Phosphorothioate synthesis is a specialized chemical process used to produce therapeutic molecules called phosphorothioate oligonucleotides. These are short, synthetic strands of nucleic acids where the natural genetic backbone has been chemically altered. The modification involves replacing a single non-bridging oxygen atom in the phosphate backbone with a sulfur atom. This alteration transforms the biological properties of the oligonucleotide, making it suitable for drug development and enabling the creation of therapeutics that can regulate specific genes within the body.
The Structural Difference and Stability Advantage
The fundamental difference between natural genetic material and a phosphorothioate molecule is a single atomic substitution in the backbone linking the nucleotide building blocks. Natural DNA and RNA use a phosphodiester linkage, which has two non-bridging oxygen atoms attached to the central phosphorus atom. In the modified phosphorothioate diester structure, one of these oxygen atoms is replaced with a sulfur atom.
This structural change yields a substantial functional benefit by dramatically increasing the molecule’s resistance to degradation by cellular enzymes called nucleases. Nucleases are enzymes that constantly patrol and break down foreign nucleic acids. The presence of the sulfur atom physically hinders the enzyme’s ability to recognize and cleave the backbone, making the oligonucleotide resistant to digestion.
This enhanced stability is the principal reason the modification is used in drug development. It allows the therapeutic oligonucleotide to remain intact and active within the body for a longer period, increasing its chance of reaching its target. The sulfur substitution also increases the molecule’s hydrophobicity, which aids in its uptake and distribution into cells.
Why Replacing Oxygen with Sulfur is Difficult
Synthesizing a precise phosphorothioate oligonucleotide is chemically challenging because the substitution of oxygen with sulfur must be tightly controlled. The complexity arises because the sulfur atom introduces a new center of asymmetry, or chirality, at the phosphorus atom in the backbone. For every phosphorothioate linkage created, two distinct three-dimensional arrangements, known as Rp and Sp stereoisomers, can be formed.
If the chemical process creates a mixture of these two forms at each linkage, the number of potential products escalates rapidly. For example, a fully modified 16-base oligonucleotide can theoretically yield $2^{15}$ distinct stereoisomers, resulting in thousands of different molecules. Since the biological activity of the oligonucleotide, such as its nuclease resistance or affinity for target RNA, can vary between these stereoisomers, a highly controlled and selective chemical reaction is necessary to ensure the final product is effective.
The Core Process of Chemical Manufacturing
Manufacturing phosphorothioate oligonucleotides relies on solid-phase synthesis, often utilizing phosphoramidite chemistry. This highly efficient and repetitive method builds the oligonucleotide chain one nucleotide at a time, starting with the first building block attached to an insoluble solid support material. The process proceeds through an automated cycle of chemical reactions to ensure high precision.
Each cycle involves four main steps:
Detritylation
This step removes a protective chemical group from the end of the growing chain, preparing it for the next incoming nucleotide.
Coupling
A new, activated nucleotide building block called a phosphoramidite is introduced, forming a temporary trivalent phosphorus linkage with the chain.
Sulfurization
This is the defining feature of the process, where the phosphorothioate bond is created. Unlike standard DNA synthesis, which uses oxidation to add an oxygen atom, phosphorothioate synthesis uses a specific sulfurizing reagent to replace the oxygen with a sulfur atom.
Capping
This final step chemically inactivates any chains that failed to couple in the preceding step, ensuring that only the full-length, correct sequence continues to grow.
This cycle is repeated until the oligonucleotide reaches its target length, at which point the completed oligonucleotide is cleaved from the solid support and purified.
Current Use in Oligonucleotide Therapeutics
The stable phosphorothioate backbone is a foundational modification for creating modern therapeutic oligonucleotides, primarily Antisense Oligonucleotides (ASOs) and small interfering RNAs (siRNA). ASOs are short, single-stranded molecules designed to bind to a specific messenger RNA (mRNA) sequence to regulate gene expression. The modification allows the ASO to survive long enough in the body to activate cellular machinery, such as the RNase H enzyme, which then degrades the target mRNA, effectively silencing the gene responsible for a disease.
This synthetic technique has led to the approval of several high-impact drugs. Examples include:
- Fomivirsen (Vitravene), the first FDA-approved phosphorothioate oligonucleotide, introduced in 1998 to treat cytomegalovirus retinitis.
- Nusinersen (Spinraza), an ASO used to treat spinal muscular atrophy (SMA), which uses the phosphorothioate backbone to modulate the splicing of the SMN2 gene.
- Mipomersen, which targets the mRNA for apolipoprotein B to lower cholesterol in patients with familial hypercholesterolemia.
For siRNA therapeutics, the modification is often strategically placed at the ends of the double-stranded molecule. This placement protects the complex from nuclease attack, ensuring it remains stable until it can shut down a target gene.