Protected nucleosides function as chemically altered nucleosides where specific functional groups like hydroxyl or amino groups receive temporary protection through protecting groups because they serve as important intermediates during nucleic acid synthesis. The modifications stop unwanted side reactions such as oxidation and alkylation or nonspecific ligations during nucleic acid assembly, which results in better synthetic efficiency and higher product purity. Solid-phase oligonucleotide synthesis relies on this protection strategy to achieve precise strand extension and complex sequence construction.
Figure 1. Structure of nucleoside phosphoramidites and protecting groups commonly used for the nucleobase and the 2'-OH. The 5'-OH and 3'-OH are protected with dimethoxytrityl (DMTr) in standard and reverse nucleoside phosphoramidites, respectively[1].
The core functions of PNS include:
A. Selective reaction control: By protecting specific sites (e.g., 5'-hydroxyl group, nucleobase amino group), it ensures that chemical reactions occur only at the target site (e.g., phosphorylation linkage of 3'-hydroxyl group).
B. Stability enhancement: Protective groups safeguard nucleobases from degradation in both acidic and basic environments (e.g., depurination). depurination).
C. Simplified synthesis steps: Automation of multi-step synthesis through staged deprotection.
Different protecting groups are used to selectively shield the functional site of a nucleoside. Below is a comparison of commonly used groups:
A. Hydroxyl Group Protected
| Protecting Group | Functional Group Protected | Stability | Common Applications |
| DMT (Dimethoxytrityl) | 5'-Hydroxyl | Acid-labile | Solid phase oligonucleotide synthesis |
| MMT (Monomethoxytrityl) | 5'-Hydroxyl | Mild acid-labile | RNA synthesis; selective deprotection |
| TBDMS (Tert-butyldimethylsilyl) | 2'-Hydroxyl, 3'-Hydroxyl | Base-stable, fluoride-sensitive | RNA synthesis; 2'-O, 3'-O protection |
| TIPDS (Tetraisopropyldisiloxane) | 2'-Hydroxyl, 3'-Hydroxyl | Highly stable | Dual protection of ribose |
| TBDPS (Tert-Butyldiphenylsilyl) | 2'-Hydroxyl, 3'-Hydroxyl | Acid stable (about 100 times more stable than TBDMS), base sensitive | RNA synthesis; 2'-O, 3'-O protection |
B. Base Protected
Each protecting group plays a specific role in stepwise synthesis leading to precise oligonucleotide assembly.
Figure 2. Example of base protection[2].
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We provide high-quality 3'-hydroxy protected nucleosides and their derivatives, some of which also have base protection.
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PNS undergoes successive coupling, oxidation, and deprotection reactions during oligonucleotide synthesis.
A. Activation and coupling: the 3'-phosphoramidite form of the protected nucleoside reacts with the growing oligonucleotide chain under acidic conditions.
B. Oxidation: Mild oxidizing agents convert the phosphite to a stable phosphate ester.
C. Deprotection: Acid, base, or fluoride treatment selectively removes the protecting group, exposing the functional portion for subsequent modification.
Figure 3. The solid-phase, four-step oligodeoxynucleotide synthesis cycle[3].
PNS are critical in the following areas:
Q1: What is the purpose of using protected nucleosides?
A1: Protected nucleosides prevent unwanted side reactions during oligonucleotide synthesis, thereby improving yield and precision.
Q2: What is the difference between DMT-protected nucleosides and MMT-protected nucleosides?
A2: DMT motifs are more stable and can be used for DNA synthesis, while MMT motifs provide selective deprotection and can be used for RNA synthesis.
Q3: Can protected nucleosides be used for therapeutic applications?
A3: Yes, they are essential for the synthesis of antisense oligonucleotides, siRNAs, and mRNA-based therapeutics.
Q4: How are the protecting groups of nucleosides removed?
A4: The deprotection process is usually carried out using specific chemical reagents: the DMT protecting group can be removed with an acid (e.g., TFA trifluoroacetic acid). The TBDMS and TOM protecting groups are usually removed with a fluorinating reagent (e.g., TEA-3HF or TBAF). Base-protecting groups (Bz, Ac, etc.) can be removed with ammonia or methanolamine.
Q5: Does Alfa Chemistry offer custom synthesis of protected nucleosides?
A5: Yes, Alfa Chemistry offers customized solutions to meet specific oligonucleotide synthesis requirements.
Alfa Chemistry delivers protected nucleosides of high quality that enable progress in nucleic acid research and therapeutic development. Our high-purity protected nucleosides serve as special components for DNA, RNA, and therapeutic oligonucleotide synthesis. Our technical staff is available to address any questions you may have.
References
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