Synthesis of Fluorogenic Polymers for Visualizing Cellular Internalization

Abstract

The binding of a polymeric ligand to a cell surface receptor can promote its internalization. Methods to track and visualize multivalent ligands within a cell can give rise to new therapeutic strategies and illuminate signaling processes. We have used the features of the ring-opening metathesis polymerization (ROMP) to develop a general strategy for synthesizing multivalent ligands equipped with a latent fluorophore. The utility of ligands of this type is highlighted by visualizing multivalent antigen internalization in live B cells.Advances in organic and polymer chemistry are providing access to new classes of bioactive polymers. 1 With the ability to generate synthetic macromolecules that vary in structure and physical properties, the applications of polymers have expanded rapidly. Some uses include as substrates for cell growth and differentiation, 2 vehicles for drug delivery, 3 therapeutics, 4 and molecules for studying cell signaling. 5 For some biological applications, bioactive polymers must be taken up into cells. Still, little is known about the cellular internalization and trafficking of polymers. Here, we present an approach for following these processes.The bioactive polymers that we focused upon were those generated by ruthenium carbeneinitiated ring-opening metathesis polymerization (ROMP). It was shown that ROMP could be used to generate biologically-active polymers over 10 years ago. 6 Since that time, ROMP has been used produce materials with a variety of biological activities. 7 Continuous advances in catalyst development and polymerization techniques have provided methods to control key features of polymer structure, including their length and the functional groups they present. 8 Ruthenium carbene initiators tolerate a diverse array of functional groups; therefore, they can be used synthesize highly functionalized polymers with tailored biological activities. ROMP initially was used to generate polymers that act on the outside of the cell, but more recently, it has been employed to assemble compounds that can be internalized by cells. Our interest in polymer uptake was prompted by our studies of B cell signaling. 9 For example, polymers generated by ROMP that display antigenic epitopes can promote antibody production in vivo. The ability of these polymers to activate this process depends upon their interactions with the antigen-specific B cell receptor (BCR), a membrane-bound immunoglobulin on the surface of B cells. These polymers bind to the specific B cell receptor to activate signaling but they also promote its internalization. We therefore anticipated that, like other antigens, these polymers would be taken up by endocytosis. To investigate polymer internalization, we envisioned using antigenic polymers equipped with a group that would report directly on internalization.Most approaches to following ligand internalization rely on appending a fluorescent dye to the molecule of interest. Uptake is detected using a discontinuous assay (typically fluorescence microscopy). Becaus...

Dye-functionalized polymers via ring opening metathesis polymerization: principal routes and applications

“…While functional group placement is limited to one unit at the end of each polymer chain, this approach is complementary to the initiator route so that both ends can be labeled if desired. Alternatively, a reactive group such as activated esters, ketones, or azides can be introduced via the above-mentioned methods which can be then further used to attach dye molecules, an approach which has been quite frequently pursued [3,[76][77][78][79].…”

Advances in Bioconjugation

“…(5 D ). The reaction has been used in an extraordinary range of contexts, including labeling proteins with small molecules [152,99,153,154], immobilizing proteins and peptides [155,118], proteomics applications [156], immobilizing carbohydrates [155], functionalizing DNA [155,157], and decorating virus particles and bioactive polymers with fluorescent molecules [158,159]. A Ru(II) catalyst leads to the 1,5-disubstituted[1,2,3]triazole [160], which mimics a cis (that is, E ) peptide bond [161], as in Fig.…”

End-labeled amino terminated monotelechelic glycopolymers generated by ROMP and Cu(I)-catalyzed azide–alkyne cycloaddition

“…ROMP polymers with pendant reactive functional groups, such as N -hydroxysuccinimide (NHS) or nitrophenyl esters, are useful materials since the pendant groups can be functionalized with various recognition elements to provide multiple final products from a common polymer intermediate in a divergent manner [7–8]. Telechelic polymers provide a means for further modifying the polymer with a variety of reporter molecules [6–7 9]. These polymers have been used as probes of biologically relevant recognition events and to functionalize surfaces [10].…”

“…ROMP of bicyclo[2.2.1]hept-5-ene-2-carboxylic acid NHS ester 4 [18] was carried out by using the rapidly initiating Grubbs 3rd generation catalyst 5 [19]. A solution of monomer 4 was cooled to −78 °C, followed by addition of a solution of catalyst 5 to initiate polymerization [9]. After TLC indicated complete consumption of the monomer 4 , the reaction was treated with an excess of the terminating agent 3 for 20 minutes to provide the end-capped polymer 6 .…”