Figure 3.

The scope and compatibility of activating and coupling modules (a) Six site-saturation peptide hydrazide and peptide amide libraries (Ac-DXSKL-N₂H₃, Ac-DFXKL-N₂H₃, Ac-DFSXL-N₂H₃, Ac-DFSKX-N₂H₃, XLKKA-NH₂ and AXKKA-NH₂) were utilized to investigate omniligase-1 substrate profiles. Ligation efficiency (blue band) was calculated by the integration of the peak areas [19,20,31] of the ligation and hydrolysis products monitored by HPLC (220 nm). (b) Two site-saturation peptide amide libraries (Ac-DFSXL-NH₂ and Ac-DFSKX-NH₂) were utilized to investigate PAM12B substrate profiles. The hydrazidation yield (blue band) was calculated by the integration of the peak areas [27] of the hydrazidation and hydrolysis products monitored by HPLC (220 nm) except for P1 = Pro or Asn. (c) Amidation and hydrazidation reactions were performed on one site-saturation peptide glycine library (DLSYXG-OH). The amidation yield (blue band) was calculated by integration of the peak areas of the amidation products and residual substrates monitored by HPLC (220 nm). The hydrazidation yield was determined as described above. (d) One-pot activation and ligation of Ac-DFSKV-G and ALKKA-NH₂. Analytical HPLC traces (220 nm) of the reaction mixture (top to bottom): substrate, 90 minutes after the addition of PHM and PAL, 10 minutes after the addition of PAM12B, before the addition of omniligase-1, 30 minutes after the addition of omniligase-1. More details can be found in the Supplementary data (Methods and results 4.2, omniligase; 4.3 to 4.5, PAM12B, PHM and PAL; and 4.6, one-pot conjugation of Ac-DFSKV-G and ALKKA-NH₂).