Fig. 4.4 Incorporation of trifluoroleucine (tfl) into the leucine zipper protein A1 at the indicated positions (L) increases the thermal stability of the dimer as shown here for a urea titration of wild type A1 (•) against variably fluorinated samples ofA1: 17% (■), 29% (□) and 92% (o) incorporated tfl; f represents the ratio of the unfolded state [19].

Similarly, Kennan [24] could increase the stability of a coiled-coil protein trimer derived from GCN4 by replacing a small alanine residue by an unnatural cyclo-hexylalanine which allows for more extensive hydrophobic contacts at the protein interfaces (Fig. 4.5). The alanine in the natural protein is too small to completely fill the empty space in between the three helices at the trimerization interface. A destabilizing void remains between the three interacting methyl groups of the alanine side chains. Hence, the peptide normally prefers to dimerize instead of forming a trimer, as the dimer interface is more closely packed. However, any increase in hydrophobic contacts at this position should further increase the stability of the trimer thereby shifting the aggregation mode from two to three strands. For example, it had already been shown that hydrophobic ligands such as cyclo-hexane or benzene present in solution can fulfill that part [25]. In solution these ligands increased the apparent thermal stability of the peptide aggregate and the oligomerization order switched to a trimer. A crystal structure of the peptide shows a single benzene molecule bound directly at the core of the trimer in between the three methyl groups. However, such a ligand with an increased hydrophobic surface can also be directly incorporated into the peptide itself. If in one helix the alanine is replaced by a cyclohexylalanine a 2:1 heterotrimer forms in which the one cyclohexyl residue partially fills the void. This again allows for more efficient and more extensive hydrophobic contacts between the three helices. Hence, the 2:1-heterotrimer is more stable than the homodimer or -trimer of the initial alanine containing peptide. Indeed, CD- and thermal melting

Fig. 4.5 Replacement of an alanine by a cyclohexylalanine (X16) within a helical peptide favors the formation of a coiled-coil 2:1 heterotrimer (B) due to increased hydrophobic contacts at protein interfaces relative to the alanine containing homotrimer (A) [24].

studies confirmed the increased thermal stability of the heterotrimer. The melting temperature of the heterotrimer increased significantly compared to the alanine containing peptide (DTm > 30 °C). However, thermal melting studies only show that an aggregate forms, but they do not allow its composition to be determined directly . That indeed a heterotrimer is formed in this case could be shown by an affinity tagging experiment. If a His-tag is attached to the cyclohexylalanine pep-tide, the heterotrimer can selectively be separated from a mixture of the two pep-tides using affinity chromatography. The retained peptide material had exactly a 2:1-composition even though the initial mixture was enriched in the alanine containing peptide. Therefore, the increased hydrophobic contacts due to cyclo-hexylalanine residue selectively stabilized the heterotrimer. On the other hand, a naphthalene ring is too large to fit into the void and consequently a peptide with a naphthylalanine residue instead of the cyclohexylalanine does not form stable heterotrimers. This is an instructive case study how specific peptide assemblies can be directed and controlled both in terms of stability as well as composition of the aggregates by fine tuning the hydrophobic contacts which are responsible for the aggregation.

116 | 4 Foldamer Hybrids: Defined Supramolecular Structures from Flexible Molecules 4.2.2

Intertwined Strands

A very common hybridization motif in Nature is the double helix as found in the structure of DNA. Two helical molecules are intertwined to form a helical double strand. In the case of DNA the two strands are held together by H-bonds and stacking interactions [16] though much debate was going on in recent years about the relative importance of these interactions for the molecular recognition of nucleobases [26]. The double helix is crucial for genetic information storage and error-free replication as it ensures the correct reproduction of the information encoded in the primary sequence of the nucleotide strand (see below) [3]. The DNA double helix is also a beautiful supramolecular structure which has intrigued many chemists to devise artificial systems that similarly form helical double strands.

A very interesting class of double helix forming foldamers is based on aromatic oligoamides as introduced by Lehn and Huc (Fig. 4.6) [33a]. These oligomers are formed from alternating 2,6-diaminopyridines and 2,6-pyridinedicarboxylic acids. An intramolecular interaction between the amide NH proton and the pyridine N-atom is responsible for a curved conformation of these molecules. A more detailed description of their conformational behavior can be found in Chapter 1. These helical oligoamides can then also further dimerize forming a stable double helix in solution. Within the double helix the two oligomer strands are held together primarily by arene-arene-interactions between pyridine rings located on top of each other, whereas H-bonds occur intramolecularly within each strand being responsible for the curvature of the helix. The formation of the double helix is accompanied by a spring-like extension of the individual helices, but the inner diameter of the central pore is not significantly affected.

In these oligomers intramolecular H-bonds pre-orientate the monomers and induce the helical structure (Fig. 4.6 A). But hybridization of such oligopyridine-dicarboxamide strands is limited to a certain length of the single strand. Huc

Fig. 4.6 Inducing helical conformations in oligoamides using intramolecular H-bonds (A). Two helical oligoamides can then dimerize to form a supramolecular double helix (B) [33c].

[27, 28], showed that with an increasing length of the single strand the enthalpic price of spring-like extension during the double helix formation is not compensated by intermolecular p-p interactions. NMR dilution experiments in CDCl3 show that at 25 °C Kdim increases from 210 for the smallest strand (5 pyridine units) to 5200 M_1 for a medium sized strand (9 pyridine units). In case of the largest strand (15 pyridine units) Kdim could not be determined because the single helix was not detected by NMR even at high concentrations. In another experiment the structure of these double helices was investigated by crystallization of pentameric oligoamides from pure DMSO and in the second case from a DMF/Et2O mixture. The comparison of both structures provides evidence that the positions of the single strands in the helix are flexible. Although the crystallo-graphic parameters (space group, unit cell parameters) of both samples indicate high resemblance to each other and even the position of incorporated water molecules differs just slightly, the superposition of both helices shows an offset of the strands of more than 1.5 A in a plane orthogonal to the helix axis. This leads to the suggestion that the interactions between the single strands are neither directional nor dependent on the distance between the strands. These results are in agreement with the assumption of a certain screw motion based on the freedom of the single strands within the helix, known from investigations in solution.

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