Fig. 2. Streptavidin-pAz showed much higher click conversion efficiency than streptavidin-N₃ in solution. (A) Reactions of synthesis of two types of amplifiers and Click conversion. Streptavidin was modified with NHS-N₃ or NHS-pAz, generating two types of amplifiers streptavidin-N₃ and streptavidin-pAz. (B) Crystal structure of a streptavidin monomer, including 4 lysine residues. (C) The deconvoluted ESI-TOF mass spectrum of the streptavidin monomer (mSA). Molecular weight (MW): 12 971 + 22n. Δm = 22 should be Na⁺. (D) Streptavidin modified with NHS-N₃. The numbers of N₃ groups on streptavidin were calculated to be 3–5. (E) Partial conversion of streptavidin-N₃ in (D) via CuAAC with biotin-PEG₄-alkyne. MWs of a–h: 13 352 + 22n (3× N₃, no biotin), 13 479 + 22n (4× N₃, no biotin), 13 809 + 22n (2× N₃, 1× biotin), 13 936 + 22n (3× N₃, 1× biotin), 14 266 + 22n (1× N₃, 2× biotin), 14 393 + 22n (2× N₃, 2× biotin), 14 723 + 22n (no N₃, 3× biotin), and 14 850 + 22n (1× N₃, 3× biotin). (F) Streptavidin modified with NHS-pAz. The numbers of pAz groups on streptavidin were calculated to be 2–5. (G) Full conversion of streptavidin-pAz in (F) via CuAAC with biotin-PEG₄-alkyne. MWs: 14 658 (no pAz, 2× biotin), 15 495 and 15 509 (no pAz, 3× biotin), and 16 345 (no pAz, 4× biotin). No intermediates, MWs: 15 038 (1× pAz, 2× biotin) and 15 874 (1× pAz, 3× biotin).