Synthesis of Homopolymers and Copolymers Containing an Active Ester of Acrylic Acid by RAFT: Scaffolds for Controlling Polyvalent Ligand Display

Abstract

We describe the synthesis of activated homopolymers and copolymers of controlled molecular weight based on the controlled radical polymerization of N-acryloyloxysuccinimide (NAS) by reversible addition fragmentation chain transfer (RAFT). We synthesized activated homopolymers in a range of molecular weights with polydispersities between 1 and 1.2. The attachment of an inhibitory peptide to the activated polymer backbone yielded a potent controlled molecular weight polyvalent inhibitor of anthrax toxin. To provide greater control over the placement of the peptides along the polymer backbone, we also used a semi-batch copolymerization method to synthesize copolymers of NAS and acrylamide (AAm). This approach enabled the synthesis of copolymers with control over the placement of peptide-reactive NAS monomers along an inert backbone; subsequent functionalization of NAS with peptide yielded well-defined polyvalent anthrax toxin inhibitors that differed in their potencies. These strategies for controlling molecular weight, ligand density, and ligand placement will be broadly applicable for designing potent polyvalent inhibitors for a variety of pathogens and toxins, and for elucidating structure-activity relationships in these systems.

Figure 1

(a) Polymerization of NAS by RAFT. i: 3-benzylsulfanylthiocarbonylsulfanylpropionic acid, DMF; ii: AICV, DMF, 70 °C, 2.5 h; iii: AIBN, DMF, 90 °C, 1.5 h. (b) Plot indicating monomer conversion as a function of time for NAS polymerization at [M]/[CTA] values of 100 (◆), 200 (●), 300 (■), and 400 (▲).

Figure 2

Inhibitory activity of polyvalent inhibitors of anthrax toxin derived from controlled molecular weight homopolymer, pNAS. (a) Inhibition of the binding of the anthrax lethal factor, (b) Inhibition of toxin-induced cytotoxicity at various concentrations of polymeric inhibitors with DP = 80 (□) and DP =120 (●). Control polymer (polyacrylamide) did not show any inhibitory activity at concentrations tested (△).

Figure 3

Characterization of polyvalent inhibitors based on poly(AAm-co-NAS) copolymers. Characterization of molecular weight during semi-batch RAFT copolymerization for polymers with an average spacing of (a) 31 and (b) 18 units between adjacent NAS blocks. (c) The inhibitory activity of the resulting peptide-functionalized polyvalent inhibitors in a cytotoxicity assay.

Scheme 1

Design of polyvalent inhibitors with control over molecular weight and ligand spacing. The linear polyvalent inhibitors displaying peptides (black ovals) are shown bound to the PA₆₃ heptamer at the peptide-binding sites (circles). The spacing between peptides on the linear scaffold is either too short (left panel) or is sufficient (right panel) to allow a polyvalent interaction.