The 5'-DMTr-3'-OH oligomer is an oligonucleotide characterized by the presence of a 4,4'-dimethoxytrityl (DMTr) protecting group on the 5'-hydroxyl group and the presence of a free 3'-hydroxyl group. The DMTr group prevents undesirable reactions of the 5'-hydroxyl group. The unprotected 3'-hydroxyl group remains open to further chemical modification.
This structural configuration makes them highly valuable in the synthesis and structural analysis of DNA and RNA, helping to facilitate genetic research and therapeutic development. Alfa Chemistry explores their properties, applications and synthetic methods in this article, emphasizing their importance in modern nucleic acid chemistry.
Properties and Role of the DMTr Group
The DMTr group is a powerful acid-destabilizing protecting group. It is widely used in oligonucleotide synthesis to protect the 5'-hydroxyl group, which is essential for maintaining the structural integrity of the oligonucleotide during synthesis. The DMTr group prevents unwanted reactions or hydrolysis at the 5'-position by forming a stabilizing ester bond with the hydroxyl group.
Figure 1. DMTr group[1].
DMTr is unique in its acid-unstable nature, which facilitates controlled removal under mild acidic conditions. This selective deprotection ensures minimal side reactions while exposing the 5'-hydroxyl group to subsequent coupling reactions. In addition, the presence of the DMTr moiety confers chromophore properties, allowing for efficient monitoring of the reaction process by colorimetric or chromatographic analysis.
Features
- Base Activation and Coupling - In the case of phosphoramidite as the activating nucleotide monomer, the DMTr-protected oligomer ensures that only the desired hydroxyl group is involved in the coupling reaction.
- Controlled Deprotection - DMTr is selectively removed to expose the 5'-hydroxyl group for subsequent coupling reactions. This precision minimizes waste and maintains oligonucleotide strand integrity.
- Easier purification - DMTr-protected oligomers can be efficiently separated using reversed-phase HPLC, and the presence of the DMTr moiety allows for clear differentiation between full-length products and failed sequences.
Synthesis of 5'-DMTr-3'-OH Oligomers
Synthesis of 5'-DMTr-3'-OH oligomers involves a series of carefully designed steps to ensure high fidelity and efficiency. These steps include protection, activation, coupling and deprotection.
| Step | Description |
| Protection | The 5'-hydroxyl is selectively protected using DMTrCl in a controlled environment to prevent side reactions. |
| Coupling Reaction | Activated phosphoramidites react with the exposed 3'-hydroxyl, extending the oligonucleotide chain. |
| Deprotection | The DMTr group is removed using agents like trichloroacetic acid (TCA) to expose the 5'-hydroxyl. |
| Purification | Post-synthesis, oligomers are purified via HPLC to isolate the desired products. |
Special Modifications
Advanced synthesis methods employ alternative protective groups, such as 2'-amino LNA monomers, to enhance oligonucleotide stability and nuclease resistance.
Figure 2. Common ribonucleoside phosphoramidite building blocks for solid-phase RNA synthesis. (a) 5 -O-DMTr-2 -O-TBDMS-3 -O-(2-cyanoethyl-N,N-diisopropylphosphoramidite); (b) 5 -O-DMTr-2 -O-TOM-3 -O-(2-cyanoethyl-N,N-diisopropylphosphoramidite); (c) 5 -O-DMTr-2 -O-Fpmp-3 -O-(2-cyanoethyl-N,N-diisopropylphosphoramidite); (d) 5 -O-DMTr-2 -O-TC-3 -O-(2-cyanoethyl-N,N-diisopropylphosphoramidite).[2].
Deprotection Methods for DMTr Removal
A variety of methods are available for removing the DMTr group, tailored to specific oligonucleotide chemistries. These methods are summarized in the following table:
- Acid-Catalyzed Deprotection:
A 3% solution of trichloroacetic acid (TCA) in dichloromethane is commonly used to remove the DMTr group, exposing the 5'-hydroxyl.
For sensitive oligonucleotides, DMTr can be removed by gentle heating at 95°C for 1.5 hours.
- Buffer-Based Deprotection:
Using a mildly acidic buffer at pH 4.5-5.0 and 40°C achieves nearly quantitative DMTr removal without compromising the oligonucleotide's integrity.
Figure 3. Mild removal of DMTr protection from the 5'-hydroxyl groups of nucleic acids[3].
Conclusion
The 5'-DMTr-3'-OH oligomer represent a critical tool in nucleic acid research and synthesis. By leveraging the unique properties of the DMTr group, scientists achieve unparalleled precision and efficiency in oligonucleotide production. Their applications span structural analysis, synthetic biology, and therapeutic development, making them indispensable in modern molecular science.
References
- Tram K.; et al. ChemInform Abstract: Further Optimization of Detritylation in Solid-Phase Oligodeoxyribonucleotide Synthesis. Nucleosides Nucleotides & Nucleic Acids. 2011, 30(1), 12-9.
- Bartosik K.; et al. Synthesis of Nucleobase-Modified RNA Oligonucleotides by Post-Synthetic Approach. Molecules. 2020, 25(15), 3344.
- Salon J.; et al. Mild Detritylation of Nucleic Acid Hydroxyl Groups by Warming-up. Nucleosides Nucleotides Nucleic Acids. 2011, 30(4), 271-279.
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