Figure 2.

Validation of phage-assisted interrogation of reactivity strategy. (a) Schematic diagram for the use of PAIR strategy in identifying reactive Hpg-encoded peptide sequences for Cu-free Sonogashira cross-coupling. M stands for methionine, and X stands for one of the following 15 amino acids: A, C, D, F, G, H, I, L, N, P, R, S, T, V, and Y (amino acid one letter code). (b) Verifying the incorporation of Hpg into phage coat protein pIII through selective reactivity in Cu-free Sonogashira cross-coupling reaction. Western blot analyses of Met-or Hpg-encoded phage lysates expressing myc-PMP-pIII proteins. 10¹¹ cfu phage was loaded into each lane based on UV–vis measurement. In anti-pIII Western blot, * denotes the full-length pIII protein while # denotes the truncated pIII. In probing biotin conjugation (panel 1), the membrane was blotted using the preformed avidin/biotinylated enzyme complex. (c) Selective enrichment of Hpg-phage after Pd-mediated cross-coupling with the biotinylated fluorescein iodide. The phage was probed with anti-pVIII-HRP antibody and quantified by adding ABTS substrate and measuring absorbance at 416 nm. ABTS = 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid).