Peptides

Several α,β,α- or α,γ,α-tripeptides, consisting of a central cyclobutane β- or γ-amino acid being flanked by two d- or l-proline residues, have been synthesized and tested as organocatalysts in asymmetric aldol additions. High yields and enantioselectivities have been achieved with α,γ,α-tripeptides, being superior to peptides containing a cyclobutane β-amino acid residue. This is probably due to their high rigidity, which hinders some of the peptide catalysts to adopt the proper active conformation. This reasoning correlates with the major conformation of the peptides in the ground state, as suggested by ¹H NMR and computational calculations. The configuration of the aldol products is controlled by the proline chirality, and consequently, the R/S configuration of aldol products can be tuned by the use of either commercially available d- or l-proline. The enantioselectivity in the aldol reactions is reversed if the reactions are carried out in the presence of water or other protic solvents such as methanol. Spectroscopic and theoretical investigations revealed that this effect is not the consequence of conformational changes in the catalyst but rather caused by the participation of a water molecule in the rate determining transition state, in such a way that the preferential nucleophilic attack is oriented to the opposite enantiotopic aldehyde face.

Four new series of diastereomeric β,γ-di- and β,γ-tetrapeptides derived from conveniently protected (1R,2S)- and (1S,2S)-2-aminocyclobutane-1-carboxylic acid and cis- and trans-γ-amino-l-proline joined in alternation have been synthesized. High resolution NMR experiments show that peptides containing trans-cyclobutane amino acid residues adopt a more folded structure in solution than those containing a cis-cyclobutane residue, which adopt a strand-like structure. The cis/trans relative configuration of the cyclobutane residue is the origin of the folding pattern of each peptide due to either intra- or inter-residue hydrogen-bonded ring formation, whereas the cis/trans isomerism of the γ-amino-l-proline residue does not have a significantly relevant role on the folding ability of these peptides.

A rapid and high-yielding cysteine labelling of peptides has been observed with the specific labelling agent for amines, N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). Interestingly, conjugation of the 4-fluorobenzoyl (FB) moiety is selectively achieved through a cysteine (Cys) thiol of the peptides with high yield (>80%) in short time (<5 min), while for a Cys amino acid derivative, a slow process has been observed. The large reactivity of these peptides for the conjugation reaction is rationalised on the basis of electrostatic interactions between the sulfhydril and the guanidinium groups of the amino acid side chains. Moreover, the stability of these novel conjugates and the biodistribution of the radiolabelled dodecapeptide by positron emission tomography (PET) in rats has been examined.

Three series of new γ‐peptides have been synthesized by starting from conveniently protected cis‐3‐amino‐2,2‐dimethylcyclobutane‐1‐carboxylic acid derivatives. The first series is constructed with only one enantiomer of this γ‐amino acid, whereas in the second one both enantiomeric cyclobutane residues are joined in alternating fashion. The high degrees of rigidity in these γ‐peptides induce the adoption of extended but sterically constrained conformations in both cases. The third series is the product of alternation of a cyclobutane residue with linear γ‐aminobutyric acid (GABA). Conformational bias in these three systems accounts for the cyclobutane being a major disrupting factor to the formation of strong intramolecular hydrogen bonds, leading to extended conformations. In contrast, investigation of cyclobutane/GABA hybrid γ‐peptides of a fourth series, in which the carbocyclic moiety is not a part of the polyamide skeleton but acts as a chiral polyfunctional platform, reveals that these peptides are able to produce intramolecular hydrogen bonds leading to well defined folded conformations.

Some hybrid tetrapeptides consisting of (1R,2S)-2-aminocyclobutane-1-carboxylic acid and glycine, β-alanine, or γ-aminobutyric acid (GABA) joined in alternation, compounds 1–3, respectively, have been investigated to gain information on the non-covalent interactions responsible for their self-assembly to form ordered aggregates, as well as on parameters such as their morphology and size. All three peptides formed nice gels in many organic solvents and significant difference in their behaviour was not observed. Scanning electron microscopy (SEM) and circular dichroism (CD) pointed out that peptide 1, which contains the shortest C₂linear residue, presented the most defined fibril network and afforded nanoscale helical aggregates. Tetrapeptide 3, with C₄linear residues in its structure, also showed bundles of fibres whereas a homogeneous spherulitic network was observed for tetrapeptide 2, with a C₃ spacer between cyclobutane residues. Computational calculations for 1 allowed us to model the self-assembly of the molecules and suggested a head-to-head arrangement to give helical structures corresponding to hydrogen-bonded single chains. These features were corroborated by a high-resolution NMR spectroscopy study of the dynamics of the gelation process in toluene-d₈ which evidenced that molecules self-assemble to afford ordered aggregates with a supramolecular chirality.

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 transform 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 beta-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.

Several labelled biomolecules, such as peptides, proteins, and antibodies, have been used as probes for in vivo optical or isotopic imaging of many physiological and pathological processes. Fast cysteine labelling of peptides promoted by an adjacent arginine has been observed with a standard labelling agent specific for amines, N-succinimidyl 4-[¹⁸F]fluorobenzoate.

Several oligomers constructed with (1R,2S)-2-aminocyclobutane-1-carboxylic acid and glycine, beta-alanine, and gamma-amino butyric acid (GABA), respectively, joined in alternation have been synthesized and studied by means of NMR and CD experiments as well as with computational calculations. Results account for the spacer length effect on folding and show that conformational preference for these hybrid peptides can be tuned from beta-sheet-like folding for those containing a C-2 or C-4 linear segment to a helical folding for those with a C-3 spacer between cyclobutane residues. The introduction of cyclic spacers between these residues does not modify the extended ribbon-type structure previously manifested in poly(cis-cyclobutane) beta-oligomers.

Two diastereomeric series of hybridc,c-pep-tides derived from conveniently protected derivatives of(1R,2S)- and (1S,2R)-3-amino-2,2-dimethylcyclobutane-1-carboxylic acid andcis-4-amino-L-proline joined in alter-nation have efficiently been prepared through convergent synthesis. High-resolution NMR experiments show that these compounds present defined conformations in solution affording very compact structures as the result of intra and inter residue hydrogen-bonded ring formation. (R,S)-cyclobutane containing peptides adopt more twisted con-formations than (S,R) diastereomers. In addition, all the sec-peptides have high tendency to aggregation providing vesicles of nanometric size, which were stable when allowed to stand for several days, as verified by transmission electron microscopy.

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 [D₈]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.

Improved methodologies are provided to synthesize (1R,2S)-2-aminocyclobutane-1-carboxylic acid derivatives and their incorporation into β-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 β-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) -dipeptides have been synthesized from appropriate derivatives ofcis- andtrans-2-aminocyclobutane-1-carboxylicacid, respectively. The predominance of eight-membered hydrogen-bonded rings has been manifested for (trans,trans)- and (trans,cis)- B-dipeptides while the formation of six-membered rings is preferred for the (cis,trans)-B-dipeptide similarly to the previously described(cis,cis)-diastereomer.