Fig. 6.

Probing NRPS substrate scope. a Scheme depicting the reaction conditions for the coupled ObiF1/F2/D/H in vitro enzyme assay using a thiol kinetic capture strategy for the β-lactone product. DTT was employed as the reactive thiol. Thioester and β-hydroxy acid product ion counts were quantified via LCMS (see Supplementary Data 1 for [M + H]⁺ m/z values and EIC traces). Heat maps represent the extracted ion counts for the amino acid (2a–h), thioester (5), and β-hydroxy acid (6) resulting from the use of variable b aldehyde (1a–h) or c benzoic acid (3a–u) substrates normalized to a phenylalanine internal standard for one trial at the 24 h reaction time point. For compounds (4–6), the first letter in the two-letter nomenclature, (a–h), represents the structure of R₁ while the second letter in the two-letter nomenclature, (a–u), represents the structure of R₂. Heat maps for additional time points (0.5 and 3 h) are provided in the Supplementary Information. The color scale to the right of each figure reflects peak counts; peaks above 1,000,000 counts are colored black