Large-scale supercritical fluid chromatography purification of unstable STING agonist intermediates

•Intermediate 1 and Intermediate 2 are two nucleoside derivatives, and required resolution by preparative supercritical fluid chromatography (SFC) to obtain the desired regioisomer as a key intermediate in a STING agonist program.•A key issue associated with interconversion between the regioisomers via silyl migration during purification was investigated in methanol, acetonitrile, and the mixture of acetonitrile and isopropanol, and the optimal organic modifier in CO2 was established to mitigate the interconversion to an acceptable level (<5%).•By extensively evaluating the effects of columns and column temperatures on the purification, an efficient large-scale SFC procedure was successfully developed and scaled up for Intermediate 1.•Rapid removal of the organic solvent from SFC fractions was another key factor in mitigating the isomer interconversion and, thus, achieving higher purity.•A similar purification strategy was applied to the regioisomeric resolution of Intermediate 2, an analog of Intermediate 1.