Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′-Amino-α‑l‑LNA Adenine Monomers: High-Affinity Targeting of Single-Stranded DNA

The development of conformationally restricted nucleotide building blocks continues to attract considerable interest because of their successful use within antisense, antigene, and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-l-LNA are two interesting examples thereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity, and enhanced enzymatic stability relative to unmodified strands. Here we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-l-LNA adenine monomers <b>W</b>–<b>Z</b>. The synthesis of the target phosphoramidites <b>1</b>–<b>4</b> is initiated from pentafuranose <b>5</b>, which upon Vorbrüggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside <b>8</b>. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts <b>8</b> into 2′-amino-α-l-LNA adenine intermediate <b>9</b>, which after a series of nontrivial protecting-group manipulations affords key intermediate <b>15</b>. Subsequent chemoselective N2′-functionalization and O3′-phosphitylation give targets <b>1</b>–<b>4</b> in ∼1–3% overall yield over 11 steps from <b>5</b>. ONs modified with pyrene-functionalized 2′-amino-α-l-LNA adenine monomers <b>X</b>–<b>Z</b> display greatly increased affinity toward DNA targets (Δ<i>T</i>_m/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2′-pyrene-functionalized 2′-amino-α-l-LNAs of considerable interest for DNA-targeting applications

C5-Alkynyl-Functionalized α‑L‑LNA: Synthesis, Thermal Denaturation Experiments and Enzymatic Stability

Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. One of the most popular strategies toward this end has been to introduce additional modifications to the sugar ring of affinity-inducing conformationally restricted nucleotide building blocks such as locked nucleic acid (LNA). In the preceding article in this issue, we introduced a different strategy toward this end, i.e., C5-functionalization of LNA uridines. In the present article, we extend this strategy to α-L-LNA: i.e., one of the most interesting diastereomers of LNA. α-L-LNA uridine monomers that are conjugated to small C5-alkynyl substituents induce significant improvements in target affinity, binding specificity, and enzymatic stability relative to conventional α-L-LNA. The results from the back-to-back articles therefore suggest that C5-functionalization of pyrimidines is a general and synthetically straightforward approach to modulate biophysical properties of oligonucleotides modified with LNA or other conformationally restricted monomers

Synthesis and Biophysical Properties of C5-Functionalized LNA (Locked Nucleic Acid)

Oligonucleotides modified with conformationally restricted nucleotides such as locked nucleic acid (LNA) monomers are used extensively in molecular biology and medicinal chemistry to modulate gene expression at the RNA level. Major efforts have been devoted to the design of LNA derivatives that induce even higher binding affinity and specificity, greater enzymatic stability, and more desirable pharmacokinetic profiles. Most of this work has focused on modifications of LNA’s oxymethylene bridge. Here, we describe an alternative approach for modulation of the properties of LNA: i.e., through functionalization of LNA nucleobases. Twelve structurally diverse C5-functionalized LNA uridine (U) phosphoramidites were synthesized and incorporated into oligodeoxyribonucleotides (ONs), which were then characterized with respect to thermal denaturation, enzymatic stability, and fluorescence properties. ONs modified with monomers that are conjugated to small alkynes display significantly improved target affinity, binding specificity, and protection against 3′-exonucleases relative to regular LNA. In contrast, ONs modified with monomers that are conjugated to bulky hydrophobic alkynes display lower target affinity yet much greater 3′-exonuclease resistance. ONs modified with C5-fluorophore-functionalized LNA-U monomers enable fluorescent discrimination of targets with single nucleotide polymorphisms (SNPs). In concert, these properties render C5-functionalized LNA as a promising class of building blocks for RNA-targeting applications and nucleic acid diagnostics

Identification and Characterization of Second-Generation Invader Locked Nucleic Acids (LNAs) for Mixed-Sequence Recognition of Double-Stranded DNA

The development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate, and modify genes. Progress has been made with triplex-forming oligonucleotides, peptide nucleic acids, and polyamides, but substantial efforts are currently devoted to the development of alternative strategies that overcome the limitations observed with the classic approaches. In 2005, we introduced Invader locked nucleic acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through modification with “+1 interstrand zippers” of 2′-<i>N</i>-(pyren-1-yl)­methyl-2′-amino-α-l-LNA monomers. Despite promising preliminary results, progress has been slow because of the synthetic complexity of the building blocks. Here we describe a study that led to the identification of two simpler classes of Invader monomers. We compare the thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2′-amino-α-l-LNA, 2′-<i>N</i>-methyl-2′-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling, and NMR spectroscopy are used to elucidate the structural factors that govern probe activation. We demonstrate that probes with +1 zippers of 2′-<i>O</i>-(pyren-1-yl)­methyl-RNA or 2′-<i>N</i>-methyl-2′-<i>N</i>-(pyren-1-yl)­methyl-2′-amino-DNA monomers recognize DNA hairpins with similar efficiency as original Invader LNAs. Access to synthetically simple monomers will accelerate the use of Invader-mediated dsDNA recognition for applications in molecular biology and nucleic acid diagnostics