Versatile fluorescent homobifunctional crosslinkers: expedient synthesis and application to di-affibody constructs...

In this contribution we report on the design of new versatile fluorescent linkers (L1-L3, L1^N) suitable for the covalent assembly of specific recognition motifs to bind biologically relevant targets such as HER2 receptors. The central fluorescent molecular platform emits in red and represents a key building block for the synthesis of conjugates and their subsequent analyses. The presence of amino groups enables straightforward functionalization with bifunctional PEG linkers (NHS, MI) of variable length for further directional bioconjugation. To demonstrate the value of this approach, the linkers were conjugated with chemically synthesized affibodies (AfB, Z_HER2:2891(M9Nle/D37E)), bearing a unique cysteine at N-terminal or C-terminal end. Indeed, this AfB binds the human epidermal growth factor receptor 2 (HER2), which is often overexpressed in different carcinomas, and it is therefore considered as a tumour marker in bioimaging and drug delivery systems. Seven AfB conjugates-three mono-AfBs (N1, N1^N, C1) and four di-AfBs (N2, N3, C2, C3)-were obtained through MI-thiol conjugation. The binding of fluorescent mono- and di-affibody (AfB) constructs was evaluated using flow cytometry to demonstrate their ability to interact with the HER2-expressing SKOV3 cells in relation with their structural properties. All tested conjugates specifically bound to HER2 receptors present on SKOV3 cells with nanomolar affinities, and only small differences between constructs were detected. The presence of our fluorescent conjugates bound to the cell surface was visualized using confocal microscopy. In addition, the internalization mechanism of AfB constructs in SKOV3 cells was investigated due to the measurement of their fluorescence following trypsin treatment. In conclusion, our innovative synthetic approach with the integration of a versatile fluorescent platform offers new perspectives for monitoring molecular therapeutics inside cell and for tuning multivalent conjugates for cancer immunotherapy.