A phosphorothioate (PS) linkage is a chemical modification to the backbone of DNA and RNA, foundational to a class of drugs known as therapeutic oligonucleotides. In natural nucleic acids, phosphodiester bonds connect neighboring sugar units. During chemical synthesis of a therapeutic oligonucleotide, a PS modification replaces one of the non-bridging oxygen atoms in this bond with a sulfur atom. This substitution is not found in nature.
The primary significance of this change is its ability to protect these molecules from being rapidly destroyed within the body. This enhanced stability and bioavailability allows them to function as effective drugs, enabling therapies for a range of genetic diseases.
The Phosphorothioate Modification
The defining feature of a phosphorothioate is the substitution of a sulfur atom for a non-bridging oxygen in the phosphate backbone. This change makes the linkage resistant to degradation by nucleases, which are enzymes that break down nucleic acids. Unmodified synthetic oligonucleotides are quickly destroyed by these enzymes, rendering them ineffective as therapeutic agents.
The sulfur atom interferes with the mechanism nucleases use to cleave phosphodiester bonds, significantly increasing the oligonucleotide’s half-life in blood serum and cells. By extending the molecule’s survival, the modification ensures it persists long enough to reach its target tissue. This enhanced stability is why most oligonucleotide-based drugs incorporate phosphorothioate linkages.
This modification also influences how the oligonucleotide interacts with proteins. After administration, PS-modified oligonucleotides bind to plasma proteins, such as albumin. This binding prevents rapid clearance of the drug by the kidneys and facilitates its distribution to various tissues.
Solid-Phase Synthesis Cycle
The creation of phosphorothioate oligonucleotides is an automated process known as solid-phase synthesis. This method builds the oligonucleotide chain one nucleotide at a time while it is attached to a solid support, typically a glass bead. The process is a cycle of four main chemical reactions: detritylation, coupling, capping, and sulfurization.
The cycle begins with detritylation, where a dimethoxytrityl (DMT) protecting group is removed from the 5′-end of the nucleotide chain anchored to the solid support. This removal exposes a hydroxyl group for the next nucleotide to be added. The coupling step introduces the next phosphoramidite building block, which reacts with the exposed hydroxyl group to extend the chain.
Following coupling, a capping step is performed to block any unreacted hydroxyl groups from participating in future cycles. This is done by acetylating these failures, which prevents the formation of shorter, incomplete sequences known as (n-1) impurities. The final step is sulfurization, where a sulfur-transfer reagent is introduced instead of using standard oxidation.
This sulfurization step converts the newly formed phosphite triester linkage into a phosphorothioate triester. Common sulfurizing agents include phenylacetyl disulfide (PADS) and the Beaucage reagent. The efficiency of this reaction is monitored, as incomplete sulfurization results in an undesirable phosphodiester linkage, impacting the final product’s purity. Once sulfurization is complete, the four-step cycle repeats until the desired oligonucleotide sequence is assembled.
Challenges of Stereochemistry and Purification
The synthesis of phosphorothioate linkages creates a chiral center at each modified phosphorus atom. Chirality means the phosphorus atom and its surrounding groups can exist in two different spatial arrangements that are mirror images of each other. These two forms, known as diastereomers, are designated as Rp and Sp. Because the synthesis process is not stereospecific, each cycle produces a nearly equal mixture of the Rp and Sp configurations.
For a therapeutic oligonucleotide that is 20 nucleotides long, there are 19 phosphorothioate linkages. This results in a product that is a complex mixture of 2^19 (524,288) different diastereomers. This is a challenge because the Rp and Sp forms can have different biological properties; the Sp isomer generally shows greater nuclease resistance, while the Rp isomer may form more stable bonds with the target RNA. This mixture can affect the overall potency and consistency of the drug.
After synthesis is complete, the full-length phosphorothioate oligonucleotide must be purified from contaminants. The primary impurities are shorter “failure” sequences that were successfully capped during the synthesis cycles. These shorter fragments must be removed to ensure the final product has a uniform length and is safe for therapeutic use.
The properties of phosphorothioate oligonucleotides make this purification demanding. The presence of numerous diastereomers and the increased hydrophobicity from the sulfur atoms cause the product to appear as broad peaks during chromatography. This makes it difficult to separate from closely related impurities, so achieving high purity often requires multiple chromatographic steps using techniques like anion exchange (AIEX) or reversed-phase HPLC.
Applications in Therapeutic Oligonucleotides
The stability provided by phosphorothioate linkages makes these molecules suited for therapeutic applications like antisense oligonucleotides (ASOs). ASOs are designed to modulate the expression of specific genes by binding to a target messenger RNA (mRNA). This binding event can prevent the mRNA from being translated into a protein or trigger its destruction, thereby reducing levels of a disease-causing protein.
This mechanism is the basis for several approved drugs. For example, nusinersen (Spinraza) is an ASO used to treat spinal muscular atrophy (SMA). It works by binding to the pre-mRNA of the SMN2 gene and modifying its splicing. Similarly, inotersen (Tegsedi) is an ASO therapy that targets the mRNA for the transthyretin (TTR) protein to treat hereditary transthyretin-mediated amyloidosis.
The utility of phosphorothioate modifications extends beyond ASOs. They are also incorporated into other nucleic acid-based platforms, such as small interfering RNAs (siRNAs) and aptamers. In siRNAs, which regulate gene expression through the RNA interference pathway, PS linkages improve stability. Aptamers, which are short oligonucleotides that fold to bind to target molecules, also benefit from the increased nuclease resistance conferred by PS modifications.