Protein nanoreactors and native enzymes for controlled/living radical polymerization

The present PhD thesis entitled ‚Protein Nanoreactors and Native Enzymes for Controlled/Living Radical Polymerization‘ began with the hypothesis that protein-catalyst conjugates are able to beneficially influence controlled/living radical polymerization, i.e. atom transfer radical polymerization (ATRP). The general motivation for this work is driven by problems that occur when transition metal catalyst are used for ATRP. These most commonly used catalyst are only biocompatible to a limited extend and the resulting polymers show unwanted coloration due to the remaining catalyst. Moreover, by conjugating the catalyst into the cavities of a protein cage, we could gain insights into the catalytic mechanism of ATRP as well as into effects of the confined space. Polymer chemistry in particular enables us to perform successful research with proteins since synthetic strategies in a biocompatible environment, i.e. aqueous solution are well established.
Inspired by problems addressed earlier in our research group, we developed a robust conjugation and purification method to attach transition metal catalysts to proteins or enclose them into protein cages using bis-aryl hydrazone linker chemistry. Successively, we determined good performing ATRP conditions that allowed for significant lower catalyst concentrations. Thus, activators regenerated by electron transfer (ARGET) ATRP was used to constantly regenerate the catalyst into its active form by the means of a reducing agent.
We described one of these protein-catalyst conjugates in full detail. The ATRP catalyst was conjugated to the globular protein bovine serum albumin (BSA) and the complex was extensively characterized using biological and physical methods. The resulting conjugate was used to polymerize N-isopropyl acrylamide (NiPAAm) and poly(ethylene glycol) methyl ether acrylate (PEGA) in aqueous solution and was subsequently analyzed upon its structural integrity after polymerization. The ARGET ATRP of NiPAAm and PEGA yielded polymers with a moderate control over the molecular weight and the polydispersity of the polymers. However, our focus was to reduce the residual copper in polymers. Thus, BSA that served as a functional handle was used to remove the copper containing catalyst effectively from solution. We showed a reduction of residual copper to ppb levels, either by precipitation or by Dynabead removal.
Further, our findings showed that some metalloproteins can mediate ATRP. Enzymes have been introduced into synthetic chemistry as green and very selective alternatives to conventional catalysts. In polymer chemistry enzymes have been used as catalysts for polycondensation, ring-opening polymerizations, free radical polymerizations of vinyl-type monomers and the polymerization of aromatic compounds by radical-induced oxidative coupling. However, controlled/living radical polymerizations catalyzed by enzymes have not been exploited. Our results and the ones from di Lena represent the first reports of biocatalytic, controlled/living radical polymerization. Bringing those enzymes into organic solution, e.g. by conjugation of end-group-reactive polymers such as PEG, poly(oxazolines) and amphiphilic blockcopolymers to the surface-exposed lysines or cysteins of the enzymes, could lead to interesting new routes towards environmentally friendly catalysis, functional materials or functional nanosystems.
The group II chaperonin thermosome (THS) from the archaea Thermoplasma acidophilum is reported as protein nanoreactor for ATRP. For that purpose, a copper catalyst was entrapped into the THS. The confined space within the protein nanoreactor favorably influenced the polymerization of NiPAAm and PEGA under ARGET ATRP conditions in comparison to polymerizations carried out with the globular protein BSA. This concept was adapted and instead of the copper-complex, we covalently entrapped an enzyme as ATRP catalyst. We demonstrated that the space constriction in the THS has similar effects on the final polymer product, as shown for THS-LxCu. Protein nanoreactors with encapsulated enzymes, i.e. ATRPases, could help to understand the reaction mechanisms behind ATRPases.
Further, we described a way to render protein cages, i.e. THS, gated nanoreactors. The gated behavior of THS driven by the hydrolysis of ATP and ATP analogues was shown by enzymatic assays. The incorporation of HRP into THS allowed to study the opening and closing of the cage by converting the non-fluorescent dihydro rhodamine 6G into its fluorescent form rhodamine 6G. In addition, nanomechanical sensing with cantilever arrays measured the surface stress induced by the opening and closing of the protein cage in response to ATP analogues. The field of gated nanoreactors is still in its infancy. Thus, gated nanoreactors could be used in the areas of medicine, sensing, synthesis of drugs and other chemical products, and as analytical tools to study reaction mechanisms in confined volumes.