Artificial Receptors for the Stabilization of b-Sheet Structures

Thomas Schrader, Markus Wehner, and Petra Rzepecki 2.4.1

b-Sheet Recognition in Nature

The ¿-sheet structure is one of the fundamental secondary structures of proteins (Box 8). It greatly stabilizes protein domains by forming an array of several parallel peptide backbone pieces five to fifteen amino acids long, which are interconnected purely by multiple hydrogen bonds. ¿-Sheets can, however, also be exposed to solvent; they then have another important role, namely as binding sites for external ligands. Therefore the active center of many enzymes contains a ¿-sheet fragment which binds to a target peptide delivering its critical functionality to the catalyti-cally active ''hot spot''. A prominent example is the discovery that peptidal substrates and inhibitors bind to diverse proteolytic enzymes as ¿-strands [1]. In other instances enzymes are only active as dimers, which by themselves represent small ¿-sheet structures. Thus two identical domains of the HIV-I protease form a four-stranded ¿-sheet at their N-termini. This in turn leads to formation of the catalyti-cally active cleavage channel which cuts the predecessor peptide into the correct pieces which automatically self-assemble to the complete virus. Such ''functional'' ¿-sheet structures are intrinsic targets for the development of new drugs aimed at blocking the active site of enzymes [2].

¿-Sheet structures can, on the other hand, pose a severe problem for the organism if they are formed spontaneously by circumventing the body's strict control mechanisms. Because of their mutual saturation of all hydrogen-bond donors and acceptors with non-polar side-chains extended to the solvent, they readily precipitate from aqueous solution, and form large aggregates. In recent years, protein folding diseases have become increasingly important; the percentage of elderly people suffering from Alzheimer's dementia, e.g., has exceeded 0.1% in the western hemisphere. European countries have been haunted by epidemics like BSE threatening humans with the related new variant of CJD. Such pathological ¿-sheets represent another medicinal challenge, calling for the development of small ligands capable of keeping these proteins in solution or even reversing plaque formation [3].

156 | 2.4 Artificial Receptors for the Stabilization of 0-Sheet Structures 2.4.2

Artificial b-Sheets and Recognition Motifs

Some twenty years ago chemists began their contributions on 0-sheet research by creating small soluble 0-sheets which could be examined structurally. Rigid templates and artificial 0-turns were adorned with growing peptide chains, and the formation of two- and three-stranded artificial 0-sheets was proven by NMR techniques and CD spectroscopy [4]. Kelly even postulated that the combination of his non-polar benzofuran template with the first lipophilic amino acids mimicked the hydrophobic cluster, which has been suggested as nucleation site for the formation of biological 0-sheets in the protein-folding process [5]. The propensity of the twenty proteinogenic amino acids for adopting the 0-sheet conformation in a pep-tide strand has been studied by various techniques and has even been extended to discrimination between parallel and antiparallel 0-sheets [6]. Recipes for the construction of larger 0-sheet peptides consisting entirely of natural amino acids have been put forward by Gellman and others, leading to the design of extended 0-sheets which remain in solution [7].

In contrast, very few external ligands have been found that are capable of docking on to an existing peptide and thereby stabilize its 0-sheet conformation. The first example was presented by our group in 1996, when we introduced amino-pyrazole derivatives for the backbone recognition of dipeptides in organic solution [8]. Later (1998) the Hamilton group truncated the diaminoquinolone motif used by Kemp et al. in their epindolidione receptors [9] for the intramolecular stabilization of short peptides in the 0-sheet conformation [10]. Bartlett recently (2002) presented the azacyclohexenone fragment for incorporation in drugs targeting 0-sheets [11]. All these recognition motifs can be characterized by a general scheme - they recognize the acceptor-donor-acceptor pattern (ADA) found in natural 0-sheets by a complementary arrangement of hydrogen-bond donors and acceptors (DAD) in a linear fashion, with optimized distances of 2.7 A and 3.7 A (Figures 2.4.1 and 2.4.2).

Fig. 2.4.1. Two illustrations for the general DAD pattern of artificial j-sheet ligands with optimized geometry.
Fig. 2.4.2. Recognition motifs following the general rule outlined in Figure 2.4.1 (A) 3-aminopyrazoles, (B) 3,6-diaminoquinolones; (C) 3-azacyclohexenones.

To avoid self-association the overall shape of larger structures has been chosen to be slightly curved, or ionic groups have been incorporated for recognition of the Nor C-terminus.

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