1 M Et 3 N in CCl 4 /Pyr/H 2 O (5:9:1) without affecting phosphorothioate or phosphoramidate linkages. For the synthesis of phosphoramidate-modified oligonucleotides, N 4-acetyl deoxycytidine-3′-H-phosphonate monomers were used to avoid transamination during the oxidation step.
Pure and Applied Chemistry, 2004
The remarkable medicinal importance of the achiral, acyclic, and uncharged aminoethylglycyl peptide nucleic acids (aegPNAs) as DNA/RNA mimics has challenged chemists to circumvent the limitations of their in vivo efficacy. In this context, we have designed conformationally restricted five-and six-membered cyclic PNA analogs by introduction of chemical bridges in aegPNAs leading to a large variety of structures with defined configurations and conformational preferences, effecting concomitant installation of a positive charge in the backbone. The synthesis and biophysical properties of these cationic aminoethylprolyl PNAs, pyrrolidine PNAs, and piperidine PNAs endowed with increased water solubility and affinity toward target nucleic acids is presented. These nucleic acid analogs as lead structures are a part of a chemical evolution process that might give rise to a synthetic nucleic acid analog having optimum properties for medicinal applications. © 2004 IUPAC, Pure and Applied Chemistry 76, 1599-1603 Conformationally restricted cationic polyamide analogs of nucleic acids 1603 Fig. 6 Piperidinyl PNA.
Nucleic Acids Research, 1998
Here we report that the poor binding of methylphosphonate oligodeoxynucleosides (MP-ODNs) to their nucleic acid targets can be improved by additional inversion of the anomeric configuration (from β to α) in the sugar moieties to give a new class of analogs, MP α-oligonucleosides. MP α-dT 12 and MP 5′ α-d(TCTTAA-CCCACA) 3′ were synthesized and their ability to form hybrids with complementary single stranded (ss)DNA and ssRNA, as well as with double stranded (ds)DNA, was evaluated. The thermal stability of hybrids formed with MP α-analogs was compared with the affinity of phosphodiester (PO) and phosphorothioate (PS) βand α-oligomers for their targets. Non-ionic MP α-oligonucleosides bound to their complementary DNA and RNA strands more tightly than their homologues with natural β-anomeric configuration did. With DNA target, MP α-oligomers formed duplexes more stable than the corresponding natural PO β-oligomer did. MP α-heteropolymer hybridized to RNA target better than PS β-oligonucleotide did but the hybrid was less stable (∆T m -0.5_C per mod.) than the hybrid formed with the natural PO β-oligomer. Only MP α-dT 12 bound to dsDNA target at low salt concentration (0.1 M NaCl).
Russian Journal of Bioorganic Chemistry, 2017
⎯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.
The Journal of Organic Chemistry, 2010
We have recently shown that combining the structural elements of 2 0 O-methoxyethyl (MOE) and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE, 2 0 ,4 0 -constrained MOE; cEt, 2 0 ,4 0 -constrained ethyl) that display improved potency over MOE and an improved therapeutic index relative to that of LNA antisense oligonucleotides. In this report we present details regarding the synthesis of the cMOE and cEt nucleoside phosphoramidites and the biophysical evaluation of oligonucleotides containing these nucleoside modifications. The synthesis of the cMOE and cEt nucleoside phosphoramidites was efficiently accomplished starting from inexpensive commercially available diacetone allofuranose. The synthesis features the use of a seldom used 2-naphthylmethyl protecting group that provides crystalline intermediates during the synthesis and can be cleanly deprotected under mild conditions. The synthesis was greatly facilitated by the crystallinity of a key mono-TBDPS-protected diol intermediate. In the case of the cEt nucleosides, the introduction of the methyl group in either configuration was accomplished in a stereoselective manner. Ring closure of the 2 0 -hydroxyl group onto a secondary mesylate leaving group with clean inversion of stereochemistry was achieved under surprisingly mild conditions. For the S-cEt modification, the synthesis of all four (thymine, 5-methylcytosine, adenine, and guanine) nucleobase-modified phosphoramidites was accomplished on a multigram scale. Biophysical evaluation of the cMOE-and cEt-containing oligonucleotides revealed that they possess hybridization and mismatch discrimination attributes similar to those of LNA but greatly improved resistance to exonuclease digestion. SCHEME 4. Synthesis of Rand S-cMOE and Rand S-cEt Uridine Phosphoramidites FIGURE 4. Structural elucidation of R-cMOE 43a, S-cMOE 43b, R-cEt 44, and S-cEt 45.
Oligonucleotides, 2006
S C and R C diastereomers of 5Ј-C-(O,O-diethyl)-phosphonylthymidine (R T and S T) were used for the synthesis of the dimers T R T and T S T, respectively. These dimers were incorporated at selected sites in oligonucleotide constructs. Melting temperature (Tm) experiments demonstrated that relative to the unmodified oligodeoxyribonucleotide, the presence of the R T moiety reduced the thermal stability of the duplexes by ϳ5.0°C per modification, whereas their S T counterparts only slightly destabilized the duplex structure (⌬Tm Յ 1°C/ modification). The stability of the triple-helical complexes containing one, two, or three modified thymidines is slightly higher than that of the parent complex. Nuclease resistance studies performed with snake venom phosphodiesterase, calf spleen phosphodiesterase, and 3-exonuclease from human plasma showed that cleavage of the oligonucleotides at the site of the modification was completely suppressed regardless of the stereochemistry of the 5-C-chiral center. The influence of the R T and S T modification in the recognition sequence of HindIII, EcoRI, and HpaI restriction endonucleases was also investigated. Although the catalytic activity of HindIII was not affected by the presence of the 5-C-ethoxyphosphonyl modification, the activities of the two remaining restriction enzymes were partially suppressed depending on the site of modification or the stereochemistry of the modification or both (R T vs. S T). FIG. 1. Structures of the 5Ј-phosphates of 5Ј-C-stereodefined phosphonothymidine units R T and S T incorporated into the DNA chain.
Bioorganic & Medicinal Chemistry, 2012
Various stereochemically pure cationic phosphorothioate oligonucleotides bearing aminoalkyl moieties were synthesized, and their duplex-forming ability against single-stranded DNA (ssDNA), single-stranded RNA (ssRNA) and triplex-forming ability against double-stranded DNA (dsDNA) were evaluated by UV melting experiments. The cationic Rp stereoisomers showed improved duplex-forming ability against ssDNA, triplex-forming ability against dsDNA and nuclease stability.
Synthesis and Biochemical Properties of Cyanuric Acid Nucleoside-Containing DNA Oligomers
Chemical Research in Toxicology, 1999
Deoxy-D-erythro-pentofuranosyl)cyanuric acid (cyanuric acid nucleoside, dY) (1) has been shown to be formed upon exposure of DNA components to ionizing radiation and excited photosensitizers. To investigate the biological and structural significance of dY residue in DNA, the latter modified 2′-deoxynucleoside was chemically prepared and then site-specifically incorporated into oligodeoxyribonucleotides (ODNs). This was achieved in good yields using the phosphoramidite approach. For this purpose, a convenient glycosylation method involving 3,5-protected 2-deoxyribofuranoside chloride and cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine) was devised. The anomeric mixture of modified 2′-deoxyribonucleosides (1/2 R/) was resolved by silica gel purification of the 5′-O-dimethoxytritylated derivatives, and then, phosphitylation afforded the desired-phosphoramidite monomer (5). After solid-phase condensation and final deprotection, the purity and the integrity of the modified synthetic DNA fragments were checked using different complementary techniques such as HPLC and polyacrylamide gel electrophoresis, together with electrospray ionization and MALDI-TOF mass spectrometry. The presence of cyanuric acid nucleoside in a 14-mer was found to have destabilizing effects on the doublestranded DNA fragment as inferred from melting temperature measurements. The piperidine test applied to dY-containing ODNs supported the high stability of cyanuric acid nucleoside inserted into the oligonucleotide chain. Several enzymatic experiments aimed at determining the biological features of such a DNA lesion were carried out. Thus, processing of dY by nuclease P 1 , snake venom phosphodiesterase (SVPDE), calf spleen phosphodiesterase (CSPDE), and repair enzymes, including Escherichia coli endonuclease III (endo III) and Fapy glycosylase (Fpg), was investigated. Finally, a 22-mer ODN bearing a cyanuric acid residue was used as a template to study the in vitro nucleotide incorporation opposite the damage by the Klenow fragment of E. coli polymerase I.