Synthesis and Hybridization Properties of Modified Oligonucleotides with PNA-DNA Dimer Blocks

Abstract

Different modified PNA-DNA dimer-analogous synthons (I and II) were synthesized as phosphoramidites. These dimer units were assembled by a 5′-modified deoxythymidine and a modified PNA monomer. These synthons were used in the routine coupling procedure for oligonucleotides. Therefore no PNA coupling chemistry is necessary to synthesize PNA-DNA chimeric oligonucleotides. Various deoxyoligonucleotides were synthesized introducing the dimer blocks I and II at different positions in the sequences. Melting temperatures of the modified oligonucleotides with their complementary DNA analogues were determined.

Backbone modifications of oligonucleotides are required in the antisense strategy for protection against endonucleolytic cleavage in biological environment. Peptide nucleic acids (PNA fragments) are known to be nuclease resistant analogues, which show stable and discriminating hybridization. For this reason we prepared chimeric PNA-DNA oligomers by incorporation of two different modified PNA-DNA dimer blocks (Scheme A) into oligonucleotides. Melting temperatures of the modified oligonucleotides with their complementary DNA were determined.     

In Search of Acyclic Analogues as Universal Nucleosides in Degenerate Probes

Abstract

Five acyclic nucleoside analogues with unnatural base moieties have been synthesized of which three successfully were incorporated into oligonucleotides. The acyclic analogue containing the base 5-nitroindazole was the least discriminating and should be further pursued for use as a universal nucleoside analogue.     

Enzymatic and Hybridization Properties of Oligonucleotide Analogues Containing Novel Phosphotriester Internucleotide Linkage

Abstract

Enhanced cellular uptake, stable and discriminating hybridization and increased stability in biological media are of particular interest for oligonucleotides of potential therapeutic application. Additionally, toxicity or immunogenicity of the oligonucleotide analogues and their biodegradation products should be minimized by minimal alteration of the biological structure and effort and cost of bulk production should be as low as possible by using a standard automated synthesis protocol. Oligonucleotide phosphotriesters with oligoethyleneglycol substituents show promise to ideally combine all these advantages. Here we describe the hybridization properties and the stability of modified oligonucleotides containing triester internucleotide linkages substituted with α,ω-dihydroxy-(3,6-dioxa)-octan-1-yl group (“triethyleneglycol triester linkages”) towards enzymatic degradation. The triester linkages are stable towards exo- and endonucleases. Regardless of number and position of triester linkages, the modified oligonucleotides showed practically no decrease of T_m in hybridization studies with complementary biological oligonucleotides. In further enzymatic studies the modified oligonucleotides were highly stable towards nucleases in human blood serum.     

Synthesis and Triplex Forming Properties of an Acyclic N7-Glycosylated Guanine Nucleoside

Abstract

A chiral acyclic nucleoside, one in which the ribose carbohydrate has been replaced with a glycerol-based linker, is prepared by glycosylating guanine at the N⁷-nitrogen. The stereochemically pure derivative is converted to a DMT-protected phosphoramidite for incorporation into DNA sequences. Sequence containing the acyclic N⁷-dG nucleoside are capable of forming DNA triplexes in which it is likely that the N¹-H and N²-amino groups of the N⁷-dG are involved in recognition of the guanine base in G-C base pairs.     

Synthesis and Enzymatic Stability of Acyclic Thymidine Dimer Analogues having a Constrained Anti-Glycosidic Conformation

Abstract

Our interest to design a novel class of antisense oligonucleotides has lead us to prepare acyclic thymidine dimer analogues having an unique constrained antiglycosidic conformation. These dimer analogues showed remarkable resistance to nuclease degradation, such as Nuclease S1, Bovine Spleen Phosphodiesterase, and Snake Venom Phosphodiesterase.     

Pyrrolidine PNA-DNA Chimeric Oligonucleotides with Extended Backbone

Abstract

Cis-D-2-hydroxy-4-thymin-1-yl-pyrrolidine propionic acid unit is used to make PNA-DNA dimer block that is incorporated in DNA sequences at selected positions. Since the amide linkage is shorter than phosphodiester linkage, insertion of an extra atom in the backbone with amide linkage seems to be better accommodated for internucleotide distance-complementarity.     

Invasion of Strongly Binding Oligonucleotides into tRNA Structure

Abstract

Interaction of yeast tRNA^Phe with oligodeoxyribonucleotides containing 5-methylcytosine, 2-aminoadenine, and 5-propynyl-2′-deoxyuridine was investigated. The modified oligonucleotides show increased binding capacity although the association rates are similar for the modified and natural oligonucleotides. The most pronounced increase in association constant (70 times) due to the incorporation of the strongly binding units was achieved in the case of oligonucleotide complementary to the sequence 65–76 of the tRNA^phe.     

Oligonucleotides containing a new type of acyclic,achiral nucleoside analogue: 1-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]thymine

An achiral, acyclic nucleoside analogue has been incorporated once or twice in oligodeoxyribonucleotides by the phosphoramidite method, and conditions found which allow deprotection of the oligonucleotides containing a sensitive modified allylic unit. The binding affinity of the modified oligonucleotides towards complementary DNA and RNA was reduced compared to unmodified DNA (DeltaT(m) -2 to -6.5 degrees C). An oligonucleotide with two modifications at the 3'-end showed considerable resistance towards cleavage with a 3'-exonuclease.     

Synthesis and biochemical evaluation of Aminopropanolyl-Thymine tri-Phosphate (ap-TTP)

Abstract

Deoxyribonucleoside triphosphates (dNTPs) are building blocks for the biosynthesis of DNA. Various modified dNTPs’ analogs have synthesized by structural changes of nucleoside’s susgar and nucleobases and employed for synthesis of modified DNA. A very few modified dNTPs have prepared from non-sugar nucleoside analogs. This report describes the synthesis of acyclic nucleoside triphosphate (NTP) analog from amino acid L-Serine as aminopropanolyl-thymine triphosphate (ap-TTP) and demonstrate its biochemical evaluation as enzymatic incorporation of ap-TTP into DNA with DNA polymerases with primer extension methods. Alanyl peptide nucleicacids (Ala-PNA) are the analogs of DNA which contains alanyl backbone. Aminopropanolyl – analogs are derivatives of alanyl back bone. Ap-TTP analog is nucleoside triphosphate analog derived from Ala-PNA. Importantly, this report also sheds light on the crystal packing arrangement of alaninyl thymine ester derivative in solid-state and reveals the formation of self-duplex assembly in anti-parallel fashion via reverse Watson-Crick hydrogen bonding and π–π interactions. Hence, ap-TTP is a useful analog which also generates the free amine functional group at the terminal of DNA oligonucleotide after incorporation.     

The Novel 6–(N-Pyrrolyl)purine Acyclic Nucleosides: 1H and 13C NMR and X-ray Structural Study†

Abstract

Synthesis of the novel nucleoside analogues containing exocyclic pyrrolo moiety and acyclic side chains attached to the purine ring at N-9 and N-7 is described. The site of alkylation was determined by ¹H and ¹³C NMR on the basis of chemical shifts, C-H coupling constants and connectivity in NOESY and HETCOR spectra. The N-9 substitution of 7 was proved by its X-ray crystallographic analysis.

     

SYNTHESIS AND PROPERTIES OF mRNA CAP ANALOGS CONTAINING PHOSPHOROTHIOATE MOIETY IN 5′,5′-TRIPHOSPHATE CHAIN

Nucleosides and oligonucleotides with an oxygen replaced by sulfur atom are an interesting class of compounds because of their improved stability toward enzymatic cleavage by nucleases. We have synthesized several dinucleotide mRNA cap analogs containing a phosphorothioate moiety in the α, β, or γ position of 5′,5′-triphosphate chain [m⁷Gp(s)ppG, m⁷Gpp(s)pG, and m⁷Gppp(s)G]. These are the first examples of the biologically important 5′mRNA cap analogs containing a phosphorothioate moiety, and these compounds may be useful in a variety of biochemical and biotechnological applications. Incorporation of a sulfur atom in the α or γ position within the dinucleotide cap analog was achieved using PSCl₃ in a nucleoside phosphorylation reaction followed by coupling the phosphorothioate of nucleoside with a second nucleotide. Synthesis of cap analogs with the phosphorothioate moiety in β position was performed using an organic phosphorothioate salt in a coupling reaction with an activated nucleotide. The structures of newly synthesized compounds was confirmed using MS and ¹H and ³¹P NMR spectroscopy. We present here the results of preliminary studies on their interaction with translation initiation factor eIF4E and enzymatic hydrolysis with human and nematode DcpS scavengers.     

Evaluation of Oligonucleotides with Novel Modifications

Abstract

Oligodeoxynucleotides modified with carboxamide linked dimeric nuclcotides and an acyclic nucleoside were prepared and investigated for their hybridization properties toward DNA.     

2-Hydroxyethoxyethylated Bases as Acyclic Analogues of 1,5-Anhydrohexitol Nucleoside Derivatives

Abstract

The synthesis and antiviral activity of a new series of acyclic nucleoside analogues containing a (2-hydroxyethoxy)ethyl moiety is discussed.     

Investigation of a Facile Synthetic Method for Phosphorothioate Dimer Synthons In Oligonucleotide Phosphorothioates Synthesis

Abstract

A facile synthetic method of a phosphorothioate dimer block was investigated. Dinucleoside phosphite triester intermediates were obtained in one-pot synthesis by the coupling of a protected nucleoside bearing free 5′-OH and a protected nucleoside bearing free 3′-OH in the presence of phosphorous trichloride (PCl₃) and 1,2,4-triazole. The intermediates were easily sulfurized to afford the desired phosphorothioate dimer blocks in 33-64% overall yields.     

Carbohydrate Modifications in Antisense Oligonucleotide Therapy: New Kids on the Block

Abstract

Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety¹ has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation^2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oligonucleotides with techniques such as capillary gel electrophoresis (CGE) and mass spectrometry help to determine the purity as well as the in vivo fate of these complex molecules. Large-scale synthesis is becoming a tangible reality for antisense oligonucleotides. Nucleic acid chemists and biologists alike are beginning to understand the structure-biological activity in terms of basic physical-organic parameters such as the gauche effect, the charge effect and conformational constraints. Synthesis of chimeric designer oligonucleotides bringing the attractive features of different modifications to a given antisense oligonucleotide sequence to generate synergistic interactions is forthcoming³⁰. These advances along with the potential availability of complete human genome sequence information promise a bright future for the widespread use of nucleic acid based therapeutics.     

Synthesis of a Carboxamide Linked UBr∗UBr Dimer–Duplex and Triplex Stabilities of the Corresponding Oligodeoxynucleotides

Abstract

A U^Br?U^Br dimer was synthesized by connecting the appropiate nucleoside monomers through a 5-atom carboxamide linkage. The dimer was incorporated in oligodeoxynucleotides and investigated for hybridization properties toward single and double stranded DNA.     

Introduction of 2-O-benzyl abasic nucleosides to the 3′-overhang regions of siRNAs greatly improves nuclease resistance

Chemically modified siRNAs containing 2-O-benzyl-1-deoxy-d-ribofuranose (R^H_OBn) in their 3′-overhang region were significantly more resistant towards serum nucleases than siRNAs possessing the natural nucleoside in this region. The knockdown efficacies and binding affinities of these modified siRNAs to the recombinant human Argonaute protein 2 (hAgo2) PAZ domain were comparable with that of siRNA with a thymidine dimer at the 3′-end.     

Solid-Phase Synthesis of Oligodeoxyribonucleotide Analogues Containing 5, 6-Dihydroimidazo [1, 2-c] Pyrimidin-5-One as a Base Moiety

Abstract

The synthesis on a polyacrylamide resin of oligonucleotides containing 5,6-dihydroimidazo [1, 2-c] pyrimidin-5-one (X) as a base residue is described. Perliminary results on enzymatic hydrolysis of the modified octanucleotide d(TXAATTCA) (21), containing. the recognition sequence for the endonuclease -ECO RI where dG is replaced by dX (1), are reported.     

Synthesis of Artificially Bent Oligonucleotides by Incorporation of Conformationally Rigid 5′-Cyclouridylic Acid Derivatives

Abstract

This paper describes the design and synthesis of a conformationally rigid dimer building block Umpc3Um having a propylene bridge linked between the uracil 5-position and 5′-phosphate group of pUm. Oligonucleotides incorporating the dimer unit with either the Sp or Rp configuration were synthesized by use of the phosphoramidite approach. The conformational properties of the dimer units and these oligonucleotides were studied in detail.     

Boranophosphate Oligonucleotides: New Synthetic Approaches

Abstract

Recently our laboratory reported a new backbone-modified class of oligonucleotides, with a borane (B₃3?) group replacing one of the non-bridging oxygen atoms. Here we present two new approaches to synthesize the boranophosphate oligonucleotides. All-stereoregular boranophosphate oligonucleotides can be prepared by enzymatic template extension reactions using nucleoside a-boranotriphosphates, which are good substrates for a number of polymerases. Larger scale synthesis of boranophosphate oligonucleotides can be carried out by effective chemical synthesis using the H-phosphonate approach, instead of previously used phosphoramidite methodology. The main advantage of H-phosphonate methodology is the ability to carry out one boronation reaction, after oligonucleotide chain elongation has been completed, using mild conditions without base damage and producing the desired boranophosphate oligonucleotides in high yield.