C5-alkynyl-functionalized α-L-LNA: synthesis, thermal denaturation experiments and enzymatic stability

Abstract

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.

Figure 1

Structures of nucleotide monomers studied herein.

Scheme 1. Synthesis of Key Intermediate 9

Abbreviations: BSA, N,O-bis(trimethylsilyl)acetamide; U, uracil-1-yl; CAN, ceric ammonium nitrate; DMTr, 4,4′-dimethoxytrityl.

Scheme 2. Synthesis of C5-Alkynyl-Functionalized α-L-LNA-U Phosphoramidites 11S–Y

Abbreviation: PCl-reagent, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.

Figure 2

3′-Exonuclease degradation of singly (top, 3′-CAC BAT ACG) and doubly modified (bottom, 3′-CAC BAB ACG) C5-functionalized α-L-LNA and reference strands. Nuclease degradation studies were conducted in magnesium buffer (50 mM Tris-HCl, 10 mM Mg²⁺, pH 9.0) using [ON] = 3.3 μM and 0.03 U of snake venom phosphodiesterase.

Figure 3

Steady-state fluorescence emission spectra of single-stranded Z1 and corresponding duplexes with complementary or mismatched DNA/RNA strands (mismatched nucleotide opposite to modification in parentheses). Conditions: λ_ex 344 nm, T = 5 °C, each oligonucleotide used in 1 μM concentration. Note that different axis scales are used.