Synthesis and characterisation of novel molecularly imprinted nanogels with aldolase Type I activity

Abstract

This research project focussed on using the molecular imprinting approach for the synthesis of nanogels capable of catalysing the cross-aldol reaction between 4-nitrobenzaldehyde and acetone. A polymerisable proline derivative was used as the functional monomer to mimic the enamine-based mechanism of aldolase type I enzymes. A 1,3-diketone template, used to create the cavity, was designed to imitate the intermediate of the aldol reaction and was bound to the functional monomer using a reversible covalent interaction prior to polymerisation. Soluble imprinted nanogels were prepared by using a high-dilution radical polymerisation, which was followed by template removal and estimation of active site concentrations by monitoring the release of 4-nitrophenolate due to the acetylation of proline units with 4-nitrophenyl acetate. Analysis by DLS and TEM confirmed an average particle size of 20 nm for the nanogel preparations, comparable to the size of natural enzymes, and an average molecular mass ranging from 258 to 288 kDa, as determined by GPLC using polymethylmethacrylate standards. The polymers were found to be soluble in DMF, DMSO and mixtures thereof, giving rise to homogeneous solutions. The kinetic characterisation of both imprinted and non-imprinted nanogels was carried out with catalyst concentrations between 0.7 and 3.5% mol. Imprinted nanogel MIP-AS147 was found to have a kcat value of 0.26 × 10-2 min-1, the highest value ever achieved with imprinted nanogels catalysing C-C bond formation. Comparison of the catalytic constants for both imprinted nanogel MIP-AS147 and non-imprinted nanogel NIP-AS133 gave a ratio of kcatMIP /kcatNIP = 20, which is indicative of good imprinting efficiency and highlights the significance of the template during the imprinting process. Analysis of the stereoselectivity of the reaction catalysed by nanogels gave 62% ee, which is comparable with the one of the “free” catalyst. This work is the first and so far only example of imprinted polymer capable of catalysing the cross-aldol addition. The kinetic data obtained provide additional evidence to the hypothesis that the nanogel format is superior to ‘bulk’ polymers for the generation of enzymelike catalysts.