GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α-O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from ...the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate–peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors.
Nucleophilic ring opening of cyclic sulfamidates derived from amino acids is a common strategy for the synthesis of lanthionine derivatives. In this work, we report the regio-, chemo-, and ...stereoselective intramolecular S-alkylation of a cysteine residue with N-sulfonyl sulfamidates for the synthesis of cyclic lanthionine-containing peptides. The strategy involves the solid-phase synthesis of sulfamidate-containing peptides followed by late-stage intramolecular cyclization. This protocol allowed for the synthesis of four full-length cytolysin S (CylLS″) analogues, two α-peptides and two hybrid α/β-peptides. Their conformational preferences and biological activities were assessed and compared with those of wild-type CylLS″.
Chemo- and diastereoselective 1,4-conjugate additions of anionic and radical C-nucleophiles to a chiral bicyclic dehydroalanine (Dha) are described. Of particular importance, radical carbon ...photolysis by a catalytic photoredox process using a simple method with a metal-free photocatalyst provides exceptional yields and selectivities at room temperature. Moreover, these 1,4-conjugate additions offer an excellent starting point for synthesizing enantiomerically pure carbon-β-substituted unnatural α-amino acids (UAAs), which could have a high potential for applications in chemical biology.
The tumor-associated carbohydrate Tn antigens include two variants, αGalNAc-O-Thr and αGalNAc-O-Ser. In solution, they exhibit dissimilar shapes and dynamics and bind differently to the same protein ...receptor. Here, we demonstrate experimentally and theoretically that their conformational preferences in the gas phase are highly similar, revealing the essential role of water. We propose that water molecules prompt the rotation around the glycosidic linkage in the threonine derivative, shielding its hydrophobic methyl group and allowing an optimal solvation of the polar region of the antigen. The unusual arrangement of αGalNAc-O-Thr features a water molecule bound into a “pocket” between the sugar and the threonine. This mechanism is supported by trapping, for the first time, such localized water in the crystal structures of an antibody bound to two glycopeptides that comprise fluorinated Tn antigens in their structure. According to several reported X-ray structures, installing oxygenated amino acids in specific regions of the receptor capable of displacing the bridging water molecule to the bulk-solvent may facilitate the molecular recognition of the Tn antigen with threonine. Overall, our data also explain how water fine-tunes the 3D structure features of similar molecules, which in turn are behind their distinct biological activities.
The first totally chemo- and diastereoselective 1,4-conjugate additions of Se-nucleophiles to a chiral bicyclic dehydroalanine (Dha) are described. The methodology is simple and does not require any ...catalyst, providing exceptional yields at room temperature, and involves the treatment of the corresponding diselenide compound with NaBH4 in the presence of the Dha. These Se-Michael additions provide an excellent channel for the synthesis of enantiomerically pure selenocysteine (Sec) derivatives, which pose high potential for chemical biology applications.
Chiral bicyclic N,O-acetal isoserine derivatives have been synthesized by an acid-catalyzed tandem N,O-acetalization/intramolecular transcarbamoylation reaction between conveniently protected ...l-isoserine and 2,2,3,3-tetramethoxybutane. The delicate balance of the steric interactions between the different functional groups on each possible diastereoisomer controls their thermodynamic stability and hence the experimental product distribution. These chiral isoserine derivatives undergo diastereoselective alkylation at the α position, proceeding with either retention or inversion of the configuration depending on the relative configuration of the stereocenters. Quantum mechanical calculations revealed that a concave-face alkylation is favored due to smaller torsional and steric interactions at the bicyclic scaffold. This synthetic methodology gives access to chiral β2,2-amino acids, attractive compounds bearing a quaternary stereocenter at the α position with applications in peptidomimetic and medicinal chemistry. Thus, enantiopure α-alkylisoserine derivatives were produced upon acidic hydrolysis of these alkylated scaffolds. In addition, α-benzylisoserine was readily transformed into a five-membered ring cyclic sulfamidate, which was ring opened regioselectively with representative nucleophiles to yield other types of enantiopure β2,2-amino acids such as α-benzyl-α-heterofunctionalized-β-alanines and α-benzylnorlanthionine derivatives.
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.
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 structure-based design of a new generation of tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated ...proline residue, such as a (4S)-4-fluoro-l-proline or 4,4-difluoro-l-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues stabilize the antigen–antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for prostate cancer.
The highly diastereoselective 1,4-conjugate additions of several nitrogen nucleophiles to chiral bicyclic dehydroalanines have been assessed effectively at room temperature in good to excellent ...yields without needing any catalyst or additional base. This methodology is general, simple, oxygen and moisture tolerant, high-yielding, totally chemo- and stereoselective. This procedure offers an efficient and practical approach for the synthesis of N β-substituted α,β-diamino acids, such as 1-isohistidine, τ-histidinoalanine, β-benzylaminoalanine, β-(piperidin-1-yl)alanine, β-(azepan-1-yl)alanine, and fluorescent and ciprofloxacin-containing amino acid derivatives.
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