Optimization of the Solid-Phase Oligonucleotide Detritylation Reaction Using In-Line IR Spectroscopy

The solid-phase synthesis is a critical step in the oligonucleotide manufacturing process, and the detritylation reaction, as an essential procedure in oligonucleotide synthesis, directly affects the purity and yield of the drug substance. However, classical in-line ultraviolet (UV) monitoring techniques suffer from issues such as a narrow signal response range, delayed monitoring, and inadequate monitoring of microreaction processes. Therefore, this study optimizes the detritylation process in oligonucleotide synthesis using in-line infrared (IR) spectroscopy. The experimental results show that under the solid-phase synthesis conditions of oligonucleotides with different lengths and sequences, the in-line IR technology can real-time and accurately reflect the kinetic changes of the dichloroacetic acid monomer (DCA-monomer, IR 1765 cm–1) and dimer (DCA-dimer, IR 1740 cm–1), acetonitrile (IR 2253 cm–1), and toluene (IR 1495 cm–1) during the detritylation process. Compared with the classical UV monitoring techniques, the in-line IR technology reduced the consumption of the detritylation reagent by about 30%, while ensuring that the overall purity and quality of depurination impurities remained unchanged. This technological advance has greatly promoted the industrial application of solid-phase synthesis in green chemistry and provides a universal platform strategy for the future process development of a variety of oligonucleotide drugs.