The structural features of MUC1‐like glycopeptides bearing the Tn antigen (α‐O‐GalNAc‐Ser/Thr) in complex with an anti MUC‐1 antibody are reported at atomic resolution. For the α‐O‐GalNAc‐Ser ...derivative, the glycosidic linkage adopts a high‐energy conformation, barely populated in the free state. This unusual structure (also observed in an α‐S‐GalNAc‐Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α‐O‐GalNAc‐Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non‐equivalence of Ser and Thr O‐glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti‐MUC1 antibodies.
Spot the difference: The structures of two Tn antigen glycopeptides, with N‐acetylgalactosamine (GalNAc, green hexagon) attached to either Ser or Thr, in complex with an anti‐MUC1 antibody are reported. The results reveal significant differences in the conformational behavior of the two glycopeptides in the bound state and demonstrate the non‐equivalence of Ser and Thr O‐glycosylation points in molecular recognition processes.
A tripartite cancer vaccine candidate, containing a quaternary amino acid (α-methylserine) in the most immunogenic domain of MUC1, has been synthesized and examined for antigenic properties in ...transgenic mice. The vaccine which is glycosylated with GalNAc at the unnatural amino acid, was capable of eliciting potent antibody responses recognizing both glycosylated and unglycosylated tumour-associated MUC1 peptides and native MUC1 antigen present on cancer cells. The peptide backbone of the novel vaccine presents the bioactive conformation in solution and is more resistant to enzymatic degradation than the natural counter part. In spite of these features, the immune response elicited by the unnatural vaccine was not improved compared to a vaccine candidate containing natural threonine. These observations were rationalized by conformational studies, indicating that the presentation and dynamics of the sugar moiety displayed by the MUC1 derivative play a critical role in immune recognition. It is clear that engineered MUC1-based vaccines bearing unnatural amino acids have to be able to emulate the conformational properties of the glycosidic linkage between the GalNAc and the threonine residues. The results described here will be helpful to the rational design of efficacious cancer vaccines.
Different behavior has been observed for the ψ torsion angle of the glycosidic linkages of d-GalNAc-Ser and d-GalNAc-Thr motifs, allowing the carbohydrate moiety to adopt a completely different ...orientation. In addition, the fact that the water pockets found in α-d-GalNAc-Thr differ from those obtained for its serine analogue could be related to the different capability that the two model glycopeptides have to structure the surrounding water. This fact could have important biological inferences (i.e., antifreeze activity).
A totally stereocontrolled C-Michael addition of serine-equivalent C-nucleophiles to tri-O-benzyl-2-nitro-d-galactal was used as the key step to synthesize several pyrano3,2-bpyrrole structures. ...These scaffolds could be regarded as conformationally restricted Tn antigen mimics, as we have demonstrated by biological assays. The pyranose rings retain their 4C1 chair conformation, as shown by molecular modeling and NMR spectroscopy. The expected bioactivity was established by a competition-tailored enzyme-linked lectin assay using both soybean and Vicia villosa agglutinins as model lectins. The facile described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational features and bioactivity demonstrate the prepared glycomimics to be promising candidates for further exploitation of this scaffold to give glycans for lectin blocking and vaccination.
Tn antigen (α-O-GalNAc-Ser/Thr) is a convenient cancer biomarker that is recognized by antibodies and lectins. This work yields remarkable results for two plant lectins in terms of epitope ...recognition and reveals that these receptors show higher affinity for Tn antigen when it is incorporated in the Pro-Asp-Thr-Arg (PDTR) peptide region of mucin MUC1. In contrast, a significant affinity loss is observed when Tn antigen is located in the Ala-His-Gly-Val-Thr-Ser-Ala (AHGVTSA) or Ala-Pro-Gly-Ser-Thr-Ala-Pro (APGSTAP) fragments. Our data indicate that the charged residues, Arg and Asp, present in the PDTR sequence establish noteworthy fundamental interactions with the lectin surface as well as fix the conformation of the peptide backbone, favoring the presentation of the sugar moiety toward the lectin. These results may help to better understand glycopeptide–lectin interactions and may contribute to engineer new binding sites, allowing novel glycosensors for Tn antigen detection to be designed.
Mucin-1 (MUC1) glycopeptides are exceptional candidates for potential cancer vaccines. However, their autoantigenic nature often results in a weak immune response. To overcome this drawback, we ...carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate an anti-MUC1 response, artificial antigens must mimic the conformational dynamics of natural antigens in solution and have an equivalent or higher binding affinity to anti-MUC1 antibodies than their natural counterparts. As a proof of concept, we have developed a glycopeptide that contains noncanonical amino acid (2S,3R)-3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. On the one hand, conformational analysis in water shows that this surrogate explores a landscape similar to that of the natural variant. On the other hand, the presence of an additional methylene group in the side chain of this analog compared to the threonine residue enhances a CH/π interaction in the antigen/antibody complex. Despite an enthalpy–entropy balance, this synthetic glycopeptide has a binding affinity slightly higher than that of its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulates the formation of specific anti-MUC1 IgG antibodies in mice and shows efficacy comparable to that of the natural derivative. The antibodies also exhibit cross-reactivity to selectively target, for example, human breast cancer cells. This investigation relied on numerous analytical (e.g., NMR spectroscopy and X-ray crystallography) and biophysical techniques and molecular dynamics simulations to characterize the antigen–antibody interactions. This workflow streamlines the synthetic process, saves time, and reduces the need for extensive, animal-intensive immunization procedures. These advances underscore the promise of structure-based rational design in the advance of cancer vaccine development.
A formal 2 + 2 cycloaddition of 2-amidoacrylates with monosubstituted donor olefins, including its asymmetric version, is described. The stereoselectivity of this reaction can be modulated by the use ...of sterically hindered aluminum aryloxides or methylaluminoxane as Lewis acids. The reaction was applied to the synthesis of both stereoisomers of 2-benzyloxycyclobutane-α-amino acid, which are protected serine analogues c4Ser(OBn).
The synthesis and conformational analysis in aqueous solution of different α‐methyl‐α‐amino acid diamides, derived from serine, threonine, β‐hydroxycyclobutane‐α‐amino acids, and their corresponding ...model β‐O‐glucopeptides, are reported. The study reveals that the presence of an α‐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 α‐methyl group and not the β‐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 Cβ 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 ψs dihedral angle. In addition, some derivatives exhibited an unusual value for the ϕs angle, which was far from a value of −60° expected for a conventional β‐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”.
En este artículo se presenta la síntesis y el análisis conformacional en disolución acuosa de diferentes diamidas de α‐metil‐α‐aminoácidos derivados de Ser, Thr y β‐hidroxiciclobutan‐α‐aminoácidos, así como de sus correspondientes β‐O‐glucopéptidos modelo. El estudio reveló que la presencia de un grupo metilo en posición α del aminoácido fuerza al péptido modelo a adoptar conformaciones plegadas de tipo hélice. Estas conformaciones plegadas son especialmente significativas para los derivados que incorporan la estructura de ciclobutano. Esta característica también fue observada para los correspondientes glucopéptidos modelo, lo cual indica que es el grupo metilo en posición α y no la β‐O‐glucosilación lo que determina en gran medida las preferencias conformacionales de la cadena peptídica en estas estructuras. Por otro lado, se observaron experimentalmente conformaciones atípicas del enlace glicosídico. Así, cuando un grupo metilo está localizado en el Cβ con configuración R, el enlace glicosídico es bastante rígido. Sin embargo, cuando dicho carbono muestra configuración S se observa un alto grado de flexibilidad en el enlace glicosídico, mostrando el ángulo diedro ψs tanto conformaciones alternadas como eclipsadas. Además, algunos derivados exhibieron valores inusuales del ángulo diedro ϕs, los cuales se apartan bastante del valor esperado de −60° para un enlace β‐O‐glicosídico convencional. En este sentido, las diferentes conformaciones exploradas por estas moléculas podrían ser utilizadas como herramientas útiles para obtener sistemas con preferencias conformacionales “à la carte”.
An à la carte menu of glucopeptides: Different α‐ and or β‐substituted α‐amino acid diamides derived from serine, threonine, and their corresponding model β‐O‐glucopeptides (see picture) were synthesized and studied in aqueous solution. The different conformations exhibited by these molecules could be a useful tool to obtain systems with conformational preferences “à la carte”.
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.
N-Acetylgalactosamine (GalNAc) α-O-linked to l-threonine (Thr) (Tn antigen) and several mimics of this Tn antigen have been synthesized to explore the impact of the underlying amino acid in the ...presentation mode of the carbohydrate moiety. The structural changes introduced in the Tn antigen mimics involve the replacement of the natural underlying Thr by non-natural amino acids while maintaining the α-O-glycosidic linkage of GalNAc or the substitution of this bond by α-C-glycosidic linkages. We also synthesized two bicyclic, conformationally restricted Tn antigen mimics. All of these compounds were subjected to a thorough conformational analysis in solution using NMR data, quantum mechanical (QM) calculations, and molecular dynamics simulations. Interestingly, in C-glycosides, the 1C4 chair conformation of the pyranose ring was predicted to be stable by QM calculations and experimentally supported by nuclear Overhauser effect cross-peaks and coupling constants observed in the NMR experiments.
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