Efficient chemical synthesis of mirror-image DNA polymerase Dpo4 assisted by one-pot multi-segment condensation

Abstract

The mirror-image DNA polymerase Dpo4 is a valuable enzymatic tool in biomedical research; however, its chemical synthesis has been hindered by low efficiency. Here, we describe an efficient chemical synthesis of D-Dpo4 using a one-pot multi-segment condensation strategy, which was achieved using Fmoc-masked peptide thioester. By minimizing the need for isolating and handling intermediates through one-pot three- and four-segment ligations, we were able to obtain D-Dpo4 with higher efficiency, achieving an improved overall yield of 9.8%, representing a twofold improvement from previous work. Our work provides a practical and streamlined approach for the synthesis of mirror-image proteins with larger sizes and underscores the utility of one-pot multi-segment condensation in chemical protein synthesis.