Anfinsen’s famous experiment showed that the restoration of catalytic activity of a completely unfolded ribonuclease A is only possible when the correct order of events is followed during the refolding process. Inspired by this work, the effect of structural constraints induced by covalent cross-links on the folding of a synthetic polymer chain via hydrogen-bonding interactions is investigated. Hereto, methacrylate-based monomers comprising either benzene-1,3,5-tricarboxamide (BTA)-based or coumarin-based pendants are copolymerized with n-butyl methacrylate in various ratios via reversible addition–fragmentation chain-transfer (RAFT) polymerization. To assess whether the folding and single-chain polymeric nanoparticle (SCPN) formation depend on the order of events, we compare two folding pathways. In the one case, we first covalently cross-link the coumarin pendants within the polymers in a solvent that prevents hydrogen bonding, after which hydrogen bonding is activated, inducing folding of the polymer. In the other case, we induce hydrogen-bonding interactions between tethered BTAs prior to covalent cross-linking of the coumarin pendants. A combinationof circular dichroism (CD) spectroscopy, UV–vis spectroscopy,size-exclusion chromatography (SEC), and dynamic light scattering(DLS) is employed to understand the effect of the structural constraintson the folding behavior of these synthetic polymers. The results showthat like in ribonuclease A, the order of events matters greatly anddetermines the outcome. Importantly, a hydrogen-bond-promoting solventprevents the formation of SCPNs upon covalent cross-linking and resultsin multichain aggregates. In contrast, covalently cross-linking thepolymer when no hydrogen bonds are present followed by inducing hydrogenbonding favors the formation of SCPNs above the UCST of the methacrylate-basedpolymer. To our surprise, the two systems show a fundamentally differentresponse to changes in temperature, indicating that also in syntheticpolymers differences in the folding pathway induce differences inthe properties of the resultant nanostructures.
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