The cellular membrane provides a barrier to the passage of large, hydrophilic molecules, such as proteins, into the cytosol. Thiol-mediated uptake, postulated to function by transporter units engaging in dynamic covalent exchange cascades with cellular thiols and disulfides, has been employed previously for the efficient cytosolic delivery of streptavidin. Using the orthogonal reactivities offered by the EBX modification developed here, we were able to functionalize streptavidin (66 kDa) with both a transporter for thiol-mediated uptake, via CuAAC 'click' chemistry with the azide handle, and a lipophilic CF3 functionalized aromatic unit, via Suzuki coupling with the hypervalent iodine motif. The functionalized proteins were still easily complexed with 2 equivalents of a red fluorescent TAMRA-biotin derivative (Figure ), and uptake of the complexes was studied in HeLa MZ cells. Compared with the protein after Suzuki coupling, but prior to click functionalization (18, R = N3), streptavidin functionalized with asparagusic acid (19, R = AspA) showed ~3-fold increase in cellular uptake whereas streptavidin functionalized with a cyclic thiosulfonate (20, R = CTO), gave ~28-fold increase in cellular uptake versus the N3 control (Scheme 6b). These results were in accordance with the relative reactivities of the two transporter motifs in thiol-disulfide exchange, and previous studies with small molecule fluorophore cargoes. Structure-activity relationships for cyclic thiosulfonate transporters and thiol-mediated uptake inhibitors revealed a strong dependence in either case on the presence of hydrophobic directing groups to target aprotic environments such as the cellular membrane and proteins, in which the proticity-dependent cascade exchange of cyclic thiosulfonates is enhanced. The ~9-fold increase in uptake efficiency observed between AspA and CTO complexes exceeds the ~3fold difference seen with small molecule fluorophore cargoes, the greater differential perhaps resulting from the additional hydrophobic directing group provided by the CF3-functionalised aromatic unit, which is known to be more important for CTO transporters. In the case of AspA and CTO, a diffuse pattern of fluorescence was observed in spinning disk confocal microscopy (SDCM) images (Figure and). In line with previous reports, this implied delivery to the cytosol rather than entrapment within endosomes following endocytosis, whereas N3 complex 18 showed a punctate fluorescence indicative of endocytosis (Figure ). These results indicate the potential of readily accessible, broadly stable cyclic thiosulfonates as transporters for the efficient cytosolic delivery of proteins via thiol-mediated uptake, and highlight the possibilities offered by the dual, orthogonal functionalizations available following EBX-tyrosine modification.