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. We used a structure-guided approach to develop unnatural glycopeptides as model antigens for tumor-associated MUC1. We designed a collection of 13 glycopeptides to bind either SM3 or 5E5, two monoclonal antibodies with distinct epitopes known to recognize tumor associated MUC1. Glycopeptide binding to SM3 or 5E5 was confirmed by surface plasmon resonance and rationalized by molecular dynamics simulations. These model antigens were conjugated to gold nanoparticles and used in a dot-blot assay to detect autoantibodies in serum samples from pancreatic cancer patients and healthy volunteers. Nanoparticle probes with glycopeptides displaying the SM3 epitope did not have diagnostic potential. Instead, nanoparticle probes displaying glycopeptides with high affinity for 5E5 could discriminate between cancer patients and healthy controls. Remarkably, the best-discriminating probes show significantly better true and false positive rates than the current clinical biomarkers CA19-9 and carcinoembryonic antigen (CEA).

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. 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.

Synthesis of Fluorescent Lanthipeptide Cytolysin S Analogues by Late-Stage Sulfamidate Ring Opening.

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/ß-peptides. Their conformational preferences and biological activities were assessed and compared with those of wild-type CylLS″.

Conformationally Restricted ß-Sheet Breaker Peptides Incorporating Cyclic α-Methylisoserine Sulfamidates.

Peptides containing variations of the ß-amyloid hydrophobic core and five-membered sulfamidates derived from ß-amino acid α-methylisoserine have been synthesized and fully characterized in the gas phase, solid state and in aqueous solution by a combination of experimental and computational techniques. The cyclic sulfamidate group effectively locks the secondary structure at the N-terminus of such hybrid peptides imposing a conformational restriction and stabilizing non-extended structures. This conformational bias, which is maintained in the gas phase, solid state and aqueous solution, is shown to be resistant to structure templating through assays of in vitro ß-amyloid aggregation, acting as ß-sheet breaker peptides with moderate activity.

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 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-ß-substituted unnatural α-amino acids (UAAs), which could have a high potential for applications in chemical biology.

Synthesis of ß2,2-Amino Acids by Stereoselective Alkylation of Isoserine Derivatives Followed by Nucleophilic Ring Opening of Quaternary Sulfamidates.

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 ß2,2-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 ß2,2-amino acids such as α-benzyl-α-heterofunctionalized-ß-alanines and α-benzylnorlanthionine derivatives.

Monitoring of the Rioja red wine production process by 1 H-NMR spectroscopy.

BACKGROUND: As an inherently quantitative and unbiased analytical technique, proton nuclear magnetic resonance (1 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. CONCLUSION: This study demonstrates the potential of NMR as a process analytical technology (PAT) tool in the wine industry, by monitoring amino acids, organic acids and three polyphenols - gallic acid, catechin and resveratrol - during the winemaking process. This study of the time course evolution of wine has been conducted in a commercial winery rather than an experimental laboratory, demonstrating the capacity of this technique in commercial winemaking production. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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 pKa promotes fragmentation even at slightly acidic pH, a common feature of several tumor environments. The Grob fragmentation can also occur under physiological conditions in living cells, highlighting the potential bioorthogonal applicability of this reaction.

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

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.

Selective modification of sulfamidate-containing peptides.

Hybrid peptides whose N-terminal residues are activated in the form of α-methylisoserine-derived cyclic sulfamidates exhibit rich reactivity as electrophiles, allowing site- and stereoselective modifications at different backbone and side chain positions. The unique properties of this scaffold allow the stereocontrolled late-stage functionalization of the peptide backbone by nucleophilic ring opening with fluorescent probes, thiocarbohydrates and tags for strain-promoted azide-alkyne cycloaddition as well as by installing labile N-terminal affinity tags (biotin) and cytotoxic drugs (chlorambucil) for pH-controlled release. Finally, an unexpected base-promoted acyl group migration from the sulfamidate N-terminus allows fast and quantitative intramolecular modification of nucleophilic side chains on the fully unprotected peptides.

Synthesis of Nß-Substituted α,ß-Diamino Acids via Stereoselective N-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 Nß-substituted α,ß-diamino acids, such as 1-isohistidine, τ-histidinoalanine, ß-benzylaminoalanine, ß-(piperidin-1-yl)alanine, ß-(azepan-1-yl)alanine, and fluorescent and ciprofloxacin-containing amino acid derivatives.

Synthesis, conformational analysis and in vivo assays of an anti-cancer vaccine that features an unnatural antigen based on an sp2-iminosugar fragment.

The Tn antigen (GalNAc-α-1-O-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 sp2-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. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.

Solvent-based strategy improves the direct determination of key parameters in edible fats and oils by 1 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. RESULTS: Through an adequate mixture of solvents, we have developed a methodology to quantify essential parameters in edible fats and oils, including 1,2-diacylglycerol, 1,3-diacylglycerol, and 1-monoacylglycerol, by using a single experiment and without the need for matrix derivatization. CONCLUSION: This methodology has been successfully applied to the analysis of olive, sunflower, corn, sesame, and peanut oils, as well as butter, walnut, salmon, and spicy pork sausage. Moreover, the evolution of thermal oxidation and lipolysis of virgin olive oil and sunflower has been analyzed. © 2020 Society of Chemical Industry.

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. 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.

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. 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.

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 Michael adducts were easily transformed into the corresponding amino acids to study their optical properties and, in some selected cases, into the corresponding N-Fmoc-d-cysteine derivatives to be used in solid-phase peptide synthesis (SPPS). To further demonstrate the utility of these non-natural Cys-derived fluorescent amino acids, the coumaryl and dansyl derivatives were incorporated into cell-penetrating peptide sequences through standard SPPS and their optical properties were studied in different cell lines. The internalization of these fluorescent peptides was monitored by fluorescence microscopy.

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.

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. Accumulation of negative charge at the NSO3 moiety in the transition state, especially when the sulfonamide NH is deprotonated, drastically raises the activation barrier for the nucleophilic attack. On the other hand, ester groups at the carboxylic position favor ring opening, whereas amides allow competition between the substitution and elimination pathways. Using pyridine as a nucleophilic probe, we have demonstrated both experimentally and computationally that a proper selection of the substitution scheme can enhance the synthetic scope of α-methylisoserine-derived sulfamidates, switching off and on the nucleophilic ring-opening in a controlled manner. This is particularly convenient for hybrid α/ß-peptide synthesis, as demonstrated recently by our group.

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. 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.

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).