Caspase-3-Responsive, Fluorogenic Bivalent Bottlebrush Polymers

Abstract

Controlling the access of proteases to cleavable peptides placed at specific locations within macromolecular architectures represents a powerful strategy for biologically responsive materials design. Here, we report the synthesis of peptide-containing bivalent bottlebrush (co)polymers (BBPs) featuring polyethylene glycol (PEG) and 7-amino-4-methylcoumarin (AMC) pendants on each backbone repeat unit. The AMCs are linked via caspase-3-cleavable peptides which, upon enzymatic cleavage, provide a "turn-on" fluorescence signal due to the release of free AMC. Time-dependent fluorscence measurements demonstrate that the caspase-3-induced peptide cleavage and AMC release from BBPs is strongly dependent on the BBP backbone length and the AMC-peptide linker location within the BBP architecture, revealing fundamental insights into the interactions of enzymes with BBPs.

Figure 1.

A) Previous works using bivalent MMs for BBP synthesis via “graft-through” ROMP. B) Bivalent, peptide-based BBPs designed to probe enzyme access quantitatively by fluorescence.

Figure 2.

Synthesis of DEVD-MM. Inset: MALDI-TOF spectrum for DEVD-MM. C₁₉₁H₃₄₄N₁₁O₈₅: Calculated: 4154.85; Found: 4154.62 [M + H+]. C₁₉₁H₃₄₃N₁₁O₈₅Na: Calculated: 4176.83; Found: 4176.60 [M + Na]⁺.

Figure 3.

A) SEC traces of DEVD-MM, DP10, DP20, and DP30 BBPs. B) Representative DLS measurements for DP10. C) SEC traces for triblock copolymers MID and END as well as each intermediate toward their synthesis. MID-1 and END-1 refer to the first blocks of MID and END, respectively; MID-2 and END-2 refer to the diblock copolymers formed after addition of a 2nd block to MID-1 and END-1, respectively. The shifts to shorter retention times upon transitioning from MID-1 to MID-2 to MID, and END-1 to END-2 to END, and the trace unreacted MM in each case, are consistent with high conversions of each block.” D) Representative DLS measurements for MID.

Figure 4.

A) Hypothesized relative rates of peptide linker cleavage (for estimates based on fraction of end-groups see Table 2S). B) Fluorescence “turn-on” versus time for all BBPs (left) and zoomed in view of the same data (right). Release conditions with and without carrier-free, recombinant human CASP3 addition were: pH 7.4, 50 mM HEPES, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1mM EDTA, 10% glycerol (CASP3 assay buffer).