H

Fig. 4.12 A six-H-bonded duplex based on a self-complementary AADADD pattern. The arrows indicate diagnostic interstrand NOEs. The pyrene units were needed to follow the dimerization using fluorescence studies.

tions, therefore larger association constants can be measured. Hence, not every spectroscopic technique is suitable for every supramolecular system. The use of a more sensitive technique such as fluorescence is sometimes hampered by the absence of appropriate reporter groups. Whereas normally most organic molecules can be analyzed by NMR, fluorescence spectroscopy requires a special chro-mophore. Therefore, sometimes synthetic modifications have to be introduced before a certain technique can be applied. Here, a pyrene labeled derivative had to be synthesized before the dimerization constant could be measured by fluorescence [38].

Gong [40] also studied several heteroduplexes in which two different molecules with complementary H-bond patterns associate to form ladder-like duplexes (Fig. 4.13). Dimer formation in these cases could be demonstrated by specific interstrand NOEs. Furthermore, as the duplex is overall less polar than the individual monomers, which cannot interact and hence possess free H-bond donors and acceptors. Hence the heterodimer shows a significantly different chromatographic behavior. Whereas the two monomers have a very low mobility on the silica gel TLC (Rf = 0.0 and 0.1 using 10% DMSO in CHCl3, respectively), a 1:1-mixture of the two monomers has a Rf value of 0.96 indicating the formation of the overall much less polar heteroduplex! Again the thermodynamic stability was too large for an accurate determination of the association constant by concentration dependent NMR studies. By isothermal titration microcalorimetry (ITC) the association constant could be estimated to be K a 109 M_1. However, as already mentioned in the introduction also the solvent plays an important role in controlling the stability of such aggregates as it dramatically affects the strength of non-covalent interactions which drive the folding or hybridization process. For exam-

Fig. 4.13 Heterodimerization of two duplex strands held together by six H-bonds which impose a significant sequence specificity (A): the introduction of a mismatch (B: to the right) leads to a decrease in binding affinity by a factor of 40 in this case.

ple, in polar solvents hydrogen bonding is significantly reduced due to the competitive solvation of donors and acceptors by individual solvent molecules. Hence, duplex formation first requires desolvation of both donors and acceptors before their intermolecular interaction can lead to the formation of a dimer. Its stability is of course only the difference of the interactions within the aggregate relative to the interaction of each monomer with the solvent. As the latter can be substantial especially in polar or aqueous solvents, the stability of H-bonded supramolecular aggregates in such solvents is significantly reduced. In this specific case of Gong's six-H-bond heteroduplex already the addition of 5% DMSO led to drop in stability of several orders of magnitude.

Also the introduction of a mismatch caused a significant decrease in the stability of the tapes. In one specific example an attractive H-bond between an amide NH and a carbonyl group was replaced by a repulsive interaction between two car-

bonyl groups. The stability of this mismatched heteroduplex was 40 times lower than of the corresponding matched pair [41]. In Nature sometimes even larger effects are observed for such mismatches. A similar chemical exchange is the reason for the upcoming resistance of bacteria towards the antibiotic Vancomycin [42]. Normally, Vancomycin forms a complex with a bacterial peptide substrate held together by five H-bonds within a hydrophobic environment. In the resistant bacteria the exchange of an amide for an ester within the substrate, replaces an attractive H-bond for a dipole repulsion. The complex stability drops by a factor of 1000. Model studies showed that the loss of the H-bond is responsible for a drop in affinity by a factor 10, whereas the repulsive dipole interaction led to another decrease by a factor of 100 [43].

This specificity of H-bonds in combination with their directionality makes H-bonds so attractive for designing supramolecular structures despite the inherent problem of their weakness in more polar solvents. The individual pattern of H-bond donors and acceptors within one monomer very specifically determines the binding partner required for stable duplex formation. Furthermore, the H-bond pattern also regulates the stability of the duplex due to attractive or repulsive secondary interactions between neighboring binding sites as initially proposed by J0rgensen in 1990 [44]. Therefore, a self-complementary AADD pattern is more stable than an ADAD pattern.

Fig. 4.14 2-ureido-4-pyrimidones exist as a rapidly interconverting mixture of three tautomers in solution with different self-assembling properties depending on their specific sequence of H-bond donor (D) and acceptor (A) sites.

Meijer et al. were one of the first groups who synthesized such quadruple hydrogen bonding motifs. Acylation of diaminotriazines and diaminopyrimi-dines led to the desired self-complementary ADAD binding motif, which was confirmed by X-ray diffraction. NMR experiments showed that this self-complementary binding motif has association constants of up to 105 in chloroform. In respect to Jorgensen a more stable quadruple hydrogen binding motif should result from an AADD array of donor and acceptor sites. Therefore Meijer et al. used 2-ureido-4-pyrimidones as basic molecules for their AADD binding site [45]. The analysis of this bonding motif became quite complicated, because of a complex equilibrium of three tautomers which all coexist in solution (Fig. 4.14). Their composition is both determined by the polarity of the solvent and the concentration of the compound itself. One tautomer has a DDA bonding pattern and can not dimerize. The other two tautomers both present self-complementary quadruple hydrogen binding patterns with either an ADAD or AADD sequence. They can form dimers but with different stabilities. The latter is favored, because of additional stabilizing secondary interactions within the dimer, which aren't possible for the ADAD pattern. Due to the fact that it was impossible to quantify an association constant for the AADD binding motif by NMR experiments, Meijer used excimer fluorescence spectroscopy as an indirect method yielding a value of 6 ■ 107 M_1. However, this example shows how complicated the situation can be

Fig. 4.15 Linear H-bonded tapes based on 2,6-triazines (A) or 2,6-pyridazines (B) as developed by Krische and coworkers.

if the self-assembling monomers do not have a specific well defined structure by themselves.

Another class of linear H-bonded tapes was designed by Krische and coworkers based on aminotriazines, which are covalently linked by flexible aminoalcohols [46]. Several oligomers were prepared, which all present an alternating repeat of an AD-DA H-bond pattern provided by the aminotriazine moiety (Fig. 4.15). This can interact with two other aminotriazine moieties in a second oligomer to form an interdigitated linear tape. ITC experiments in 1,2-dichloroethane at 20 °C showed an increasing association constant Ka from the monomer (4.7 M_1) over the dimer (5 x 103 M_1) to the trimer (6.9 x 108 M_1). The tetramer instead had a lower association constant Ka of 1.1 x 103 M-1, even lower than the dimer. This value resembles the association constants for duplex dimer formation, which suggests an intramolecular folding of the tetramer. Related duplex strands based on 3,6-diaminopyridazines were introduced by Krische later (Fig. 4.15 B) [47]. The association behavior was measured in analogy by ITC under the same conditions mentioned above, resulting in association constants of 5 M_1 for the monomer, 870 M_1 for the dimer and 8 x 105 M_1 for trimer.

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