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Synthesis of antisense oligonucleotides with minimum depurination
Achim H Krotz1, Douglas L Cole, Vasulinga T Ravikumar
1Isis Pharmaceuticals, Inc., Carlsbad, California 92008, USA. akrotz@isisph.com
Nucleosides, Nucleotides & Nucleic Acids
|May 15, 2003
Summary
Optimizing oligonucleotide synthesis involves balancing 4,4'-dimethoxytrityl (DMTr) group removal with purine glycosidic linkage stability. A mildly acidic sodium acetate buffer (pH 3.0-3.2) minimizes depurination during DMTr deprotection, improving oligonucleotide purity.
Area of Science:
- Oligonucleotide Chemistry
- Synthetic Organic Chemistry
Background:
- Acid-labile protecting groups like 4,4 '-dimethoxytrityl (DMTr) are crucial in oligonucleotide synthesis.
- Low pH conditions required for DMTr removal can cause unwanted depurination of purine bases.
Purpose of the Study:
- To resolve the conflict between efficient DMTr group removal and minimizing acid-induced depurination in preparative oligonucleotide synthesis.
- To establish optimal conditions for DMTr deprotection that preserve oligonucleotide integrity.
Main Methods:
- Utilizing a mildly acidic sodium acetate (NaOAc) buffer (10 mM, pH 3.0-3.2).
- Monitoring DMTr removal progress via High-Performance Liquid Chromatography (HPLC).
- Calculating optimum reaction times based on HPLC monitoring.
Main Results:
- A pH range of 3.0-3.2 was identified as optimal for DMTr removal.
- HPLC monitoring allowed for precise determination of reaction endpoints.
- Oligonucleotides were obtained with significantly reduced depurination.
Conclusions:
- Mildly acidic NaOAc buffer (pH 3.0-3.2) effectively balances DMTr deprotection and purine stability.
- This method enhances the purity and yield of synthetic oligonucleotides.
- Optimized deprotection conditions are critical for preparative oligonucleotide chemistry.