Abstract:Molecularly imprinted polymers (MIPs) have the potential to complement antibodies in bioanalysis, are more stable under harsh conditions, and are potentially cheaper to produce. However, the affinity and especially the selectivity of MIPs are in general lower than those of their biological pendants. Enzymes are useful tools for the preparation of MIPs for both low and high-molecular weight targets: As a green alternative to the well-established methods of chemical polymerization, enzyme-initiated polymerization has been introduced and the removal of protein templates by proteases has been successfully applied. Furthermore, MIPs have been coupled with enzymes in order to enhance the analytical performance of biomimetic sensors: Enzymes have been used in MIP-sensors as "tracers" for the generation and amplification of the measuring signal. In addition, enzymatic pretreatment of an analyte can extend the analyte spectrum and eliminate interferences.

Isothermal titration calorimetry (ITC) is widely used to study protein-ligand, DNA-drug and/or protein-protein interactions but its application for small molecule complexation remains limited namely when the titration is performed in organic solvents. Compared to other dedicated spectroscopic techniques like nuclear magnetic resonance, infrared spectrometry or fluorimetry, which require a series of experiments to extract site-specific stoichiometry and affinity information, ITC provides in a single experiment a complete thermodynamic picture of the overall interaction mechanism. This chapter presents examples that support the high potential of ITC to probe interactions between small molecules in methanol, acetonitrile and methanol/water mixture on a Nano ITC Low Volume device (TA Instruments), with an emphasis on both simple (1:1) and more complex (1:1 and 1:2) interaction mechanisms.

Rutin was enzymatically acylated with hexadecanedioic acid, in tert-amyl alcohol, by an immobilized lipase from Candida antartica "Novozym 435". The effect of different techniques of water removal, temperature, concentration of rutin and diacid/rutin molar ratio was investigated. The obtained results indicated that drying the media by adding the molecular sieves in the outer loop of the reactor was the most efficient method leading to water content lower than 200 ppm. The highest performances (conversion yield and initial rate) were reached at 90°C, 131 mM of rutin, and 118 mM of acid. Depending on the water content and the diacid/rutin molar ratio, only rutin 4‴-hexadecanedioate or both rutin 4‴-hexadecanedioate and dirutin 4‴, 4‴-hexadecanedioate were synthesized.