Optimized Coupling Protocols for the Incorporation of Cys Derivatives during Fmoc-SPPS
Incorporation of cysteine during Fmoc-SPPS can result in significant racemization depending on the coupling conditions [1, 2, 3, 4]. During the preparation of cysteine containing peptides as APIs, we identified the necessity to investigate coupling conditions for Fmoc-Cys(Trt)-OH and Fmoc-Cys(Acm)-OH based on previous results of other groups [2, 3, 4] in more detail.
Using the model peptide Z-Ala-Cys(PG)-Pro-OH, we studied the influence of coupling reagent, solvent, activation time, and temperature during preactivation and coupling on the racemization of Cys. SPPS was performed on H-Pro-2-chlorotrityl resin with 3 equiv. of amino acid derivative / coupling reagent / base or additive. Peptides were cleaved from the resin with 1% TFA in DCM. After evaporation, the residue was analyzed on HPLC (C18; gradient of ACN in 0.1% TFA). The content of the LDL-isomer was determined using the relative peak areas A from HPLC as: A(LDL-isomer) / [A(LDL-isomer) + A(LLL-isomer)].
KeywordsAcid Derivative Polar Solvent Clear Increase Relative Peak Weak Base