Reversible coordinative binding of Lewis basic donors to iminodiacetato (IDA) and nitrilotriacetato (NTA) metal complexes is widely used for the design of synthetic receptors binding to peptides, proteins or enzymes at physiological conditions. However, no data on the affinity of M(II)-NTA (M = Cu, Ni, Zn) to a single histidine or imidazole moiety are available. We herein report (chapter 1) the investigation of the binding affinity and thermodynamics of copper(II), nickel(II) and zinc(II) NTA complexes to histidine, imidazole and hen egg white lysozyme, bearing a single surface exposed histidine unit, by isothermal titration calorimetry at physiological conditions. Further, we describe a peptide-metal complex hybrid approach to enhance the binding affinity of Cu(II)-NTA to lysozyme.
In the second part of this work (chapter 2) the rationally design of a selective peptide receptor is described. The combination of copper(II)nitrilotriacetato (NTA) complex with an ammonium-ion sensitive and luminescent benzocrown ether revealed a peptide receptor with a micromolar affinity and selectivity for glycine and histidine containing peptide sequences. This affinity closely resembles that of copper(II) ion peptide binding: The two free coordination sites of the copper(II) NTA complex bind to imidazole and amido nitrogen atoms, retracing the initial coordination steps of non-complexed copper(II) ions. The benzocrown ether recognizes intramolecularly the N-terminal amino moiety and the significantly increased emission intensity signals the binding event, as only if prior coordination of the peptide has taken place, the intramolecular ammonium ion � benzocrown ether interaction is of sufficient strength in water to trigger an emission signal. Intermolecular ammonium ion � benzocrown ether binding is not observed. Isothermal titration calorimetry confirmed the binding constants derived from emission titrations. Thus, as deduced from peptide coordination studies, the combination of a truncated copper(II) coordination sphere and a luminescent benzocrown ether allows for the more rational design of sequence selective peptide receptor. Suitable combinations of an affinity tag and an artificial probe are useful for non-covalent protein labelling. Several of such peptide tag � probe pairs have been developed and reported in the literature. The most prominent example is the His-tag � Ni(II)-NTA (nitrilotriacetic acid) pair. Recently, the Hamachi group reported a genetically encodable oligo-aspartate sequence (D4-tag) and a corresponding oligonuclear Zn(II) dipicolyl¬amine (Zn(II)-Dpa) complex as new peptide tag � probe pair, which is orthogonal to the His-tag � Ni(II)-NTA pair. We describe in the third part of this work (chapter 3) the preparation of fluorescent 1,4,7,10-tetraazacyclododecane (cyclen) Zn(II) complexes and their application as an alternative artificial probe for the D4-tag system. The binding affinities of the new complexes to the affinity tags were investigated by emission and UV-vis titration. Tetranuclear Zn(II)-cyclen complexes respond to the presence of oligo-aspartate, oligo-glutamate and oligo-aspartate dimers in aqueous solution at micromolar concentrations by a strong spectroscopic change. Based on the high binding affinities due to strong electrostatic interactions and Job´s plot analysis, we propose the formation of receptor�peptide tag aggregates. The results clearly show the potential of Zn(II)-cyclen complexes for applications as non-covalent protein markers, although their optical properties require further optimization for practical use.
The fourth part of this work (chapter 4) deals with the syntheses of new amphiphilic 1,4,7,10-tetraazacyclododecane Zn(II) complexes for a template guided cooperative self-assembly of nucleotides at interfaces fabricated by combination of self-assembly monolayer technique (SAM) and Langmuir Blodgett technique (LB). Three amphiphilic Zn(II)-cyclen complexes were synthesized as binding sites at interfaces prepared by a combination of SAM and LB film approaches or in vesicles. Detailed investigations of the binding properties of surfaces incorporating the new amphiphilic complexes are in progress.
The fifth part of this work (chapter 5) deals with the preparation of self assembled vesicular polydiacetylene (PDA) particles with embedded metal complex receptor sites. The particles respond to the presence of ATP and PPi (pyrophosphate) in buffered aqueous solution by visible changes of their color and emission properties. Blue PDA vesicles of uniform size were obtained upon UV irradiation from mono- and dinuclear zinc(II)-cyclen and iminodiacetato copper [Cu(II)-IDA] modified diacetylenes, embedded in amphiphilic diacetylene monomers. Addition of ATP and PPi to the PDA vesicle solution induces a color change from blue to red observable by the naked eye. The binding of ATP and PPi changes the emission intensity. Other anions like ADP, AMP, H2PO4¯, CH3COO¯, F¯, Cl¯, Br¯ and I¯ failed to induce any spectral changes. The zinc(II)-cyclen nanoparticles are useful for the facile detection of PPi and ATP in millimolar concentrations in neutral aqueous solutions, while Cu(II)-IDA modified vesicular PDA receptors are able to selectively discriminate between ATP and PPi.
In the sixth chapter (chapter 6) we report a new methodology for the preparation of a artificial phosphate receptors. Phosphate anion probes typically consist of a binding site and a luminescent reporter group. The luminescent moiety is either part of the chemosensor in close proximity of the analyte binding site or in indicator displacement assays non-covalently bound to the binding site and displaced by the analyte. We report here the preparation and binding properties of 80 nm vesicular synthetic receptors, which contain amphiphilic 1,4,7,10-tetraazacyclododecane (cyclen) Zn(II) complexes as phosphate anion binding sites and amphiphilic coumarin derivatives as fluorescent reporter groups. By colocalization of binding sites and reporter groups in the vesicle they respond to the presence of phosphate anions in aqueous solution at micromolar concentrations by a strong emission decrease. The technique avoids the covalent synthesis of labelled analyte binding sites and allows the rapid and versatile preparation of luminescent nanometer size synthetic receptors.
In the last part of this work (chapter 7) the syntheses of new non-fluorescent and fluorescent amphiphilic Lewis acidic metal complexes (metal chelating artificial lipids) based on 1,4,7,10-tetraazacyclododecane Zn(II) complexes, dipicolylamine (Dpa) complexes and a nitrilotriacetato acid (NTA) complex are described. The prepared metal chelating artificial lipids will be used for fabrication of complex self-assembled supramolecular surfaces by one or more different chemosensors for molecular recognition at interfaces
