Improving 2-Chlorotrityl Chloride (2-CTC) Resin Activation

Abstract

Used in solid-phase peptide synthesis (SPPS) for peptides with an acid termination, the 2-chlorotrityl chloride (2-CTC) resin is highly susceptible to moisture, leading to reduced resin loading and lower synthetic yields. It is therefore recommended that the resin be activated with thionyl chloride (SOCl₂) before peptide assembly. Here we present an optimized procedure for resin activation that minimizes the use of SOCl₂ as the activation reagent and reduces the activation time. Additionally, we demonstrate the feasibility of reusing the 2-CTC resin when following the activation protocol, achieving comparable results to the first usage of the resin. Moreover, we achieved different degrees of resin activation by varying the amount of SOCl₂. For instance, the use of 2% SOCl₂ in anhydrous dichloromethane (DCM) allowed up to 44% activation of the resin, thereby making it suitable for the synthesis of longer peptides. Alternatively, employing 25% SOCl₂ in anhydrous DCM resulted in up to 80% activation with a reaction time of only 5 min in both cases.

Keywords: 2-CTC resin activation; 2-CTC resin reutilization; resin loading; solid-phase peptide synthesis.

Figure 1

DCM drying process with the use of a basic aluminum oxide column.

Figure 2

Loading of activated 2-CTC resins obtained under different % SOCl₂ and activation times. The significant differences between the % of SOCl₂ used was analyzed by Tukey’s multiple comparison test, are indicated in bold as follows: **** p < 0.0001. Significant differences in activation time, analyzed by Tukey´s multiple comparison test, are indicated in bold as follows: ## p < 0.01, and # p < 0.05.

Figure 3

Analysis by reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) of crude Fmoc-Gly-Lys(Boc)-Thr(tBu)-OH, using a 50 to 100% acetonitrile gradient in 8 min for RP-HPLC and 50 to 100% acetonitrile gradient in 3.5 min for LC-MS.