Development of Silyl‐Protected Phosphoramidite Building Blocks for Short <scp>ssDNA</scp> Synthesis^†

Comprehensive Summary To overcome the depurination challenges associated with classical DNA synthesis methods, this study introduces a novel approach using fluoride‐sensitive, triisopropylsilyl (TIPS)‐protected phosphoramidite monomers for the efficient synthesis of short single‐stranded DNA. The bulky TIPS group selectively protects the 5'‐hydroxyl group of nucleosides and can be rapidly removed under mild, non‐acidic conditions using fluoride ions, minimizing side reactions. Four novel TIPS‐protected phosphoramidite monomers were synthesized and characterized, achieving &gt;99% deprotection efficiency. These monomers were applied to the solid‐phase synthesis of a 5‐mer ssDNA sequence, yielding an overall efficiency of 99% for each cycle. The strategy demonstrates significant potential for improving the reliability and scalability of oligonucleotide synthesis, offering a promising alternative for applications in synthetic biology and nucleic acid research.