Surface-Controlled Sialoside-Based Biosensing of Viral and Bacterial Neuraminidases.

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.

Accelerated Solid Phase Glycan Synthesis: ASGS.

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 are completed in minutes.

Unexpected Nucleophile Masking in Acyl Transfer to Sterically Crowded and Conformationally Restricted Galactosides.

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.

Accelerated solid-phase synthesis of glycopeptides containing multiple N-glycosylated sites.

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.

Impedimetric Bacterial Detection Using Random Antimicrobial Peptide Mixtures.

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.

Sulfation Pattern Dependent Iron(III) Mediated Interleukin-8 Glycan Binding.

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.

Profiling Heparan Sulfate-Heavy Metal Ions Interaction Using Electrochemical Techniques.

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.

Effect of Interfacial Properties on Impedimetric Biosensing of the Sialylation Process with a Biantennary N-Glycan-Based Monolayer.

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.

Determining the structure and binding mechanism of oxytocin-Cu2+ complex using paramagnetic relaxation enhancement NMR analysis.

Oxytocin is a neuropeptide that binds copper ions in nature. The structure of oxytocin in interaction with Cu2+ was determined here by NMR, showing which atoms of the peptide are involved in binding. Paramagnetic relaxation enhancement NMR analyses indicated a binding mechanism where the amino terminus was required for binding and subsequently Tyr2, Ile3 and Gln4 bound in that order. The aromatic ring of Tyr2 formed a π-cation interaction with Cu2+. Oxytocin copper complex structure revealed by paramagnetic relaxation enhancement NMR analyses.

Multiphosphorylated peptides: importance, synthetic strategies, and applications for studying biological mechanisms.

Unraveling the role of post-translational modification (PTM) patterns is one of the most urgent and unresolved issues facing the scientific community. Attempts to crack the phosphorylation bio-barcode led to significant findings, which suggest that many proteins cannot be regarded as a single entity but exist as several forms which differ in their phosphorylation patterns and their functions. While protein regions that do not contain PTMs can be rather simply mimicked using peptide libraries, heavily phosphorylated regions are much harder to study using the same tools. The differences between the syntheses of simple mono-, di- and tri-phosphopeptides and the synthesis of multiphosphopeptides are dramatic. While simple phosphopeptides can be synthesized using almost standard SPPS strategies, the synthesis of multiphosphopeptides is to date a major synthetic challenge. Synthesis of multiphosphopeptides requires the insertion of several phosphate groups simultaneously or sequentially into various positions on the peptide in the presence of many other potential modification sites. These groups are bulky, unstable and cannot be easily introduced when in close proximity. Moreover, since the same protein region can possess many alternative multiphosphorylation patterns, libraries comprising a large number of peptides with different degrees and positions of phosphorylation are essential. Many strategies have been developed to provide routes to enable the preparation of multiphosphopeptides. These methods are essentially different from the methods used for the preparation of simple phosphopeptides. In this review, we specifically emphasize the challenges and importance of synthesizing multiphosphopeptides and their libraries. The historical perspective and state of the art strategies are described. We demonstrate here how the different synthetic approaches attempt to address the special problems associated with the synthesis of multiphosphopeptides. The advantages and disadvantages of each strategy are discussed in order to provide a roadmap for the synthesis of such libraries. An overview of the existing strategies and some comments regarding future directions are provided. Applications of multiphosphopeptide libraries as tools to study the effect of phosphorylation patterns on the biological function of proteins are also described.

The breaking beads approach for photocleavage from solid support.

Photocleavage from polystyrene beads is a pivotal reaction for solid phase synthesis that relies on photolabile linkers. Photocleavage from intact porous polystyrene beads is not optimal because light cannot penetrate into the beads and the surface area exposed to irradiation is limited. Thus, hazardous, technically challenging and expensive setups are used for photocleavage from intact beads. We developed a new concept in which grinding the beads during or prior to irradiation is employed as an essential part of the photocleavage process. By grinding the beads we are exposing more surface area to the light source, hence, photocleavage can be performed even using a simple benchtop LED setup. This approach proved very efficient for photocleavage of various model compounds including fully protected oligosaccharides.

Automated glycan assembly using the Glyconeer 2.1 synthesizer.

Reliable and rapid access to defined biopolymers by automated DNA and peptide synthesis has fundamentally altered biological research and medical practice. Similarly, the procurement of defined glycans is key to establishing structure-activity relationships and thereby progress in the glycosciences. Here, we describe the rapid assembly of oligosaccharides using the commercially available Glyconeer 2.1 automated glycan synthesizer, monosaccharide building blocks, and a linker-functionalized polystyrene solid support. Purification and quality-control protocols for the oligosaccharide products have been standardized. Synthetic glycans prepared in this way are useful reagents as the basis for glycan arrays, diagnostics, and carbohydrate-based vaccines.

Sulfation Patterns of Saccharides and Heavy Metal Ion Binding.

Sulfated saccharides are an essential part of extracellular matrices, and they are involved in a large number of interactions. Sulfated saccharide matrices in organisms accumulate heavy metal ions in addition to other essential metal ions. Accumulation of heavy metal ions alters the function of the organisms and cells, resulting in severe and irreversible damage. The effect of the sulfation pattern of saccharides on heavy metal binding preferences is enigmatic because the accessibility to structurally defined sulfated saccharides is limited and because standard analytical techniques cannot be used to quantify these interactions. We developed a new strategy that combines enzymatic and chemical synthesis with surface chemistry and label-free electrochemical sensing to study the interactions between well-defined sulfated saccharides and heavy metal ions. By using these tools we showed that the sulfation pattern of hyaluronic acid governs their heavy metal ions binding preferences.

Direct Assembly and Metal-Ion Binding Properties of Oxytocin Monolayer on Gold Surfaces.

Peptides are very common recognition entities that are usually attached to surfaces using multistep processes. These processes require modification of the native peptides and of the substrates. Using functional groups in native peptides for their assembly on surfaces without affecting their biological activity can facilitate the preparation of biosensors. Herein, we present a simple single-step formation of native oxytocin monolayer on gold surface. These surfaces were characterized by atomic force spectroscopy, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy. We took advantage of the native disulfide bridge of the oxytocin for anchoring the peptide to the Au surface, while preserving the metal-ion binding properties. Self-assembled oxytocin monolayer was used by electrochemical impedance spectroscopy for metal-ion sensing leading to subnanomolar sensitivities for zinc or copper ions.

A targeted approach for the synthesis of multi-phosphorylated peptides: a tool for studying the role of phosphorylation patterns in proteins.

Protein phosphorylation barcodes, clusters of several phosphorylation sites within a short unfolded region, control many cellular processes. Existing biochemical methods used to study the roles of these barcodes suffer from low selectivity and provide only qualitative data. Chemically synthesized multiphosphopeptide libraries are selective and specific, but their synthesis is extremely difficult using the current peptide synthesis methods. Here we describe a new microwave assisted approach for synthesizing a library of multiphosphopeptides, using the C-terminus of rhodopsin as a proof of concept. Our approach utilizes multiple protocols for synthesizing libraries of multiphosphopeptides instead of the inefficient single protocol methods currently used. Using our approach we demonstrated the synthesis with up to seven phosphorylated amino acids, sometimes next to each other, an accomplishment that was impractical before. Synthesizing the Rhodopsin derived multiphosphopeptide library enabled dissecting the precise phosphorylation barcode required for the recruitment, activation and modulation of the conformation of Arrestin. Since phosphorylation barcodes modulate the activity of hundreds of GPCRs, synthesizing libraries of multiphosphopeptides is the method of choice for studying their molecular mechanisms of action. Our approach provides an invaluable tool for evaluating how protein phosphorylation barcodes regulate their activity.

Detection of Cu2+ Ions with GGH Peptide Realized with Si-Nanoribbon ISFET.

The presence of heavy metal ions such as copper in the human body at certain concentrations and specific conditions can lead to the development of different diseases. The currently available analytical detection methods remain expensive, time-consuming, and often require sample pre-treatment. The development of specific and quantitative, easy-in-operation, and cost-effective devices, capable of monitoring the level of Cu2+ ions in environmental and physiological media, is necessary. We use silicon nanoribbon (SiNR) ion-sensitive field effect transistor (ISFET) devices modified with a Gly-Gly-His peptide for the detection of copper ions in a large concentration range. The specific binding of copper ions causes a conformational change of the ligand, and a deprotonation of secondary amine groups. By performing differential measurements, we gain a deeper insight into the details of the ion-ligand interaction. We highlight in particular the importance of considering non-specific interactions to explain the sensors' response.

DMAP-assisted sulfonylation as an efficient step for the methylation of primary amine motifs on solid support.

Several multistep strategies were developed to ensure single methylation of amines on solid support. These strategies rely on the introduction of the o-NBS protecting/activating group as a key step. We found that the state-of-the-art strategies fail for the methylation of several primary amine motifs, largely due to inefficient sulfonylation. Here we show that using the superior nucleophilic base DMAP instead of the commonly used base collidine as a sulfonylation additive is essential for the introduction of the o-NBS group to these amine motifs. DFT calculations provide an explanation by showing that the energy barrier of the DMAP intermediate is significantly lower than the one of the collidine. We demonstrate that using DMAP as a sole additive in the sulfonylation step results in an overall effective and regioselective N-methylation. The method presented herein proved highly efficient in solid-phase synthesis of a somatostatin analogue bearing three Nα-methylation sites that could not be synthesized using the previously described state-of-the-art methods.

Automated glycan assembly of a S. pneumoniae serotype 3 CPS antigen.

Vaccines against S. pneumoniae, one of the most prevalent bacterial infections causing severe disease, rely on isolated capsular polysaccharide (CPS) that are conjugated to proteins. Such isolates contain a heterogeneous oligosaccharide mixture of different chain lengths and frame shifts. Access to defined synthetic S. pneumoniae CPS structures is desirable. Known syntheses of S. pneumoniae serotype 3 CPS rely on a time-consuming and low-yielding late-stage oxidation step, or use disaccharide building blocks which limits variability. Herein, we report the first iterative automated glycan assembly (AGA) of a conjugation-ready S. pneumoniae serotype 3 CPS trisaccharide. This oligosaccharide was assembled using a novel glucuronic acid building block to circumvent the need for a late-stage oxidation. The introduction of a washing step with the activator prior to each glycosylation cycle greatly increased the yields by neutralizing any residual base from deprotection steps in the synthetic cycle. This process improvement is applicable to AGA of many other oligosaccharides.

Backbone cyclic helix mimetic of chemokine (C-C motif) receptor 2: a rational approach for inhibiting dimerization of G protein-coupled receptors.

The transmembrane helical bundle of G protein-coupled receptors (GPCRs) dimerize through helix-helix interactions in response to inflammatory stimulation. A strategy was developed to target the helical dimerization site of GPCRs by peptidomimetics with drug like properties. The concept was demonstrated by selecting a potent backbone cyclic helix mimetic from a library that derived from the dimerization region of chemokine (C-C motif) receptor 2 (CCR2) that is a key player in Multiple Sclerosis. We showed that CCR2 based backbone cyclic peptide having a stable helix structure inhibits specific CCR2-mediated chemotactic migration.

Biocatalysis versus Molecular Recognition in Sialoside-Selective Neuraminidase Biosensing.

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.