Modification of methylaluminoxane-activated ansa-zirconocene catalysts with triisobutylaluminum-transformations of reactive cations studied by NMR spectroscopy

When triisobutylaluminum (AliBu(3)) is added to solutions containing methylaluminoxane (MAO) and rac-[Me(2)Si(ind)(2)ZrCl(2)] (ind: indenyl) in C(6)D(6), NMR spectra show that methyl-bridged mixed-alkylaluminum dimers Al(mu-Me)(2)Me(4-x)iBu(x) predominate. These dimers react with MAO under partial transfer of isobutyl groups and induce a conversion of the initially prevailing cationic trimethylaluminum adduct rac-[Me(2)Si(ind)(2)Zr(mu-Me)(2)AlMe(2) (+)] to rac-[Me(2)Si(ind)(2)Zr(mu-Me)(2)AlMeiBu(+)] and rac-[Me(2)Si(ind)(2)Zr(mu-Me)(2)AliBu(2) (+)]. These species are unstable and release isobutene under formation of zirconocene hydrides.     

The interaction of highly helical structural mutants with the NOP receptor discloses the role of the address domain of nociceptin/orphanin FQ

Nociceptin is a heptadecapeptide whose sequence is similar to that of Dynorphin A, sharing a message domain characterized by two glycines and two aromatic residues, and a highly basic C-terminal address domain but, in spite of these similarities, displays no opioid activity. Establishing the relative importance of the message and address domains of nociceptin has so far been hampered by its extreme conformational flexibility. Here we show that mutants of this peptide, designed to increase the helical content in the address domain, can be employed to explain the mode of interaction with the NOP receptor. Nociceptin analogues in which Ala residues are substituted with aminoisobutyric acid (Aib) show a substantial increment of activity in their interaction with the NOP receptor. The increment of biological activity was attributed to the well-documented ability of Aib to induce helicity. Here we have verified this working hypothesis by a conformational investigation extended to new analogues in which the role of Aib is taken up by Leu. The NMR conformational analysis confirms that all Ala/Aib peptides as well as [Leu(7,11)]-N/OFQ-amide and [Leu(11,15)]-N/OFQ-amide mutants (N/OFQ=nociceptin/orphanin FQ) have comparable helix content in helix-promoting media. We show that the helical address domain of nociceptin can place key basic residues at an optimal distance from complementary acidic groups of the EL(2) loop of the receptor. Our structural data are used to rationalize pharmacological data which show that although [Leu(11,15)]-N/OFQ-amide has an activity comparable to those of Ala/Aib peptides, [Leu(7,11)]-N/OFQ-amide is less active than N/OFQ-amide. We hypothesize that bulky residues cannot be hosted in or near the hinge region (Thr(5)-Gly(6)-Ala(7)) without severe steric clash with the receptor. This hypothesis is also consistent with previous data on this hinge region obtained by systematic substitution of Thr, Gly, and Ala with Pro.     

NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors

Binding events of ligands to receptors are the key for an understanding of biological processes. Gaining insight into protein-protein and protein-ligand interactions in solution has recently become possible on an atomic level by new NMR spectroscopic techniques. These experiments identify binding events either by looking at the resonance signals of the ligand or the protein. Ideally, both techniques together deliver a complete picture of ligand binding to a receptor. The approaches discussed in this review allow screening of compound libraries as well as a detailed identification of the groups involved in the binding events. Also, characterization of the binding strength and kinetics is possible, competitive binding as well as allosteric effects can be identified, and it has even been possible to identify ligand binding to intact viruses and membrane-bound proteins.     

SAR by ILOEs: an NMR-based approach to reverse chemical genetics

Reverse chemical genetics is an emerging technique that makes use of small molecule inhibitors to characterize how a protein functions. In this regard, we have developed an NMR-based approach (SAR by ILOEs) that enables the identification of high affinity ligands for a given protein target without the need of a specific assay. Our approach is of general applicability and could result very powerful in reverse chemical-genetics studies, target validation, and lead discovery. We report a recent application on the design and synthesis of compounds that inhibit protein-membrane interactions.     

Rapid screening of binding constants by calibrated competitive 1H NMR spectroscopy

A calibrated competitive NMR method has been developed that is appropriate for the rapid screening of binding constants. This method involves the initial characterisation of a receptor-substrate binding event for which the (1)H NMR spectrum of a given receptor (calibrant) is modified by the substrate of interest at a range of concentrations. For all subsequent "unknown" receptors, K(a) values are then determined by using a competition assay (in the presence of the calibrant receptor) by measuring a single standard (1)H NMR spectrum. This enables a rapid assessment of the recognition properties of a library of potential receptors. Only the calibrant receptor needs to be NMR active, while the library of putative receptors, as well as the substrate, can be NMR silent. This method assumes the formation of complexes of 1:1 stoichiometry. To demonstrate this methodology, the binding of a number of crown ether type compounds with K+ ions has been studied. Comparison of the binding strengths obtained by using this approach with those in the literature shows excellent agreement. A range of new compounds that have recently been synthesised within our group has also been screened in order to illustrate how this approach can rapidly assess binding ability. This method has significance for chemists working in the fields of combinatorial receptor/substrate design and supramolecular chemistry as a means of rapid optimisation of binding strength.     

Structural studies of {Li} 2-lithiopyrrolidines using NMR spectroscopy

A selection of N-substituted 2-lithiopyrrolidines were prepared and their structures were investigated by ⁶Li and ¹³C NMR spectroscopy. Evidence is presented for aggregation and dynamic solvation effects, depending on the nature of the N-substituent and substituents on the pyrrolidine ring. Studies were performed with N-Boc (coordinating carbonyl group), N-methoxyethyl (coordinating methoxy group) and N-alkyl (no coordinating group) heterocycles to represent three different classes of organolithiums: dipole-stabilized, unstabilized and chelated, and unstabilized.