Optimization of glycosylation temperatures is essential for automated solid phase glycan assembly. Constructing a reliable optimization system that mimics key reaction components is crucial for increasing the atom economy and the energy efficiency of the process. Although glycosyl acceptors play a pivotal role in glycosylation, evaluating their effect on glycosylation efficiency is nontrivial. While screening various glycosyl acceptors for optimization is impractical, compromising for simple alkyl acceptors produces results of dubious relevance. We demonstrate that optimization with a modified trans-4-aminocyclohexanol-based acceptor accurately reflects the optimal glycosylation temperature of several glycosyl acceptors, overcoming previous limitations. The contextual use of both n-alkyl and glycan-mimetic acceptors for automated optimization enabled translation of optimized glycosylation temperature to highly efficient solid phase synthesis of disaccharides.

Specific phosphorylation patterns regulate the activity of proteins and play a central role in protein self-assembly. In Tau, such patterns drive the formation of disease-related condensates and aggregates. Understanding their functional impact is essential for studying Tauopathies such as Alzheimer’s Disease. Here we show how specific phosphorylation patterns regulate Tau self-assembly and control the interplay between its aggregation and condensation, using a peptide-based approach that allows systematic analysis of libraries of specific phosphorylation patterns at the domain level and is complementary to the current protein-level methods. We applied our methodology to study the effect of specific phosphorylations on the aggregation and condensation of the R4 domain of Tau that is pivotal for its self-assembly, forming the β-helix motif that is common to various Tau patient fibrils. Using advanced phosphopeptide synthesis methods developed in our labs, we generated a library of multi-phosphorylated peptides derived from Tau R4. We found that phosphorylation at Ser341 promotes aggregation, while Ser352 enhances condensation. Phosphorylation at Ser356 inhibits both processes. The source of these different outcomes is the distinct microenvironments around each phosphorylated site. Our results provide a residue-level resolution of how the decision between Tau condensation and aggregation is being made. This was possible by using our peptide-based approach, which is complementary to protein-level method and enables efficient identification of active phosphorylation patterns. These can later be studied at the protein level.

Heparan sulfate (HS) interactions with interleukin 8 (IL-8) are crucial for immune system response. The structural features of the HS and the environmental entities, such as metal ions, can regulate these interactions. However, it is challenging to evaluate the effect of each parameter on the interactions because of low accessibility to well-defined saccharides and the lack of characteristic features to be determined by analytical tools. We evaluated the effect of the HS structural features on IL-8 binding affinity utilizing electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS). We showed that the metal ions Ca²⁺ and Mg²⁺ dissimilarly mediate the interactions of HS and IL-8 in structure-dependent manner of the HS. We showed that in all glycans, a positive synergistic effect on IL-8 binding was observed. For several glycans, the presence of ions resulted in a dramatic increase in the affinity to IL-8, while for other glycans, a milder effect was observed. This demonstrated that both structural motifs and environmental features are crucial for maintaining the interactions between the HS and IL-8.

Streptococcus pneumoniae is a pathogenic bacterium that contains the surface-bound neuraminidase, NanA. NanA has two domains that interact with sialosides. It is hard to determine the contribution of each domain separately on catalysis or binding. In this work, we used biochemical methods to obtain the separated domains, applied electrochemical and surface analysis approaches, and determined the catalytic and binding preferences toward a surface-bound library of sialosides. Impedimetric studies on two different surfaces revealed that protein–surface interactions provide a tool for distinguishing the unique contribution of each domain at the interface affecting the substrate preference of the enzyme in different surroundings. We showed that each domain has a sialoside-specific affinity. Furthermore, while the interaction of the sialoside-covered surface with the carbohydrate-binding domain results in an increase in impedance and binding, the catalytic domain adheres to the surface at high concentrations but retains its catalytic activity at low concentrations.

Synthesis of complex glycans is extremely challenging. In the current issue of Device, Seeberger and co-workers introduce a new oligosaccharide synthesizer. The introduced technology integrates innovative solutions to overcome difficulties associated with solidphase oligosaccharide synthesis. The focus on an energy efficient, smaller, and user-friendly device forecasts exciting advances in glycochemistry.

Neuraminidases (NA) are sialic acid-cleaving enzymes that are used by both bacteria and viruses. These enzymes have sialoside structure-related binding and cleaving preferences. Differentiating between these enzymes requires using a large array of hard-to-access sialosides. In this work, we used electrochemical impedimetric biosensing to differentiate among several pathogene-related NAs. We used a limited set of sialosides and tailored the surface properties. Various sialosides were grafted on two different surfaces with unique properties. Electrografting on glassy carbon electrodes provided low-density sialoside-functionalized surfaces with a hydrophobic submonolayer. A two-step assembly on gold electrodes provided a denser sialoside layer on a negatively charged submonolayer. The synthesis of each sialoside required dozens of laborious steps. Utilizing the unique protein–electrode interaction modes resulted in richer biodata without increasing the synthetic load. These principles allowed for profiling NAs and determining the efficacy of various antiviral inhibitors.

Sialic acid recognition and hydrolysis are essential parts of cellular function and pathogen infectivity. Neuraminidases are enzymes that detach sialic acid from sialosides, and their inhibition is a prime target for viral infection treatment. The connectivity and type of sialic acid influence the recognition and hydrolysis activity of the many different neuraminidases. The common strategies to evaluate neuraminidase activity, recognition, and inhibition rely on extensive labeling and require a large amount of sialylated glycans. The above limitations make the effort of finding viral inhibitors extremely difficult. We used synthetic sialylated glycans and developed a label-free electrochemical method to show that sialoside structural features lead to selective neuraminidase biosensing. We compared Neu5Ac to Neu5Gc sialosides to evaluate the organism-dependent neuraminidase selectivity–sensitivity relationship. We demonstrated that the type of surface and the glycan monolayer density direct the response to either binding or enzymatic activity. We proved that while the hydrophobic glassy carbon surface increases the interaction with the enzyme hydrophobic interface, the negatively charged interface of the lipoic acid monolayer on gold repels the protein and enables biocatalysis. We showed that the sialoside monolayers can serve as tools to evaluate the inhibition of neuraminidases both by biocatalysis and molecular recognition.

Design and synthesis of orthogonally protected monosaccharide building blocks are crucial for the preparation of well-defined oligosaccharides in a stereo- and regiocontrolled manner. Selective introduction of protecting groups to partially protected monosaccharides is nontrivial due to the often unpredictable electronic, steric, and conformational effects of the substituents. Abolished reactivity toward a commonly used Lewis base-catalyzed acylation of O-2 was observed in conformationally restricted 4,6-O-benzylidene-3-O-Nap galactoside. Investigation of analogous systems, crystallographic characterization, and quantum chemical calculations highlighted the overlooked conformational and steric considerations, the combination of which produces a unique passivity of the 2-OH nucleophile. Evaluating the role of electrophile counterion and auxiliary base in the acylation of the sterically crowded and conformationally restricted galactoside system revealed an alternative Brønsted base-driven reaction pathway via nucleophilic activation. Insights gained from this model system were utilized to access the target galactoside intermediate within the envisioned synthetic route. The acylation strategy described herein can be implemented in future syntheses of key monomeric building blocks with unique protecting group hierarchies.

Solid phase synthesis is the most dominant approach for the preparation of biological oligomers as it enables the introduction of monomers iteratively. Accelerated solid phase synthesis of biological oligomers is crucial for chemical biology, but its application to the synthesis of oligosaccharides is not trivial. Solid-phase oligosaccharide assembly is a slow process performed in a variety of conditions and temperatures, requires an inert gas atmosphere, and demands high excess of glycosyl donors. The process is done in special synthesizers and poor mixing of the solid support increases the risk of diffusion-independent hydrolysis of the activated donors. High shear stirring is a new way to accelerate solid phase synthesis. The efficient mixing ensures that reactive intermediates can diffuse faster to the solid support thereby increasing the kinetics of the reactions. We report here a stirring-based accelerated solid-phase oligosaccharide synthesis. We harnessed high shear mixing to perform diffusion-dependent glycosylation in a short reaction time. We minimized the use of glycosyl donors and the need to use an inert atmosphere. We showed that by tailoring the deprotection and glycosylation conditions to the same temperature, assembly steps are performed continuously, and full glycosylation cycles is completed in minutes.

The biosensing of bacterial pathogens is of a high priority. Electrochemical biosensors are an important future tool for rapid bacteria detection. A monolayer of bacterial-binding peptides can serve as a recognition layer in such detection devices. Here, we explore the potential of random peptide mixtures (RPMs) composed of phenylalanine and lysine in random sequences and of controlled length, to form a monolayer that can be utilized for sensing. RPMs were found to assemble in a thin and diluted layer that attracts various bacteria. Faradaic electrochemical impedance spectroscopy was used with modified gold electrodes to measure the charge-transfer resistance (RCT) caused due to the binding of bacteria to RPMs. Pseudomonas aeruginosa was found to cause the most prominent increase in RCT compared to other model bacteria. We show that the combination of highly accessible antimicrobial RPMs and electrochemical analysis can be used to generate a new promising line of bacterial biosensors.

 

Peptide fragments of glycoproteins containing multiple N-glycosylated sites are essential biochemical tools not only to investigate protein–protein interactions but also to develop glycopeptide-based diagnostics and immunotherapy. However, solid-phase synthesis of glycopeptides containing multiple N-glycosylated sites is hampered by difficult couplings, which results in a substantial drop in yield. To increase the final yield, large amounts of reagents but also time-consuming steps are required. Therefore, we propose herein to utilize heating and stirring in combination with low-loading solid supports to set up an accelerated route to obtain, by an efficient High-Temperature Fast Stirring Peptide Synthesis (HTFS-PS), glycopeptides containing multiple N-glycosylated sites using equimolar excess of the precious glycosylated building blocks.

 

Optimizing glycosylation conditions for automated glycan assembly is highly challenging, demand wasteful use of precious building blocks and rely on nontrivial analyses. We developed a semi-quantitative method for automated optimization of glycosylation temperature that utilized minute quantities of donors and translated those conditions to solid-phase glycan assembly.

 

Preparing phosphorylated peptides with multiple adjacent phosphorylations is synthetically difficult, leads to β-elimination, results in low yields, and is extremely slow. We combined synthetic chemical methodologies with computational studies and engineering approaches to develop a strategy that takes advantage of fast stirring, high temperature, and a very low concentration of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to produce multiphosphorylated peptides at an extremely rapid time and high purity.

 

Heparan sulfate glycosaminoglycans provides extracellular matrix defense against heavy metals cytotoxicity. Identifying the precise glycan sequences that bind a particular heavy metal ion is a key for understanding those interactions. Here, electrochemical and surface characterization techniques were used to elucidate the relation between the glycans structural motifs, uronic acid stereochemistry, and sulfation regiochemistry to heavy metal ions binding. A divergent strategy was employed to access a small library of structurally well-defined tetrasaccharides analogs with different sulfation patterns and uronic acid compositions. These tetrasaccharides were electrochemically grafted onto glassy carbon electrodes and their response to heavy metal ions was monitored by electrochemical impedance spectroscopy. Key differences in the binding of Hg(II), Cd(II), and Pb(II) were associated with a combination of the uronic acid type and the sulfation pattern

 

Short proteoglycan fragments are of great importance for biochemical research. The solid-phase synthesis of such glycopeptides relies on excessive use of glycosylated amino acids, extended reaction times, and additional post-assembly deprotection protocols. We employed high-shear mixing for expedient and equimolar O-glycopeptide assembly. We further developed a stirring-based deprotection on the solid support, thus completing the synthesis of a glycopeptide library in a minimal amount of time and purification hurdles. Publisher's Version

 

 

 

 

Sensing enzymatic sialylation provides new tools for the evaluation of pathological events and pathogen invasion. Enzymatic sialylation is usually monitored via fluorescence or metabolic labeling, which requires relatively large amounts of the glycan substrate with limited availability. Using a label-free biosensor requires smaller quantities of substrates because the interactions induce measurable changes to an interface, which can be translated into a signal. The downside of label-free biosensors is that they are very sensitive to changes at the interface, and the properties of the surface layer can play a major role. Electrochemical impedance spectroscopy was used here to follow the enzymatic sialylation of a biantennary N-glycan acceptor in mixed monolayers. The surfaces contained either neutral, positively or negatively charged, or zwitterionic functional groups. The systems were characterized by contact potential difference, ellipsometry, and contact angle analyses. We found that the characteristics of the mixed monolayer have a profound effect on the biosensing of the enzymatic sialylation. Positively charged layers were found to adsorb the enzyme under the reaction conditions. Negatively charged and zwitterionic surfaces were nonresponsive to enzymatic sialylation. Only the neutral mixed monolayers provided signals that were related directly to enzymatic sialylation. This work demonstrates the importance of appropriate interface properties for monitoring enzymatic sialylation processes. 

 

 

 

Accelerating solid-phase synthesis is crucial for accessing a large number of peptides in a short time. Since standard peptide synthesis is usually done under poor diffusion conditions with slow or no mixing of the solid support, acceleration of the process is achieved by applying a large excess of reagents. In this work, overhead stirring and heating were combined to provide accelerated solid-phase peptide synthesis without using an excess of reagent. A new setup that allows both heating and fast stirring was designed specifically for research laboratory-scale peptide synthesis. By increasing the diffusion of both reagents and beads in a narrow dimension reactor, solid-phase reactions were done in seconds and medium-size peptides were synthesized in minutes. 

 

 

 

 

Cytokines such as interleukin-8 activate the immune system during infection and interact with sulfated glycosaminoglycans with specific sulfation patterns. In some cases, these interactions are mediated by metal ion binding which can be used to tune surface-based glycan-protein interactions. We evaluated the effect of both hyaluronan sulfation degree and Fe3+ on interleukin-8 binding by electrochemical impedance spectroscopy and surface characterizations. Our results show that sulfation degree and metal ion interactions have a synergistic effect in tuning the electrochemical response of the glycated surfaces to the cytokine.