Suppression of Simultaneous Fmoc-His(Trt)-OH Racemization and N^α‑DIC-Endcapping in Solid-Phase Peptide Synthesis through Design of Experiments and Its Implication for an Amino Acid Activation Strategy in Peptide Synthesis

Coupling Fmoc-His­(Trt)-OH in solid-phase peptide synthesis is frequently accompanied by significant racemization. Histidine is among the most susceptible amino acid residues inclined to racemize in peptide syntheses. In this study, the His racemized impurity could not be effectively purged by the applied chromatographic purification. Consequently, a Taguchi design of experiment (DOE) was first applied to screen the critical process parameters affecting the histidine racemization. Optimization of the DOE is subsequently performed to search for the optimum. The derived DOE models reveal that the conditions of Fmoc-His­(Trt)-OH carboxylate pre-activation prior to its coupling to growing peptide chains are critical for the subject histidine racemization. Intensive Fmoc-His­(Trt)-OH pre-activation stimulates this side reaction. On the other hand, without the amino acid pre-activation, by adopting the in situ Fmoc-Xaa-OH activation, another side reaction, that is, peptide N^α endcapping by N,N-diisopropylcarbodiimide, is boosted. A conflicting relationship between histidine racemization and peptide N^α endcapping has been detected through the DOE investigation. Significant models are established for the histidine racemization and peptide N^α endcapping, and reconciliation to balance these two side reactions is accomplished on this basis.