Split-and-mix peptide libraries have been encoded by capping cleavable, laddered sequences with p-bromobenzoic acid, allowing rapid analysis of the cleaved ladder fragments by mass spectrometry. The encoding methodology has been applied to the synthesis of libraries of peptide receptors and a library of tripeptides functionalised with a bicyclic guanidinium unit has been successfully screened to identify binding partners for Val-Val-Ile-Ala, the C-terminal tetrapeptide sequence of the amyloid-β protein, Aβ(1–42). (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

A new macrocyclic receptor incorporating a thiourea moiety has been synthesised. Crystal structures of the macrocycle showed that the receptor has a rigid backbone but the thiourea moiety can orientate itself to bind to a DMSO solvent molecule. Force-field (MMFFs) calculations were performed to model the macrocycle and its binding properties with respect to N-protected amino acids, which were measured experimentally by NMR titration. Binding free energies were calculated by using the mode integration algorithm (MINTA) or free-energy perturbation (FEP). Excellent qualitative agreement with experiment was obtained. To further exploit the accuracy of the free-energy predictions for this system, the faster free-energy algorithm MINTA was used as a prediction tool to test the binding affinity of the macrocycle towards a series of several other amino acid derivatives, which speeded up considerably the screening process and reduced laboratory costs.

As a result of the accurate agreement between computation and experiment obtained using default forcefield parameters, the MMFFs forcefield together with a Monte Carlo conformational search method (MCMM/LMCS) and the MINTA free-energy calculation algorithm has been used to probe the enantioselective potential of a new macrocyclic receptor, hence saving time and money on costly experimental procedures. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Libraries of “unsymmetrical” tweezer receptors, featuring a guanidinium head group as a carboxylate binding site and two independently synthesized peptidic arms, have been prepared and screened to identify receptors for the N-Ac-Lys-D-Ala-D-Ala tripeptide sequence. The binding properties of one such receptor structure, with dye-labeled N-Ac-Lys-D-Ala-D-Ala, were investigated. These studies demonstrated that when attached to the solid-phase, the receptor binds dye-labeled N-Ac-Lys-D-Ala-D-Ala, in buffered aqueous media, with mM binding affinity.

A series of chiral bisthiourea macrocycles 1–4 have been prepared and their binding properties with various dicarboxylate salts have been examined by using NMR titration and isothermal calorimetry experiments. Macrocycle 1, in particular, favours the 1:1 binding of N-protected L-glutamate and aspartate, but favours 1:2 binding of the corresponding D-amino acids in polar solvents (dimethyl sulfoxide and acetonitrile). The macrocycles, however, do not bind carboxylates at all in the less competitive solvent chloroform. The binding properties of these macrocyles are sensitive to small structural changes as demonstrated by the altered binding properties of macrocycles 2–4 compared with 1.

Solvation of the macrocycle by polar solvents leads to strong binding of an N-protected excitatory amino acid neurotransmitter. A 1:1 complex forms highly enantioselectively between the macrocycle and N-Boc-L-glutamate (as the dicarboxylate anion, see picture) in CH₃CN and DMSO (with a larger entropic driving force for binding), but no binding occurs in the less competitive solvent CDCl₃.

Solvatation des Makrocyclus durch polare Lösungsmittel ermöglicht die starke Bindung eines N-geschützten anregenden Aminosäureneurotransmitters. Während sich in CH₃CN und DMSO mit hoher Enantioselektivität (und mit einer größeren entropischen Triebkraft zur Bindung) ein 1:1-Komplex aus Makrocyclus und N-Boc-L-glutamat (in Form des Dicarboxylatanions, siehe Grafik) bildet, beobachtet man keine Bindung im schwächer koordinierenden CDCl₃.

A library of “tweezer” receptors, incorporating a guanidinium “head group” and two peptide derived side arms has been prepared on the solid-phase using an orthogonally protected guanidinium scaffold 12. The library was screened with various tripeptide derivatives in an aqueous solvent system. A tweezer receptor 25 for the side chain protected tripeptide 19 was identified from the screening experiments. Receptor 25 was resynthesised and solution binding studies were carried out, which revealed that 25 binds to tripeptide 19 with K_a=8.2×10⁴±2.5×10⁴ (15 % DMSO/H₂O, pH 8.75) and with appreciable selectivity over the tripeptide enantiomer 22 and the side chain deprotected tripeptide 20.

Split-and-mix libraries of resin-bound “tweezer” receptors have been prepared and screened to identify receptors for dye-labelled tripeptides. The receptors incorporate a diamidopyridine unit to serve as a specific recognition site for the CO₂H group, leading to strong and selective receptors for peptide guests with a CO₂H terminus. The role of the dye-label, attached to the peptide guest to allow visualisation of selective recognition events in the screening experiments, has also been examined and was found to have a significant influence on the binding selectivities.

Eine kombinatorische Bibliothek von pinzettenförmigen Rezeptoren, in die eine Diamidopyridin-Einheit als spezifische Bindungsstelle für eine CO₂H-Gruppe eingebaut ist (siehe Bild), wurde durchmustert, um einen sequenzspezifischen Rezeptor für ausgewählte Peptidliganden mit einem CO₂H-Terminus zu identifizieren.

A combinatorial library of “tweezer” receptors, which incorporate a diamidopyridine unit to provide a specific binding site for a CO₂H group (see picture), can be screened to identify sequence-selective receptors for chosen peptide guests with a CO₂H terminus.