Improved methodologies are provided to synthesize (1R,2S)-2-aminocyclobutane-1-carboxylic acid derivatives and their incorporation into beta-peptides of 2-8 residues bearing different N-protecting groups. The conformational analysis of these oligomers has been carried out by using experimental techniques along with theoretical calculations. This study shows that these oligomers adopt preferentially a strand-type conformation in solution induced by the formation of intra-residue six-membered hydrogen-bonded rings, affording cis-fused [4.2.0]octane structural units that confer high rigidity on these beta-peptides. Moreover, all of them are prone to self-assemble producing nano-sized fibres, as evidenced by TEM, AFM and SPFM, and, in some instances, they also form gels. These techniques and molecular modelling allowed us to suggest an aggregation model for the assembly structures in which a parallel molecular-arrangement is preferred and the conformation is similar to that observed in solution. According to this model, both hydrogen-bonding and hydrophobic interactions would account for formation of the assemblies.

Three new bis(cyclobutane) beta-dipeptides have been synthesized from appropriate derivatives of cis- and trans-2-aminocyclobutane-1-carboxylic acid, respectively. The predominance of eight-membered hydrogen-bonded rings has been manifested for (trans,trans)- and (trans,cis)-beta-dipeptides while the formation of six-membered rings is preferred for the (cis,trans)- beta-dipeptide similarly to the previously described (cis,cis)-diastereomer.

Two chiral synthetic β-dipeptides have been constructed, one with two trans-cyclobutane residues and the other with one trans and one cis fragment, 1 and 2, respectively, and investigated to get insight into the non-covalent interactions responsible for their self-assembly to form ordered aggregates, as well into parameters such as their morphology and size. Experimental evidence of the formation of these assemblies was provided by spectroscopy, microscopy and X-ray diffraction experiments that suggest the formation of nanoscale helical aggregates. This process involves a conformational change in the molecules of each dipeptide with respect to the preferred conformation of the isolated molecules in solution. A high-resolution NMR spectroscopy study allowed the determination of the dynamics of the gelation process in [D(8)]toluene and the sol-gel transition temperature, which was around 270 K in this solvent at a concentration of 15 mM. NMR spectroscopy experiments also provided some information about conformational changes involved in the sol-gel transition and also suggested a different gel packing for each dipeptide. These observations have been nicely explained by computational studies. The self-assembly of the molecules has been modelled and suggested a head-to-head molecular arrangement for 1 and a head-to-tail arrangement for 2 to give helical structures corresponding to hydrogen-bonded single chains. These chains interact with one another in an antiparallel way to afford bundles, the significant geometry parameters of which fit well to the main peaks observed in wide-angle X-ray diffraction spectra of the aggregates in the solid state.

Chirality of the monomeric residues controls and determines the prevalent folding of small oligopeptides (from di- to tetramers) composed of 2-aminocyclobutane-1-carboxylic acid (ACBA) derivatives with the same or different absolute and relative configuration. The cis-form of the monomeric ACBA gives rise to two conformers, namely, Z6 and Z8, while the trans-form manifests uniquely as an H8 structure. By combining these subunits in oligo- and polypeptides, their local structural preference remains, thus allowing the rational design of new short foldamers. A lego-type molecular architecture evolves; the overall look depends only on the conformational properties of the structural building units. A versatile and efficient method to predict the backbone folds of designed cyclobutane β-peptides is based on QM calculations. Predictions are corroborated by high-resolution NMR studies on selected stereoisomers, most of them being new foldamers that have been synthesized and characterized for the first time. Thus, the chiral expression of monomeric building units results in the defined secondary structures of small oligomers. As a result of this study, a new set of chirality controlled foldamers is provided to probe as biocompatible biopolymers.

Two generations of hybrid γ,γ-peptides containing cyclobutane amino acids and cis-γ-amino-L-proline joined in alternation have been synthesized and their capacity to cross the eukaryotic cell membrane has been evaluated. The first generation consists of di-, tetra- and hexapeptides, and their properties have been analyzed as well as the influence of peptide length and chirality of the cyclobutane residues. Results have shown that the absolute configuration of the cyclobutane amino acid does not have a relevant influence. The second generation consists of hybrid γ,γ-hexapeptides with a common backbone and distinct side chains introduced with different linkage types through the α-amino group (N(α)) of the proline monomers. These peptides have been shown to be non-toxic towards HeLa cells and to internalize them effectively, the best results being obtained for the peptides with a spacer of five carbons between the N(α) atom and the guanidinium group. The introduction of cyclobutane residues inside the sequence affords a good balance between charge and hydrophobicity, reducing the number of positive charges. This results in lower toxicity and similar cell-uptake properties when compared to previously described peptide agents.