Small nucleic acid-based drugs, namely oligonucleotide drugs, are short-chain nucleic acids consisting of dozens of nucleotides in series. At present, most oligonucleotide drugs are obtained through solid-phase chemical synthesis by phosphite amide. And the preparation of the core material nucleoside phosphite monomer (nucleoside monomer) is the key technology that has become a main technical barrier.

1. Basic structure of nucleoside monomer

The phosphite monomer used to synthesize DNA is mainly composed of base, deoxyribose, 5″-DMT, 2-cyanoethyl at the 3” end, and diisopropylamine, while the RNA phosphite monomer needs to protect 2″-OH. In order to prevent the cross-reaction of 2″-OH and 5″-OH in solid-phase synthesis, the protective group is generally TBDMS. In addition, due to the presence of primary amino groups in adenine, cytosine, guanine, and some artificial bases, it is also necessary to protect them with appropriate protective groups in the unit.

2. Chemical modification of nucleoside monomers to facilitate the development of small nucleic acid-based drugs

Since the structure of the original small nucleic acid is easily degraded by nucleases in vivo and has poor pharmacokinetic properties, it can not be directly used as a drug in general. It needs to undergo multi-site chemical modifications to improve affinity, stability, and metabolic properties. After decades of accumulation, three generations of technologies have been developed.

2.1 The first generation technology — phosphoric acid modification

Phosphoric acid modification is the basic chemical modification, and its most used skeleton modification is thiophosphoric acid, that is, a sulfur atom replaces the non-bridging oxygen atom of phosphodiester bond (P-S replaces P-O), which can reduce the hydrophilicity of oligonucleotides, increase the resistance to nuclease degradation, and improve the stability and half-life of drugs.

2.2 The second generation technology — ribose modification

It is mainly a chemical modification of the hydroxyl group of the ribose structure. Ribose modification can affect the affinity of small nucleic acid and target RNA, the stability to ribozyme, and the properties after binding with RNA. Since the structure of RNA differs from that of DNA by only a 2′ hydroxyl group, a small change in this hydroxyl group is sufficient to cause significant effects, and may also determine the configuration of the glycosyl portion of RNA, ultimately impacting the affinity to the target RNA. Hydroxyl modification can also change the sensitivity of phosphoric acid to nuclease and affect the stability of the drug in vivo. Common modifications include 2′-O-methylation, 2′-O-methoxyethyl modification (2′-MOE), and fluorine (F) substitution.

2.3 The third generation technology — Ribose five-membered ring modification

The main purpose is to make more chemical modifications to the mother ring in the structure of the ribose to improve the nature of drugs, including LNA (locked nucleic acid), PNA (peptide nucleic acid), and PMO (phosphorodiamidate morpholino oligomer). LNA is a classical nucleotide bridging modification technology, which fixes its conformation through the bridging between the second and fourth carbon atoms of nucleotide, enhancing the resistance of drugs to nucleases and the affinity of drugs to target mRNA. Modification of PNA and PMO can also improve the resistance, affinity, and specificity of nucleic acid-based drugs to nuclease.

3. Marketed small nucleic acid-based drugs

At present, there are 12 small nucleic acid-based drugs on the market, and the nucleoside monomer composition of each drug is shown in the table below. The continuous growth of small nucleic acid-based drug market scale will drive the growth of nucleoside monomer market as well.