Detection of Tumor-Associated Autoantibodies in the Sera of Pancreatic Cancer Patients Using Engineered MUC1 Glycopeptide Nanoparticle Probes.

Pancreatic cancer is one of the deadliest cancers worldwide, mainly due to late diagnosis. Therefore, there is an urgent need for novel diagnostic approaches to identify the disease as early as possible. We have developed a diagnostic assay for pancreatic cancer based on the detection of naturally occurring tumor associated autoantibodies against Mucin-1 (MUC1) using engineered glycopeptides on nanoparticle probes.

Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses.

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.

Towards Enantiomerically Pure Unnatural α-Amino Acids via Photoredox Catalytic 1,4-Additions to a Chiral Dehydroalanine.

Chemo- and diastereoselective 1,4-conjugate additions of anionic and radical -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.

Synthesis of β-Amino Acids by Stereoselective Alkylation of Isoserine Derivatives Followed by Nucleophilic Ring Opening of Quaternary Sulfamidates.

Chiral bicyclic -acetal isoserine derivatives have been synthesized by an acid-catalyzed tandem ,-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.

Monitoring of the Rioja red wine production process by H-NMR spectroscopy.

Background: As an inherently quantitative and unbiased analytical technique, proton nuclear magnetic resonance ( H-NMR) provides an excellent method to monitor the quality of food and beverages, and a sensitive and informative tool to study the winemaking process.

Results: By using NMR, it is possible to monitor quantitative changes in wine metabolites (amino acids, organic acids and some phenolic compounds) during the winemaking process, including wine ageing. This study shows an increase in the concentration of the phenols at the beginning of alcoholic fermentation, as well as a stabilization and slight increase in gallic acid and a slight decrease in resveratrol during the oak barrel ageing step.

Bioorthogonal Self-Immolative Linker Based on Grob Fragmentation.

A self-immolative bioorthogonal conditionally cleavable linker based on Grob fragmentation is described. It is derived from 1,3-aminocyclohexanols and allows the release of sulfonate-containing compounds in aqueous media. Modulation of the amine p promotes fragmentation even at slightly acidic pH, a common feature of several tumor environments.

Structure-based Design of Anti-cancer Vaccines: The Significance of Antigen Presentation to Boost the Immune Response.

Immunotherapy, alone or in combination with other therapies, is widely used against cancer. Glycoprotein Mucin 1 (MUC1), which is overexpressed and aberrantly glycosylated in tumor cells, is one of the most promising candidates to engineer new cancer vaccines. In this context, the development of stable antigens that can elicit a robust immune response is mandatory.

Toward Enantiomerically Pure β-Seleno-α-amino Acids via Stereoselective -Michael Additions to Chiral Dehydroalanines.

The first totally chemo- and diastereoselective 1,4-conjugate additions of -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 NaBH in the presence of the Dha. These -Michael additions provide an excellent channel for the synthesis of enantiomerically pure selenocysteine (Sec) derivatives, which pose high potential for chemical biology applications.

Synthesis, conformational analysis and assays of an anti-cancer vaccine that features an unnatural antigen based on an sp-iminosugar fragment.

The Tn antigen (GalNAc-α-1--Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography.

Synthesis of -Substituted α,β-Diamino Acids via Stereoselective -Michael Additions to a Chiral Bicyclic Dehydroalanine.

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 -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.

Solvent-based strategy improves the direct determination of key parameters in edible fats and oils by H NMR.

Background: Edible fats and oils are very important in nutrition and as a main source of energy and are also essential nutrients. There are several methods for the analysis of edible fats and oils, but nowadays nuclear magnetic resonance (NMR) is emerging as a powerful tool (albeit complex and high-tech demanding) to identify, quantify, and differentiate many types of food, including fats and oils. In this sense, the challenges of this technique are the simplification of methodology and taking advantage of a 400 MHz NMR instrument.

Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage.

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.

Water Sculpts the Distinctive Shapes and Dynamics of the Tumor-Associated Carbohydrate Tn Antigens: Implications for Their Molecular Recognition.

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.

Tn Antigen Mimics by Ring-Opening of Chiral Cyclic Sulfamidates with Carbohydrate C1- S- and C1- O-Nucleophiles.

Starting from commercially available ( S)-isoserine and effectively accessible ( S)-α-methylserine, enantiopure cyclic sulfamidates have been prepared as chiral building blocks for the synthesis of various S- and O-glycosylated amino acid derivatives, including unnatural variants of the Tn antigen, through highly chemo-, regio-, and stereoselective nucleophilic ring-opening reactions with carbohydrate C1- S- and C1- O-nucleophiles.

Cell-Penetrating Peptides Containing Fluorescent d-Cysteines.

A series of fluorescent d-cysteines (Cys) has been synthesized and their optical properties were studied. The key synthetic step is the highly diastereoselective 1,4-conjugate addition of aryl thiols to a chiral bicyclic dehydroalanine recently developed by our group. This reaction is fast at room temperature and proceeds with total chemo- and stereoselectivity.

The Use of Fluoroproline in MUC1 Antigen Enables Efficient Detection of Antibodies in Patients with Prostate Cancer.

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.

Substituent Effects on the Reactivity of Cyclic Tertiary Sulfamidates.

The reactivity of cyclic tertiary sulfamidates derived from α-methylisoserine strongly depends on the substitution at the C and N termini. These substrates are one of the very few examples able to undergo nucleophilic ring opening at a quaternary carbon with complete inversion of the configuration, as demonstrated both experimentally and computationally. When the sulfonamide is unprotected, the characteristic ring-opening reaction is completely silenced, which explains that the majority of the ring-opening reactions reported in the literature invoke N-alkyl or N-carbonyl-protected sulfamidates.

Tn Antigen Mimics Based on sp(2)-Iminosugars with Affinity for an anti-MUC1 Antibody.

The first examples of amino acid (Ser/Thr)-sp(2)-iminosugar glycomimetic conjugates featuring an α-O-linked pseudoanomeric linkage are reported. The key synthetic step involves the completely diastereoselective α-glycosylation of Ser/Thr due to strong stereoelectronic and conformational bias imposed by the bicyclic sp(2)-iminosugar scaffold. Mucin-related glycopeptides incorporating these motifs were recognized by the monoclonal antibody (mAb) scFv-SM3, with activities depending on both the hydroxylation pattern (Glc/Gal/GlcNAc/GalNAc) of the sp(2)-iminosugar and the peptide aglycone structure (Ser/Thr).

Design of α-S-Neoglycopeptides Derived from MUC1 with a Flexible and Solvent-Exposed Sugar Moiety.

The use of vaccines based on MUC1 glycopeptides is a promising approach to treat cancer. We present herein several sulfa-Tn antigens incorporated in MUC1 sequences that possess a variable linker between the carbohydrate (GalNAc) and the peptide backbone. The main conformations of these molecules in solution have been evaluated by combining NMR experiments and molecular dynamics simulations.

Bifunctional Chiral Dehydroalanines for Peptide Coupling and Stereoselective S-Michael Addition.

A second generation of chiral bicyclic dehydroalanines easily accessible from serine has been developed. These scaffolds behaved as excellent S-Michael acceptors when tri-O-acetyl-2-acetamido-2-deoxy-1-thio-α-d-galactopyranose (abbreviated as GalNAc-α-SH) was used as a nucleophile. This addition proceeds with total chemo- and stereoselectivity, complete atom economy, quickly, and at room temperature, making it a true click reaction.

Mucin architecture behind the immune response: design, evaluation and conformational analysis of an antitumor vaccine derived from an unnatural MUC1 fragment.

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.

Synthesis of Mixed α/β2,2-Peptides by Site-Selective Ring-Opening of Cyclic Quaternary Sulfamidates.

A method for site- and stereoselective peptide modification using a cyclic sulfamidate scaffold containing peptides is described. A peptide synthesis strategy allowing the rapid generation of mixed α/β-peptides incorporating a sulfamidate residue, derived from 2-methylisoserine, has been generalized. The unique electrophilic nature of this scaffold for nucleophilic substitution at a quaternary center with total inversion of its configuration, which was demonstrated computationally, allows for site-selective conjugation with various nucleophiles, such as anomeric thiocarbohydrates and pyridines.

Nuclear Magnetic Resonance (NMR) as a tool for the study of the metabolism of Rickettsia slovaca.

Rickettsial infections are caused by intracellular bacteria. They do not grow in standard culture media so there are limitations in routine practice to study their metabolism. Nuclear Magnetic Resonance (NMR) spectroscopy is used for identification of metabolites in biological samples.

Deciphering the Non-Equivalence of Serine and Threonine O-Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti-MUC1 Antibody.

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.