How a 1987 Chemistry Breakthrough Paved the Way for Modern Medicine
A simple chemical twist on DNA's backbone opened the door to a new world of therapeutics.
Year: 1987
Scientists: Sudhir Agrawal & John Goodchild
Journal: Tetrahedron Letters
Key Innovation: N-pent-4-enoyl protecting group
Imagine a drug that doesn't just temporarily treat a disease's symptoms but can precisely silence the genetic instructions that cause it. This is the promise of antisense therapy, a revolutionary approach to medicine. Yet, in the 1980s, turning this promise into reality was hampered by a fundamental problem: the body's enzymes rapidly destroy natural genetic material.
This article explores the pivotal 1987 work by scientists Sudhir Agrawal and John Goodchild, whose clever chemistry created a more stable, drug-like oligonucleotide, helping to launch the entire field of oligonucleotide therapeutics.
The concept of antisense therapy is elegantly simple. If a disease is caused by a specific harmful protein, scientists can design a short, synthetic strand of DNA or RNA—an oligonucleotide—that is complementary to the messenger RNA (mRNA) blueprint for that protein. When introduced into cells, this "antisense" oligonucleotide binds perfectly to its target mRNA, effectively shutting down production of the problematic protein 1 .
However, the first generation of these oligonucleotides had a natural phosphodiester (PO) backbone, which acted like a welcome mat for nucleases—the body's dedicated molecular scissors. These enzymes would rapidly chop the therapeutic oligonucleotides into useless fragments before they could do their job 1 .
Furthermore, these early molecules were often poor at entering cells, making them ineffective as drugs. The dual challenge of stability and delivery needed to be solved for antisense therapy to become a viable treatment approach.
Agrawal and Goodchild's key insight, published in the journal Tetrahedron Letters, was to develop a novel protecting group strategy for the chemical synthesis of a specific type of modified oligonucleotide: the methylphosphonate 2 .
In oligonucleotide synthesis, building blocks called phosphoramidites are stitched together. To prevent unwanted side reactions, certain reactive parts of these building blocks must be "protected" by temporary chemical groups. Agrawal and Goodchild introduced the N-pent-4-enoyl group as a new protecting group for the nucleobases (A, C, G, T) during the synthesis of nucleoside methylphosphonates 2 .
This was a significant improvement because it allowed for the more efficient and rapid synthesis of oligonucleotides where the standard phosphate backbone oxygen was replaced by a methyl group (methylphosphonate), making the linkage neutral in charge and, crucially, more resistant to nucleases 2 1 .
A novel protecting group that enabled efficient synthesis of methylphosphonate oligonucleotides
The goal of the featured experiment was to synthesize oligonucleoside methylphosphonates using new N-pent-4-enoyl-protected nucleoside phosphonamidite building blocks.
| Reagent/Component | Function in the Experiment |
|---|---|
| N-pent-4-enoyl protected nucleoside phosphonamidites | Novel building blocks for oligonucleotide synthesis, enabling the incorporation of methylphosphonate linkages 2 . |
| Solid-Phase Support (e.g., CPG) | A porous glass bead that serves as an anchor, allowing the oligonucleotide to be built one unit at a time and easily purified 3 . |
| Diisopropylcarbodiimide (DIC) & 1-Hydroxybenzotriazole (HOBT) | Efficient activators and catalysts used to load the first nucleoside onto the solid support, a critical first step 3 . |
| Acetonitrile | A safer solvent alternative to toxic pyridine, used to dissolve reactants and wash away impurities 3 . |
The solid-phase support (CPG) was functionalized with linker molecules to provide attachment points.
The first nucleoside, protected with the N-pent-4-enoyl group, was attached to the support using the efficient DIC/HOBT activation method 3 .
The synthesis cycle began:
After the full sequence was assembled, the completed oligonucleotide was cleaved from the support. The N-pent-4-enoyl protecting groups were then removed under specific chemical conditions, yielding the final, pure product 2 .
The successful development of these new synthons was a milestone in chemistry. It provided researchers with a facile and rapid method to synthesize not just pure methylphosphonate oligonucleotides, but also chimeric analogs (mixed-backbone oligonucleotides) and methylphosphonothioates 2 . This opened up a world of possibilities for tailoring the properties of therapeutic oligonucleotides.
| Oligonucleotide Type | Backbone Charge | Nuclease Resistance | RNase H Activation? | Key Challenge |
|---|---|---|---|---|
| Phosphodiester (PO) | Negative (Ionic) | Low | Yes | Rapidly degraded in biological fluids 1 . |
| Methylphosphonate (P-ME) | Neutral (Non-ionic) | High | No | Poor solubility; could not harness catalytic RNase H mechanism 1 . |
| Phosphorothioate (PS) | Negative (Ionic) | Moderate | Yes | Became the "first-generation" choice due to RNase H activation and good solubility 1 . |
The most critical finding from subsequent cellular experiments was the mechanism of action. When Agrawal and others tested these modified oligonucleotides, they discovered a crucial difference:
When bound to their target RNA, could activate RNase H. This cellular enzyme acts as molecular scissors, cutting the target RNA and allowing a single antisense molecule to destroy multiple RNA targets, a catalytic and highly potent effect 1 .
While stable and good at binding, could not activate RNase H. They worked only by steric hindrance, which was a less potent mechanism 1 .
This understanding led directly to the next big idea: Mixed-Backbone Oligonucleotides (MBOs). Scientists realized they could combine the best features of different modifications. For example, an MBO might have a central "window" of phosphorothioate DNA to activate RNase H, flanked by segments of 2'-O-methyl or methylphosphonate nucleotides to boost binding affinity and nuclease stability 4 5 .
| Property | Phosphorothioate (PS) Oligo | Mixed-Backbone (MBO) | Advantage of MBO |
|---|---|---|---|
| Anti-HIV-1 Activity (IC90) | 0.8 μM | 0.2 μM | 4x more potent antiviral effect |
| Complement Activation | 50% at 0.4 mg/mL | 50% at >1.0 mg/mL | Reduced immune-related side effects |
| aPTT Coagulation Effect | 50% prolongation at 48 μM | 50% prolongation at 96 μM | Reduced interference with blood clotting |
The 1987 work by Agrawal and Goodchild was more than just a synthetic chemistry achievement. It was a critical step in the evolution of oligonucleotides from a laboratory tool to a legitimate therapeutic modality. By providing a robust method to create stable, modified oligonucleotides, it helped researchers unravel the fundamental rules of what makes an effective antisense drug.
Today, the lessons learned from these early experiments are embedded in multiple approved drugs. Medicines for spinal muscular atrophy, Duchenne muscular dystrophy, and hereditary transthyretin amyloidosis, among others, all rely on chemically modified oligonucleotides whose design can be traced back to this foundational work 1 .
The journey from a protective chemical group in a 1987 paper to life-changing medicines stands as a powerful testament to the importance of basic chemical research. The field of oligonucleotide therapeutics continues to expand, with new modifications and delivery systems being developed to treat an ever-widening range of diseases.