The Nature and Sequence of the Amino Acid Aglycone Strongly Modulates the Conformation and Dynamics Effects of Tn Antigen's Clusters

All in a cluster : The selected sequence has a crucial influence on both the orientation and flexibility of the carbohydrate moiety in mucin‐type glycopeptides (see picture). This feature can be explained through the varied conformational behavior of the glycosidic linkage of the Thr residue when compared with the Ser residue (see figure). On this basis, and taking into account that these carbohydrates presumably interact with components of the immune system, these findings could be helpful in the design of new cancer vaccines.

     

Non-natural amino acids as modulating agents of the conformational space of model glycopeptides

The synthesis and conformational analysis in aqueous solution of different alpha-methyl-alpha-amino acid diamides, derived from serine, threonine, beta-hydroxycyclobutane-alpha-amino acids, and their corresponding model beta-O-glucopeptides, are reported. The study reveals that the presence of an alpha-methyl group forces the model peptides to adopt helix-like conformations. These folded conformations are especially significant for cyclobutane derivatives. Interestingly, this feature was also observed in the corresponding model glucopeptides, thus indicating that the alpha-methyl group and not the beta-O-glucosylation process largely determines the conformational preference of the backbone in these structures. On the other hand, atypical conformations of the glycosidic linkage were experimentally determined. Therefore, when a methyl group was located at the Cbeta atom with an R configuration, the glycosidic linkage was rather rigid. Nevertheless, when the S configuration was displayed, a significant degree of flexibility was observed for the glycosidic linkage, thus showing both alternate and eclipsed conformations of the psi(s) dihedral angle. In addition, some derivatives exhibited an unusual value for the phi(s) angle, which was far from a value of -60 degrees expected for a conventional beta-O-glycosidic linkage. In this sense, the different conformations exhibited by these molecules could be a useful tool in obtaining systems with conformational preferences "à la carte".     

Coexistence of hydrogen-bonded loop and extended tetrapeptide conformations

The conformations of a protected tetrathiopeptide have been analysed by isotope labelling and two-dimensional infrared spectroscopy (2D-IR). It has been found that Boc-Ala-Gly(=S)-Ala-Aib-OMe in acetonitrile, as well as its oxopeptide analogue, can adopt a hydrogen-bonded loop conformation in coexistence with less restricted structures. The two types of conformations interconvert too quickly to be resolved on the nuclear magnetic resonance (NMR) timescale, but give rise to different cross peaks in two-dimensional infrared spectra. The hydrogen bond between the Boc terminal group and the amide proton of Aib can be broken by photoisomerisation of the thioamide bond.     

Engineering O-glycosylation points in non-extended peptides: implications for the molecular recognition of short tumor-associated glycopeptides

The ties that bind: The incorporation of non-natural residues in the peptide backbone allows the design of O-glycosylation points in helical segments. This strategy could help to modulate the binding properties between glycopeptides and their protein receptors, such as lectins and antibodies.     

Preferred conformations of peptides containing tert-leucine, a sterically demanding, lipophilic alpha-amino acid with a quaternary side-chain Cbeta atom

Terminally protected homopeptides of tert-leucine, from the dimer to the hexamer, co-oligopeptides of tert-leucine in combination with alpha-aminoisobutyric acid or glycine residues up to the hexamer level, and simple dipeptides representing known scaffolds for catalysts in asymmetric organic reactions were prepared by solution methods and fully characterized. The results of conformation analysis, performed by use of FT-IR absorption, NMR, CD, and X-ray diffraction techniques, indicate that this hydrophobic alpha-amino acid with tetrasubstitution at the Cbeta atom is structurally versatile. We show that it prefers extended or semiextended conformations, but can also be accommodated in folded structures, provided that these are biased by the presence of helicogenic residues. The current large-scale production of Tle, combined with its conformational preferences unravelled in this work, should make this bulky, hydrophobic, Calpha-trisubstituted alpha-amino acid a regular building block of any strategy seeking to tailor peptides with improved catalytic and pharmacological properties.     

Effect of the Substituents of the Neighboring Ring in the Conformational Equilibrium of Iduronate in Heparin‐like Trisaccharides

Based on the structure of the regular heparin, we have prepared a smart library of heparin‐like trisaccharides by incorporating some sulfate groups in the sequence α‐D ‐GlcNS‐ (1‐4)‐α‐L ‐Ido2S‐(1‐4)‐α‐D ‐GlcN. According to the 3D structure of heparin, which features one helix turn every four residues, this fragment corresponds to the minimum binding motif. We have performed a complete NMR study and found that the trisaccharides have a similar 3D structure to regular heparin itself, but their spectral properties are such that allow to extract very detailed information about distances and coupling constants as they are isotropic molecules. The characteristic conformational equilibrium of the central iduronate ring has been analyzed combining NMR and molecular dynamics and the populations of the conformers of the central iduronate ring have been calculated. We have found that in those compounds lacking the sulfate group at position 6 of the reducing end glucosamine, the population of ²S₀ of the central iduronate residue is sensitive to the temperature decreasing to 19 % at 278 K. On the contrary, the trisaccharides with 6‐O‐sulfate in the reducing end glucosamine keep the level of population constant with temperature circa 40 % of ²S₀ similar to that observed at room temperature. Another structural feature that has been revealed through this analysis is the larger flexibility of the L ‐IdoAS‐ D ‐GlcN glycosidic linkage, compared with the D ‐GlcNS‐L ‐IdoA. We propose that this is the point where the heparin chain is bended to form structures far from the regular helix known as kink that have been proposed to play an important role in the specificity of the heparin–protein interaction.     

Molecular Dynamics Simulations of Small Peptides: Can One Derive Conformational Preferences from ROESY Spectra?

Folding properties of beta-peptides were investigated by means of NMR experiments and MD simulations of beta-dipeptides, which serve as small test systems to study the influence of stereocenters and side chains on hydrogen-bond and consequently on secondary-structure formation. Two stereoisomers, SR and SS, of a Val-Phe dipeptide, and of the corresponding Ala-Ala dipeptide, and a Gly-Gly dipeptide were simulated in methanol for 40 ns. In agreement with experiment, the isomers of the Val-Phe dipeptide adopt quite different conformers at 298 K, the differences being reduced at 340 K. Interestingly, the SR isomer shows enhanced hydrogen bonding at the higher temperature. The adopted conformations are primarily determined by the R or S side chain substitution, and less by the type of side chain. Back-calculation of (1)H ROESY spectra and (3)J coupling constants from the MD simulations and comparison with the experimental data for the Val-Phe dipeptides shows good agreement between simulation and experiment, and reveals possible problems and pitfalls, when deriving structural properties of a small and extremely flexible molecule from NMR data only. Inclusion of all aspects of internal dynamics is essential to the correct prediction of the NMR spectra of these small molecules. Cross comparison of calculated with experimental spectra for both isomers shows that only a few out of many ROESY peaks reflect the sizeable conformational differences between the isomers at 298 K.     

Cyclic beta-tetra- and pentapeptides: synthesis through on-resin cyclization and conformational studies by X-ray, NMR and CD spectroscopy and theoretical calculations

The solution-phase synthesis of the simplest cyclic beta-tetrapeptide, cyclo(beta-Ala)4 (4), as well as the solid-phase syntheses through side chain anchoring and on-resin cyclization of the cyclic beta3-tetrapeptide cyclo(-beta3hPhe-beta3hLeu-beta3hLys-beta3hGln-) (14) and the first cyclic beta3-pentapeptide cyclo(-beta3hVal-beta3hPhe-beta3hLeu-beta3hLys-beta3hLys-) (19) are reported. Extensive computational as well as spectroscopic studies, including X-ray and NMR spectroscopy, were undertaken to determine the preferred conformations of these unnatural oligomers in solution and in the solid state. cyclo(beta-Ala)4 (4) with no chiral side chains is shown to exist as a mixture of rapidly interchanging conformers in solution, whereas inclusion of chiral side chains in the cyclo-beta3-tetrapeptide causes stabilization of one dominating conformer. The cyclic beta3-pentapeptide on the other hand shows larger conformational freedom. The X-ray structure of achiral cyclo(beta-Ala)4 (4) displays a Ci-symmetrical 16-membered ring with adjacent C=O and N-H atoms pointing pair wise up and down with respect to the ring plane. CD spectroscopic examinations of all cyclic beta-peptides were undertaken and revealed results valuable as starting point for further structural investigations of these entities.     

On the influence of charged side chains on the folding-unfolding equilibrium of beta-peptides: a molecular dynamics simulation study

The influence of charged side chains on the folding-unfolding equilibrium of beta-peptides was investigated by means of molecular dynamics simulations. Four different peptides containing only negatively charged side chains, positively charged side chains, both types of charged side chains (with the ability to form stabilizing salt bridges) or no charged side chains were studied under various conditions (different simulation temperatures, starting structures and solvent environment). The NMR solution structure in methanol of one of the peptides (A) has already been published; the synthesis and NMR analysis of another peptide (B) is described here. The other peptides (C and D) studied herein have hitherto not been synthesized. All four peptides A-D are expected to adopt a left-handed 3(14)-helix in solution as well as in the simulations. The resulting ensembles of structures were analyzed in terms of conformational space sampled by the peptides, folding behavior, structural properties such as hydrogen bonding, side chain-side chain and side chain-backbone interactions and in terms of the level of agreement with the NMR data available for two of the peptides. It was found that the presence of charged side chains significantly slows down the folding process in methanol solution due to the stabilization of intermediate conformers with side chain-backbone interactions. In water, where the solvent competes with the solute-solute polar interactions, the folding process to the 3(14)-helix is faster in the simulations.     

Serine versus Threonine Glycosylation with α‐O‐GalNAc: Unexpected Selectivity in Their Molecular Recognition with Lectins

The molecular recognition of several glycopeptides bearing Tn antigen (α‐O‐GalNAc‐Ser or α‐O‐GalNAc‐Thr) in their structure by three lectins with affinity for this determinant has been analysed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the molecular recognition. In fact, while Soybean agglutinin and Vicia villosa agglutinin lectins prefer Tn‐threonine, Helix pomatia agglutinin shows a higher affinity for the glycopeptides carrying Tn‐serine. The different conformational behaviour of the two Tn biological entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topology of the binding site of the lectins are at the origin of these differences.     

Gas‐Phase Peptide Structures Unraveled by Far‐IR Spectroscopy: Combining IR‐UV Ion‐Dip Experiments with Born–Oppenheimer Molecular Dynamics Simulations

Vibrational spectroscopy provides an important probe of the three‐dimensional structures of peptides. With increasing size, these IR spectra become very complex and to extract structural information, comparison with theoretical spectra is essential. Harmonic DFT calculations have become a common workhorse for predicting vibrational frequencies of small neutral and ionized gaseous peptides. ¹ Although the far‐IR region (<500 cm^?1) may contain a wealth of structural information, as recognized in condensed phase studies, ² DFT often performs poorly in predicting the far‐IR spectra of peptides. Here, Born–Oppenheimer molecular dynamics (BOMD) is applied to predict the far‐IR signatures of two γ‐turn peptides. Combining experiments and simulations, far‐IR spectra can provide structural information on gas‐phase peptides superior to that extracted from mid‐IR and amide A features.     

Hemicarbasucrose: Turning off the Exoanomeric Effect Induces Less Flexibility

Hemicarbasucrose, a close congener of sucrose in which the endocyclic oxygen atom of the glucose moiety is replaced by a methylene group was synthesized for the first time. The conformational behaviour of hemicarbasucrose was studied by a combination of molecular mechanics and NMR spectroscopy (J and NOE data). It was shown that the carbadisaccharide populates two distinct conformational families in solution, the normal syn‐ψ conformation, which is the predominating conformation of the parent natural O‐glycoside, and the anti‐ψ conformation, which has not been detected for the O‐disaccharide. Interestingly, the hemicarbasucrose is less flexible than its natural congener.     

Solution structure of quinoline- and pyridine-derived oligoamide foldamers

The unambiguous elucidation of a new folded structure in solution may prove to be a very challenging task. The NMR protocols developed for solving the solution structures of alpha-peptides have been applied to aliphatic beta- and gamma-peptides but are not directly applicable to aromatic oligomers. In particular, the string of spin systems in an aromatic sequence cannot be reconstituted solely from correlations between protons. For aromatic oligomers, it is shown that the assignment of a large part of the 13C NMR spectrum through HMBC and HSQC experiments allows to unambiguously assign proton NMR spectra and in turn to interpret NOE correlations. This has been implemented both with quinoline- and pyridine-derived oligoamide foldamers, and should be applicable to a wide range of oligomers including various combinations of monomers. The NOE correlations allow the unambiguous solution structure elucidation of helical conformations of oligoamides derived from pyridine and quinoline monomers showing that, in these series, the solution structures correspond very well to the structures observed in the solid state.     

Stereoselective Fluorination Alters the Geometry of a Cyclic Peptide: Exploration of Backbone‐Fluorinated Analogues of Unguisin A

New methods for enhancing the efficiency of peptide cyclization, and for fine‐tuning the conformations of cyclic peptides, are valuable from a drug development perspective. Herein stereoselective fluorination is investigated as a new strategy for achieving these goals. Four vicinal difluorinated analogues of the natural cyclic heptapeptide unguisin A have been efficiently synthesized. The analogues are found to adopt dramatically different secondary structures, controlled by the fluorine stereochemistry.