MULTIFUNCTIONAL CHEMICAL CROSS-LINKERS FOR QUANTIFYING AND VISUALIZING INTRACELLULAR PROCESSES

Abstract

Chemical cross-linkers have proven valuable for a diversity of biological applications such as protein enrichment, nanoparticle surface functionalization, and conjugate-based drug delivery. Two valuable subclasses of cross-linkers include cleavable and heteromultifunctional linkers. The introduction of cleavage sites within a linker is important for drug delivery applications, as it enables programmable and spatiotemporal release of tethered molecules or therapeutic cargo. Heteromultifunctional linkers that allow the orthogonal attachment of multiple biomolecules or chemical moieties are important for the synthesis of heterovalent ligands and molecular probes. Due to synthetic limitations, a vast majority of cleavable linkers have been limited to a single cleavage site. Traditional approaches for synthesizing heteromultifunctional linkers are limited to three functional groups and lack the functional diversity to allow for orthogonal cleavage via nonenzymatic cues. Further, approaches that utilize natural amino acids and peptidyl scaffolds are sensitive to proteolysis in the biological environment. An emerging paradigm in polymer chemistry is the synthesis of sequence-defined polymers. Oligothioetheramides (oligoTEAs), a subset of sequence-defined polymers, have recently been reported by our research group. The oligoTEA synthesis approach utilizes an orthogonally reactive N-allylacrylamide monomer, which can undergo alternating photoinitiated thiol-ene “click” reactions and phosphine-catalyzed thiol-Michael additions. Aside from their facile synthesis, oligoTEAs have several benefits including the incorporation of a diverse panel of pendant and backbone functionalities. Furthermore, oligoTEAs are stable to proteolytic degradation and as such are stable in the biological environment. We have leveraged the iterative nature of oligoTEA synthesis to address many of the limitations of heteromultifunctional cross-linker synthesis. Herein, I will describe the development of oligoTEAs as a platform for synthesizing cleavable heteromultifunctional cross-linkers. We have taken this class of cross-linkers and applied it towards quantifying the intracellular processing of stimuli-responsive drug carriers. In combination with a kinetic model, we have extracted the rate constant for intracellular disulfide bond degradation in the HER2 receptor endocytic pathway. In collaboration with the Paszek group, we have also used our synthesis platform to design probes to visualize the glycocalyx via expansion microscopy. These probes selectively modify metabolically-labelled cell surface glycans via bioorthogonal “click” chemistry. Taken together, these works highlight the utility of oligoTEAs as a platform to design heteromultifunctional cross-linkers for a range of biological applications.