REGULATION OF PROTEIN BINDING ACTIVITIES OF MOLECULARLY IMPRINTED POLYMER BY SINGLE OR MULTI-STEP POST-IMPRINTING MODIFICATIONS

Molecularly imprinted polymers (MIPs) are synthetic polymer receptors and expected to be alternatives of naturally occurring materials such as antibodies and enzymes because of their intrinsic stability, simple procedures of preparation and a wide range of choice of monomers. Conventionally, MIPs are prepared by copolymerization of a functional monomer(s), a comonomer(s) and a crosslinker(s) in the presence of target molecule [1]. Recently, we have developed a new strategy for preparing multifunctional MIPs, so-called post-imprinting modifications (PIMs), which inspired by post-translational modifications for protein bio-synthesis [2]. PIM was designed as a site-directed chemical modification of functional monomer residues within a cavity. Functional monomers bearing a modifiable moiety (e.g. disulphide bond or imine bond) were used to prepare MIPs. As PIMs, the functional monomer residues in imprinted cavities formed after the removal of the template molecule, were transformed via disulphide exchange reaction or formation of imine bond, resulting in the MIP with desired functions such as fluorescent signalling ability and photo-responsibility.

In this study, we demonstrated the regulation of protein binding activities of MIPs by single or multi-step PIMs. For the PIM treatment, a functional monomer containing a disulphide bond as a modifiable moiety, ({[2-(2-Methacrylamido)-ethyldithio]-ethylcarbamoyl}-methoxy)-acetic acid (MDTA)[3,4] was designed and copolymerized with acrylamide and N, N’-methylenebisacrylamide in the presence of a target protein. After reduction of the disulphide linkage derived from MDTA, various functional groups including carboxy, amino, sulfonate and ethyleneoxide group were introduced by disulphide exchange reaction using the PIM reagents bearing a pyridyldisulphide moiety, which can react with a thiol group under mild conditions. Protein binding activity of PIM-treated polymers was examined by surface plasmon resonance sensing system. The reversible change of protein binding activity of MIP was confirmed by exchanging the functional groups. Furthermore, multi-step PIMs introducing plural functional groups into the cavities were demonstrated on the improvement of protein selectivity. Detailed characteristics in the PIM-based MIPs will be discussed in this presentation.

References

[1] Takeuchi T, Hayashi T, Ichikawa S, Kaji A, Masui M, Matsumoto H, Sasao R (2016) Chromatography 37, 43-64. [Open Access].

[2] Takeuchi T, Sunayama H, Takano E, Kitayama Y (2015) Post-imprinting and In-Cavity Functionalization in Molecularly Imprinted Polymers in Biotechnology, Mattiasson B, Ye L (Eds), Springer Berlin Heidelberg: 95-106.

[3] Sunayama H, Takeuchi T (2014) Molecularly imprinted protein recognition cavities bearing exchangeable binding sites for postimprinting site-directed introduction of reporter molecules for readout of binding events. ACS Applied Materials and Interfaces 6: 20003-20009.

[4] Sunayama H, Kitayama Y, Takeuchi T (2018) Regulation of protein binding activities of molecularly imprinted polymers via post-imprinting modifications to exchange functional groups within the imprinted cavity. Journal of Molecular Recognition 31: e2633.