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. 2019 Mar 6;7:113. doi: 10.3389/fchem.2019.00113

Figure 5.

Figure 5

N- and C-terminal labeling of ISEr scaffold for detection. (A) A fluorescent dye DY680 (yellow star) was coupled via NHS-ester to the N-terminus of the effector-PEG27 scaffold and two integrin α3β1 binding peptides were ligated using CuAAC ligation. MS and analytical HPLC (with fluorescence detection at 690 nm) traces are shown for the purified product: MWcalc: 6889.0 Da, MWobs: 6890.5 Da. (B) CuAAC ligation of cMET binders to N-terminal biotinylated effector-PEG27 scaffold. Biotin is represented by a white hexagon. The reaction was monitored by RP-HPLC and the purified product was analyzed by MS: MWcalc: 9836.4 Da, MWobs: 9837.0 Da. (C) C-terminal labeling of ISErs using orthogonal chemoselective ligations. Integrin αvβ6-targeting binder peptides were ligated to the effector-PEG27 scaffold by CuAAC ligation. After removal of the S-tBu cysteine protecting group with TCEP, the fluorescent dye maleimide was coupled to the free thiol. MS and analytical HPLC trace of purified Cy5-labeled ISEr (MWcalc: 7513.5 Da, MWobs: 7514.0 Da). Fluorescence microscopy image showing binding of a commercial anti-human integrin β6-allophycocyanain-labeled antibody (left) and Cy5-labeled ISEr (right) to HT-29 cells, detected at 647 nm (top left, magenta), cell cytoskeleton stained with phalloidin (top right, green), nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI, bottom left, blue) and overlay (bottom right). Scale bar = 10 μm.