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Fig. 2.2.4. Crystal structure of 5.3 H2O.

moments [23]. The picolinic acid subunits in 5 should therefore stabilize peptide conformations with all amide protons pointing into the cavity center, ideally pre-organized for anion binding. This arrangement was indeed found. The preferred conformation of 5 in solution and the solid state differs significantly from those observed for other peptides, such as, for example, 3 in its iodide complex (Figure 2.2.3), however [22].

The reason for this difference are cis amide conformations at the three proline subunits of 5. Figure 2.2.4 shows that the presence of these amide conformations causes the three aromatic subunits of the peptide to be oriented almost parallel to the C3 axis of the macrocycle, and the characteristic dish-shaped cavity observed for all other peptides to disappear. Despite this overall conformation, 5 is still able to interact with anions [22]. On addition of salts such as BTMA+ tosylate to a solution of 5 in d6-DMSO, for example, the NH signals of the peptides shift downfield in the NMR spectrum, a typical indication of hydrogen-bonding interactions between the NH protons and the anion. Almost no shift of the corresponding BTMA+ cation signals could be detected, and the cation is thus not involved in anion binding. Complex formation strongly affects the resonance of the peptide H(a) protons, however. The corresponding downfield shift of the signal accounts for the close proximity of the peptide H(a) protons to the negative charge density of the guest in the complex, and is thus another indication of complex formation. It enabled us to investigate anion binding not only in aprotic solvents but also in polar, protic solvents such as water-methanol mixtures, in which the peptide NH protons are in rapid exchange with the protons of the solvent molecules.

To our surprise, we found that 5 binds anions such as halides or sulfate even in 80% D2O-CD3OD, which is remarkable considering that, despite the large excess of water molecules in these mixtures, the hydrogen-bonding interactions between the anions and the peptide are still efficient [22]. Assignment of the structure of the anion complexes of5 revealed the reason for the unusual receptor properties of this peptide. We could show that 5 preferentially forms 2:1 sandwich-type complexes with suitable anions in which the guests are bound in a cavity located be-

Fig. 2.2.5. Top and side views of the crystal structure of the iodide complex of 5.

tween two almost perfectly interlocking cyclopeptide moieties. The crystal structure of the corresponding iodide complex is depicted in Figure 2.2.5.

This crystal structure shows that the iodide forms hydrogen bonds to all six NH of the two peptide moieties in the complex. It also demonstrates how effectively the anion is embedded between the cyclopeptides. Complex formation thus shields the guest from surrounding solvent molecules, an effect that strengthens receptor-substrate interactions; this might be one reason for the anion affinity of 5 in aqueous solution.

To improve the aqueous solubility of 5 we also synthesized a cyclopeptide 6 containing hydroxyproline subunits [24]. Although this compound is very water-soluble, and in solution adopts a similar conformation to 5, it only forms 1:1 complexes with anions. A reason for the inability of 7 to form 2:1 complexes could be that the hydroxyproline subunits in 6 are better solvated than prolines in aqueous solution, and the desolvation required for aggregation of two cyclopeptide molecules thus occurs less readily. Steric hindrance of hydroxyl groups from different cyclopeptide moieties in the a dimeric complex of 6 could, moreover, also make aggregation difficult. Although peptide 6 cannot form sandwich-type complexes, it proved to be valuable for a quantitative determination of the anion affinity of 5, the results of which are summarized in Table 2.2.3 [24].

This table shows that, for a given anion, the stability constants of the 1:1 complexes of both peptides are comparable, and that the stability of the 1:1 complexes increases in the order Cl_ > Br~ > I" > SO42~. This order can be rationalized in terms of the size of the ions, with larger ions forming more stable complexes because they fit better into the available peptide cavity. For sulfate, an additional contribution to complex stability from the higher charge of this anion must be considered.

The large stability constants K2 of the 2:1 complexes of 5 indicate that, once formed, the 1:1 complexes of this peptide have a strong tendency to bind the sec-

Tab. 2.2.3. Stability constants Ka and maximum chemical shifts Admax of some anion complexes of 5 and 6 (80% D2O-CD3OD; T = 298 K; K1 and K2 in m_1; Ka in m"2; Admax maximum chemical shift of the peptide H(a) protons in ppm; error limits of the stability constants of the complexes of 6 < 20%, and 5 < 40%).

Tab. 2.2.3. Stability constants Ka and maximum chemical shifts Admax of some anion complexes of 5 and 6 (80% D2O-CD3OD; T = 298 K; K1 and K2 in m_1; Ka in m"2; Admax maximum chemical shift of the peptide H(a) protons in ppm; error limits of the stability constants of the complexes of 6 < 20%, and 5 < 40%).

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