An approach to peptide-based ATP receptors by a combination of random selection, rational design, and molecular imprinting.

Random selection, rational design and molecular imprinting were cooperatively utilized to develop peptide-based ATP synthetic receptors. In this fusion strategy, combinatorial chemistry was utilized for screening a precursor peptide useful for construction of ATP receptors, and rational design was employed in modification of the selected precursor peptide for higher affinity and selectivity. Finally, molecular imprinting was used for pre-organizing the conformation of the precursor peptide as complementary to a target molecule ATP. The fusion strategy appeared to have advantage to sole use of the individual strategy: (1) a low hit-rate of combinatorial chemistry will be improved by customizing a higher order structure of a selected peptide by molecular imprinting, (2) combinatorial chemistry allows us to semi-automatically select components of water-compatible synthetic receptors, (3) rational design improves the selected peptide sequence for better molecularly imprinted receptors. A peptide consisting of a randomly selected sequence and a rationally designed sequence (Resin-Lys-Gly-Arg-Gly-Lys-Gly-Gly-Gly-Glu-Lys-Tyr-Leu-Lys-NHAc) was designed and synthesized as a precursor peptide. The rational design was made according to the sequence of the adenine binding site of biotin carboxylase. The on-beads peptide was cross-linked with dimethyl adipimidate in the presence of ATP. In the saturation binding tests, the cross-linked on-beads peptide showed 5.3 times higher affinity compared to the non-cross-linked peptide with the same sequence. Furthermore, the cross-linked peptide showed improved selectivity; the ratios of binding constants, K((ATP))/K((ADP)) and K((ATP))/K((GTP)), were increased from 2.4 to 19, and from 0.8 to 10, respectively. It would be notable that the peptide without the rationally designed sequence showed no discrimination between ATP and GTP (K((ATP))/K((GTP)) as 0.9), suggesting that the rationally designed site was successfully engaged for recognition of the adenine base.

Face-to-face porphyrin moieties assembled with spacing for pyrazine recognition in molecularly imprinted polymers.

A strategy for arranging two porphyrin moieties in a face-to-face fashion in polymeric material was demonstrated by molecular imprinting, whereby porphyrin Zn(II) complex monomers were cross-linked with ethylene glycol dimethacrylate in the presence of pyrazine or 1,5-naphthyridine as a template molecule. In chromatographic studies using the resultant imprinted polymers as stationary phase, both the polymers showed selectivity for the original template molecule, suggesting that two zinc porphyrin moieties were immobilized in the face-to-face fashion, and were center-aligned for pyrazine recognition and offset-arranged for 1,5-naphthyridine recognition. The imprinted polymer with porphyrin moieties also showed a decrease in its fluorescence intensity in response to the concentration of the target molecule, suggesting the potential utility as sensing material.

Molecularly imprinted nanocomposites for highly sensitive SPR detection of a non-aqueous atrazine sample.

A surface plasmon resonance (SPR) sensor highly sensitive and selective for the herbicide atrazine was composed by immobilizing atrazine-imprinted polymer with gold nanoparticles on a gold thin film as a sensor chip. In the detection, the atrazine-imprinted polymer was expected to work as synthetic receptor for selectively capturing atrazine in organic solvent, and the gold nanoparticles were expected to exhibit a coupling effect with the gold thin film to enhance the local electromagnetic field between the nanoparticles and the gold film, making the sensor chip highly sensitive for changes in microenvironmental polarity. Thus, a combination of the atrazine-imprinted polymer and gold nanoparticles enabled us to compose an SPR sensor demonstrating the detection of 5 pM atrazine in acetonitrile.

Molecular imprinting under molecular crowding conditions: an aid to the synthesis of a high-capacity polymeric sorbent for triazine herbicides.

Molecular crowding, an important feature of the molecular environments in biological cells, was applied to the synthesis of antibody-mimic polymers selective for a group of biologically active compounds, the triazine herbicides. Synthesis of these polymers was conducted using molecular imprinting under molecular crowding conditions, whereby atrazine (a template molecule) was complexed with methacrylic acid (a functional monomer) in the presence of a macromolecular crowding agent (either poly(methyl methacrylate) (PMMA) or polystyrene (PS)) followed by cross-linking with ethylene glycol dimethacrylate. After removal of atrazine from the polymer matrix, the retention properties and selectivity of the resultant polymers were assessed by chromatographic tests. The addition of a crowding-inducing agent resulted in polymers with superior retention properties and excellent selectivity for triazine herbicides, as compared to polymers prepared without addition of a crowding-inducing agent. An imprinted polymer prepared in the presence of PS as a crowding agent exhibited a retention factor for atrazine an order of magnitude larger than that of an imprinted polymer prepared in the absence of a crowding agent. NMR results suggest that the crowding agent is capable of promoting hydrogen bond formation between atrazine and methacrylic acid, which could account for the effect of crowding on molecular imprinting.

Molecularly-imprinted polymeric logic gates selective for predetermined chemical input species.

Selective AND and OR logic gates were fabricated by molecular imprinting with simultaneous use of two kinds of template species, with the resultant binding behaviour (AND or OR) of the imprinted polymers governed by intermolecular interactions between the two template species.

SPR sensor chip for detection of small molecules using molecularly imprinted polymer with embedded gold nanoparticles.

Molecularly imprinted polymer gel with embedded gold nanoparticle was prepared on a gold substrate of a chip for a surface plasmon resonance (SPR) sensor for fabricating an SPR sensor sensitive to a low molecular weight analyte. The sensing is based on swelling of the imprinted polymer gel that is triggered by an analyte binding event within the polymer gel. The swelling causes greater distance between the gold nanoparticles and substrate, shifting a dip of an SPR curve to a higher SPR angle. The polymer synthesis was conducted by radical polymerization of a mixture of acrylic acid, N-isopropylacrylamide, N,N'-methylenebisacrylamide, and gold nanoparticles in the presence of dopamine as model template species on a sensor chip coated with allyl mercaptan. The modified sensor chip showed an increasing SPR angle in response to dopamine concentration, which agrees with the expected sensing mechanism. Furthermore, the gold nanoparticles were shown to be effective for enhancing the signal intensity (the change of SPR angle) by comparison with a sensor chip immobilizing no gold nanoparticles. The analyte binding process and the consequent swelling appeared to be reversible, allowing one the repeated use of the presented sensor chip.

Synthetic cinchonidine receptors obtained by cross-linking linear poly(methacrylic acid) derivatives as an alternative molecular imprinting technique.

A molecular imprinting approach to construct synthetic receptors was examined, wherein a linear pre-polymer bearing functional groups for intermolecular interaction with a given molecule is cross-linked in the presence of the molecule as a template, and subsequent removal of the template from the resultant network-polymer is expected to leave a complementary binding site. Poly(methacrylic acid) (PMAA) derivatized with a vinylbenzyl group as a cross-linkable side chain was utilized as the pre-polymer for the molecular imprinting of a model template, (-)-cinchonidine. Selectivity of the imprinted polymer was evaluated by comparing the retentions of the original template, (-)-cinchonidine and its antipode (+)-cinchonine in chromatographic tests, exhibiting a selectivity factor up to 2.4. By assessment of the imprinted polymers in a batch mode, a dissociation constant at 20 degrees C for (-)-cinchonidine was estimated to be K (d) = 2.35 x 10(-6) M (the number of binding sites: 4.54 x 10(-6) mol/g-dry polymer). The displayed affinity and selectivity appeared comparable to those of an imprinted polymer prepared by a conventional monomer-based protocol, thus showing that the pre-polymer, which can be densely cross-linked, is an alternative imprinter for developing template-selective materials. (-)-Cinchonidine-imprinted polymers were prepared and assessed using the pre-polymers bearing different densities of the vinylbenzyl group and different amounts of the cross-linking agent to examine the appropriate density of the cross-linking side chain that was crucial for developing the high affinity and selectivity of the imprinted polymers.

Composite of Au nanoparticles and molecularly imprinted polymer as a sensing material.

A molecularly imprinted polymer with immobilized Au nanoparticles (Au-MIP) is reported as a novel type of sensing material. The sensing mechanism is based upon the variable proximity of the Au nanoparticles immobilized in the imprinted polymer, which exhibits selective binding of a given analyte accompanied by swelling that causes a blue-shift in the plasmon absorption band of the immobilized Au nanoparticles. Using adrenaline as the model analyte, it was shown that molecular imprinting effectively enhanced the sensitivity and selectivity, and accordingly, Au-MIP selectively detects the analyte at 5 microM. The combination of molecular imprinting and the Au nanoparticle-based sensing system was shown to be a general strategy for constructing sensing materials in a tailor-made fashion due to wide applicability of the imprinting technique and the independence of the sensing mechanism from the analyte recognition system.