Oligonucleotide Synthesis Building Blocks

Liang Z, et al. European Journal of Organic Chemistry, 2023, e202300614.

5'-O-(2-Isopropoxyprop-2-yl)-protected Phosphoramidite Building Blocks (IIP-protected) represent an innovative approach in the liquid phase oligonucleotide synthesis (LPOS) for protecting the 5'-OH functionality of nucleosides. This protection method was achieved through an acid-catalyzed transacetalization with 2,2-diisopropoxypropane, providing high yields and efficient 5'-O protection. The IIP protection was applied to the phosphoramidite building blocks of nucleosides (dA, dG, dC, and dT) using a tetrapodal precipitative soluble support.
The utility of IIP protection in LPOS was demonstrated by employing standard nucleobase protecting groups, tetrazole as the activator, and m-chloroperbenzoic acid for oxidation, facilitating efficient coupling. The IIP group exhibited several advantages, such as fast cleavage with formic acid (half-life<10 s in 6% HCOOH in dichloromethane/methanol at room temperature), resulting in the formation of volatile byproducts—acetone and isopropanol. These byproducts enhance atom economy and minimize residual waste, making the IIP-protected phosphoramidite building blocks a sustainable option for oligonucleotide synthesis.
The IIP protection group's small size and its ability to be removed quickly without leaving significant non-volatile residues further contribute to the efficiency and practicality of the method. This protection strategy can be integrated into current phosphoramidite chemistry workflows, offering a highly effective means of synthesizing short 2'-deoxyoligonucleotides while maintaining high yield and purity.

Schmidtgall B, et al. Beilstein Journal of Organic Chemistry, 2024, 11, 50-60.

The development of oligonucleotide-derived bioactive agents relies on modifications to the nucleic acid backbone. A notable advancement in this field is the NAA-modification, a synthetic approach that introduces positive charges at specific sites within the typically polyanionic DNA backbone. This modification utilizes a novel internucleotide linkage that enables the creation of zwitterionic oligonucleotides, offering new avenues for designing bioactive oligonucleotide analogues.
In this study, the synthesis of NAA-modified DNA oligonucleotides was achieved using "dimeric" A–T phosphoramidite building blocks (S)-7 and (R)-7, marking a significant step towards expanding the versatility of NAA-modified oligonucleotide synthesis. These stereoselectively synthesized phosphoramidites allow for the introduction of NAA-linkages at specific T–T and X–T motifs (X = A, C, G), enabling fine-tuned control over the placement of positive charges within the oligonucleotide sequence.
The (S)- and (R)-configured NAA-motifs were synthesized with high diastereoselectivity, facilitating their incorporation into automated solid-phase DNA synthesis. The resulting NAA-modified oligonucleotides demonstrated stable duplex formation with both native DNA and RNA strands, maintaining the typical B-form (DNA/DNA) and A-form (DNA/RNA) helices, despite a slight destabilization in duplex stability. Importantly, these modified oligonucleotides retained the ability to discriminate mismatches, showcasing their potential in various applications, including gene regulation and diagnostics.

Radi M, et al. Tetrahedron Letters, 2005, 46(25), 4361-4364.

The synthesis of 6-vinylcytidine derivatives has gained attention for their potential in enhancing the binding affinity and nuclease resistance of oligonucleotides used in antisense therapies. A key synthetic strategy to achieve this involves C-6 formylation of cytidine followed by a Wittig reaction, resulting in the formation of reactive 6-vinyl groups that can improve interactions with target sequences.
In this work, a 6-vinylcytidine derivative (1) was synthesized through a two-step process: initial C-6 formylation of cytidine followed by a Wittig reaction to introduce the vinyl group. This vinyl moiety was shown to possess excellent Michael acceptor properties, which could facilitate binding to nucleophilic targets through a Michael addition reaction, enhancing the oligonucleotide's ability to interact with complementary sequences. Additionally, the 2′-O-MOE moiety was incorporated to provide nuclease resistance, further improving the stability and efficacy of the resulting oligonucleotide analogues.
The incorporation of a vinyl group at the C-6 position of the pyrimidine ring constrains the nucleoside into a non-natural syn conformation. This stereochemical modification is significant as it allows for more efficient cross-linking with guanosine residues in target sequences, promoting enhanced duplex destabilization and selective targeting. Although 6-substituted pyrimidine oligonucleotides have been explored, the introduction of reactive moieties at the C-6 position offers promising new insights into their base interactions and potential therapeutic applications.
Overall, the combination of C-6 formylation and Wittig reaction provides an effective route to synthesize 6-vinylcytidine derivatives, which could play a crucial role in overcoming key challenges in antisense oligonucleotide therapies, including nuclease stability and binding affinity, particularly in the treatment of cancer, cardiovascular, and infectious diseases.