Looking Inside the SPPS Reactor through a Refractometer: Online Quantification of the Resin Loading

The methodology of solid-phase peptide synthesis (SPPS) has been a key driver behind the significant advancements and growing interest in peptides for drug discovery. SPPS has many advantages, including short production times, automation compatibility, and versatility. However, it is like a black box as the intermediates are not isolated, and the results are unknown until the end of the entire process unless the synthesis is stopped, samples are taken, and analyses are performed to know the course of the synthesis. However, this is time-consuming and impacts cost-effectiveness. A key aspect of SPPS is accurately determining the initial loading of the resin. Overestimating the loading compared to the actual value leads to the use of a greater excess of reagents, which can enhance the purity of the final product but incurs higher economic costs. In contrast, underestimation of loading can lead to the formation of deletion peptides. The most widely used method to calculate resin loading is via the incorporation of an Fmoc derivative and then removal of the Fmoc group with piperidine, followed by the UV spectrophotometric determination of the dibenzofulvene-piperidine adduct. This operation requires halting the synthetic process, and it is time-consuming. Herein, the quantitative use of the refractometry index is proposed for the online determination of resin loading. This approach enables real-time monitoring of the reaction, allowing the process to be stopped when the desired loading is achieved or to add more coupling reagents to improve loading.