Internationale Ausgabe: DOI: 10.1002/anie.201612667 Molecularly Imprinted Polymers Deutsche Ausgabe: DOI: 10.1002/ange.201612667 Enzyme-Initiated Free-Radical Polymerization of Molecularly Imprinted Polymer Nanogels on a Solid Phase with an Immobilized Radical Source Mira Daoud Attieh, Yi Zhao, Assem Elkak, Aude Falcimaigne-Cordin,* and Karsten Haupt* Abstract: An enzyme-mediated synthetic approach is de- scribed for the preparation of molecularly imprinted polymer nanoparticles (MIP-NPs) in aqueous media. Horseradish peroxidase (HRP) was used to initiate the polymerization of methacrylate or vinyl monomers and cross-linkers by catalyz- ing the generation of free radicals. To prevent entrapment of the enzyme in the cross-linked polymer, and to enable it to be reused, HRP was immobilized on a solid support. MIPs based on 4-vinylpyridine and 1,4-bis(acryloyl)piperazine for the recognition of 2,4-dichlorophenoxyacetic acid (2,4-D) and salicylic acid were synthesized in an aqueous medium. MIPs for the protein trypsin were also synthesized. MIP nano- particles with sizes between 50 and 300 nm were obtained with good binding properties, a good imprinting effect, and high selectivity for the target molecule. The reusability of immobi- lized HRP for MIP synthesis was shown for several batches. Molecular imprinting is a rapidly developing technique for the preparation of functional polymers with specific molec- ular-recognition properties. These materials, sometimes re- ferred to as “plastic antibodies”, result from the copolymer- ization of appropriate functional and cross-linking monomers in the presence of a template molecule. The subsequent removal of the template reveals binding cavities in the polymeric matrix that are capable of specifically recognizing and binding the template or a structurally very similar target molecule. [1, 2] Owing to this specificity but also to their physical and chemical stability, MIPs have been used as antibody mimics in many applications, such as immuno- assays, [3] sensing, [4] bioseparation, [5] catalysis, and more recently, drug delivery [6] and even as drugs. [7] Free-radical polymerization (FRP) is the most common technique used for MIP synthesis. FRP is normally triggered by the photochemically, thermally, or electrochemically induced decomposition of a chemical initiator, depending on the nature of the initiator. Controlled/living radical polymerization methods, such as atom transfer radical polymerization (ATRP) [8] and reversible addition–fragmen- tation chain transfer (RAFT) polymerization, [9] have been also applied to the synthesis of MIPs, thus allowing improve- ment of their molecular-recognition properties and enabling the synthesis of hierarchical materials and nanocompo- sites. [10, 11] However, all these radical polymerization tech- niques employ toxic reagents for the initiation step or lead to the incorporation of toxic by-products into the polymeric network. Even in small amounts in polymers, these toxic compounds and moieties will greatly limit the use of the material in biomedical, pharmaceutical, cosmetics, and envi- ronmental applications, which require a high level of safety and biocompatibility. Furthermore, many companies and producers of cosmetic products want to avoid even a theoret- ical risk, and therefore will forbid the use of certain precursors, even if they can be completely eliminated from the final product, and even if that product is shown to be nontoxic. In the context of cross-linked polymer nanoparticles, such as MIPs, self-initiated polymerization without initiators is possible in some cases, although this method is not compatible with all monomers. [12] To broaden the application of these materials, we propose herein a novel strategy to produce MIPs in aqueous media by a “greener” process, in which molecularly imprinted polymer nanoparticles (MIP-NPs) are synthesized by enzyme-mediated free-radical polymerization; an immobilized horseradish peroxidase (HRP) is used to catalyze the formation of free radicals (Figure 1 A). Indeed, enzymes are an environmentally friendly and nontoxic alternative to the traditional chemical and metal catalysts used as initiators for free-radical or controlled/living radical polymerization techniques. Several oxidoreductases, for example, laccases and peroxidases, have been used to produce polymers from vinyl and aromatic compounds. [13–15] These enzymes are transition-metal-containing oxidases that cata- lyze electron-transfer reactions, which generate radicals capable of initiating polymerization. Among them, HRP has attracted particular attention and has been employed pre- dominantly as a catalyst for free-radical polymerization. [13] Its natural function in plants is defense against pathogens through the formation of reactive radical species. [14] Although HRP has been used for the synthesis of hydrogels by cross- linking biopolymers [15] and for the synthesis of multilayer materials, [16] the direct synthesis of cross-linked polymer particles, such as MIPs and other microgels, by radical polymerization has not been reported. The main problem with cross-linked polymers is the possible entrapment of the initiator enzyme. We propose to solve this problem by the use [*] Dr. M. DaoudAttieh, Dr. Y. Zhao, Dr. A. Falcimaigne-Cordin, Prof. K. Haupt Sorbonne UniversitØs—UniversitØ de Technologie de Compigne CNRS Enzyme and Cell Engineering Laboratory CS 60319, 60203 Compigne cedex (France) E-mail: aude.cordin@utc.fr karsten.haupt@utc.fr Dr. M. Daoud Attieh, Prof. A. Elkak UniversitØ Libanaise, Laboratoire de Valorisation des Ressources Naturelles et Produits de SantØ, CitØ Universitaire Rafic Hariri Hadath (Lebanon) Supporting information for this article can be found under: http://dx.doi.org/10.1002/anie.201612667. Angewandte Chemie Zuschriften 3387 Angew. Chem. 2017, 129, 3387 –3391 # 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim