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

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.

Thiol-mediated anchoring of ligands to self-assembled monolayers for studies of biospecific interactions.

We report a method to immobilize thiol-containing ligands onto self-assembled monolayers (SAMs) of alkanethiolates presenting chloracetylated hexa(ethylene glycol) groups. The chloroacetyl groups react with thiols under mild basic conditions, enabling the stable immobilization of biologically active ligands in a well-defined orientation. These SAMs on gold are well suited for studies of biospecific interactions of immobilized ligands with proteins and cells. As a demonstration, we functionalized these SAMs with thiol-containing derivatives of biotin and benzene sulfonamide and observed the specific binding of neutravidin and carbonic anhydrase, respectively. We also used this method to generate mixed SAMs presenting the Arg-Gly-Asp (RGD) peptide sequence and demonstrated the integrin-mediated adhesion of fibroblast cells to these SAMs. This approach would allow the immobilization of proteins and other sensitive biomolecules and ligands for a wide variety of applications in biotechnology.

Synthesis of polyvalent inhibitors of controlled molecular weight: structure-activity relationship for inhibitors of anthrax toxin.

We describe a novel method to synthesize activated polymers of controlled molecular weight and apply this method to investigate the relationship between the structure and activity of polyvalent inhibitors of anthrax toxin. In particular, we observe an initial sharp increase in potency with increasing ligand density, followed by a plateau where potency is independent of ligand density. Our simple strategy for designing polyvalent inhibitors of controlled molecular weight and ligand density will be broadly applicable for designing inhibitors for a variety of pathogens and toxins, and for elucidating structure-activity relationships in these systems. Our results also demonstrate a role for kinetics in influencing inhibitory potency in polyvalent systems. Finally, our work presents a synthetic route to polyvalent inhibitors that are more structurally defined and effective in vivo. This control over inhibitor composition will be generally useful for the optimization of inhibitor potency and pharmacokinetics, and for the eventual application of these molecules in vivo.

Synthesis of copolymers containing an active ester of methacrylic acid by RAFT: controlled molecular weight scaffolds for biofunctionalization.

We report the controlled radical copolymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with a monomer containing an active ester, N-methacryloyloxysuccinimide (NMS), by reversible addition fragmentation chain transfer (RAFT). The large difference in the reactivity ratios of HPMA and NMS resulted in significant variations in copolymer composition with increasing conversion during batch copolymerization. The use of a semi-batch copolymerization method, involving the gradual addition of the more reactive NMS, allowed uniformity of copolymer composition to be maintained during the polymerization. We synthesized polymers in a wide range of molecular weights (M(n) = 3000-50,000 Da) with low polydispersities (1.1-1.3). The effect of the ratio of monomer to chain transfer agent (CTA) on the molecular weight of the polymer was investigated. Given the numerous applications of poly(HPMA)-based conjugates in designing polymeric therapeutics, these controlled molecular weight activated polymers represent attractive scaffolds for biofunctionalization. As a demonstration, we attached a peptide to the activated polymer backbone to synthesize a potent controlled molecular weight polyvalent inhibitor of anthrax toxin.

Design of water-soluble, thiol-reactive polymers of controlled molecular weight: a novel multivalent scaffold.

Multivalent molecules, i.e. scaffolds presenting multiple copies of a suitable ligand, constitute an emerging class of nanoscale therapeutics. We present a novel approach for the design of multivalent ligands, which allows the biofunctionalization of polymers with proteins or peptides in a controlled orientation. It consists of the synthesis of water-soluble, activated polymer scaffolds of controlled molecular weight, which can be biofunctionalized with various thiolated ligands in aqueous media under mild conditions. These polymers were synthesized by ring-opening metathesis polymerization (ROMP) and further modified to make them water-soluble. The incorporation of chloride groups activated the polymers to react with thiol-containing peptides or proteins, and the formation of multivalent ligands in aqueous media was demonstrated. This strategy represents a convenient route for synthesizing multivalent ligands of controlled dimensions and valency.