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.

 

Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin 1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: `key sites' required for arrestin binding and activation, an `inhibitory site' that abrogates arrestin binding, and `modulator sites' that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.

 

We report how the electron transport through a solid-state metal/Gly-Gly-His (GGH) tripeptide monolayer/metal junction and the metal/GGH work function (WF) are modified by the GGH complexation with Cu2+ ions. Conducting atomic force microscopy is used to measure the current-voltage histograms. The WF is characterized by combining macroscopic Kelvin probe and Kelvin probe force microscopy at the nanoscale. We observe that the complexation of Cu2+ ions with the GGH monolayer is highly dependent on the molecular surface density and results in opposite trends. In the case of a high-density monolayer the conformational changes are hindered by the proximity of the neighboring peptides, hence forming an insulating layer in response to copper complexation. However, the monolayers of a slightly lower density allow for the conformational change to a looped peptide wrapping the Cu-ion, which results in a more conductive monolayer. Copper-ion complexation to the high- and low-density monolayers systematically induces an increase of the WFs. Copper-ion complexation to the low-density monolayer induces an increase of electron-transport efficiency, whereas the copper-ion complexation to the high-density monolayer results in a slight decrease of electron transport. Both of the observed trends agree with first-principle calculations. Complexation of copper to the low-density GGH monolayer induces a new gap state slightly above the Au Fermi energy that is absent in the high-density monolayer.

 

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.

 

Mixing of polystyrene resins in solid-phase synthesis is performed by shaking or gentle agitation of the reaction vessel to avoid breaking the brittle beads. These mixing strategies result in poor diffusion to and into the beads. Using a large excess of reagents is the common way to compensate for these deficiencies. We use fast overhead stirring for performing coupling reactions on a solid support. We show that fast overhead stirring enhances the efficiency of amide bond formation on the solid support compared to the state-of-the-art mixing method, while preserving the integrity of the beads. We find that fast overhead stirring minimizes the effect of decomposition of the activated species by increasing the diffusion-dependent coupling reaction. This allows decreasing the excess of reagents used for the multistep synthesis of peptides, thus providing a greener and more sustainable alternative for peptide synthesis on solid supports.

 

A sulfation-controlled binding of heavy metal ions to glycans was realized using electrochemical analysis. The artwork presents glassy carbon electrodes modified by a series of hyaluronans with similar sizes but different sulfation patterns. The binding of heavy metal ions to these surfaces resulted in changes of the layer density that produced impedimetric response. The impedimetric response revealed that the selectivity of the hyaluronans to a specific metal ion depends on their sulfation pattern. More information can be found in the Full Paper by J. Rademann, S. Yitzchaik, M. Hurevich, et al. (DOI: 10.1002/chem.201901538).

 

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.

 

The brittle nature of the polymer beads used in SPPS dictates mild mixing techniques with low mass transfer. We demonstrate that vigorous overhead mechanical stirring with superior mass transfer properties kept the beads intact and significantly accelerates reaction kinetics and efficiency.

 

We report the modulation of the specific metal gation properties of a peptide and demonstrate a highly selective sensor for copper(II) ion. The neuropeptide oxytocin (OT) is reported for its high affinity towards Zn2+ and Cu2+ at physiological pH. The binding of the metal ions to OT is tuned by altering the pH of the medium. OT was self-assembled on glassy carbon electrode using surface chemistry, and electrochemical impedance spectroscopy (EIS) was used to probe the binding of Cu2+. Our results clearly indicate that at pH 10.0, the binding of Cu2+ to OT is increased compared to that at pH 7.0, while the binding to Zn2+ becomes almost negligible. This proves that the selectivity of OT towards each of the ions can be regulated simply by controlling the pH of the medium and hence allows the preparation of a sensing device with selectivity to Cu2+.

 

Permanent protecting groups are essential for oligosaccharide synthesis. However, the removal of the traditionally used protecting groups is not trivial and demands considerable expertise. Using photolabile protecting groups as permanent protection for glycan can overcome many limitations associated with the traditional oligosaccharide synthesis approach. It is demonstrated here that up to eight photolabile protecting groups can be readily removed from a single glycan using a benchtop LED setup that is very easy to operate. This report suggests that further development of the strategy will offer an attractive alternative for oligosaccharide synthesis.

 

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.

 

Copper ions play a major role in biological processes. Abnormal Cu2+ ions concentrations are associated with various diseases, hence, can be used as diagnostic target. Monitoring copper ion is currently performed by non-portable, expensive and complicated to use equipment. We present a label free and a highly sensitive electrochemical ion-detecting biosensor based on a Gly-Gly-His tripeptide layer that chelate with Cu2+ ions. The proposed sensing mechanism is that the chelation results in conformational changes in the peptide that forms a denser insulating layer that prevents RedOx species transfer to the surface. This chelation event was monitored using various electrochemical methods and surface chemistry analysis and supported by theoretical calculations. We propose a highly sensitive ion-detection biosensor that can detect Cu2+ ions in the pM range with high SNR parameter.

 

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 Na-methylation sites that could not be synthesized using the previously described state-of-the-art methods.

 

Zinc and copper are essential metal ions for numerous biological processes. Their levels are tightly maintained in all body organs. Impairment of the Zn2+ to Cu2+ ratio in serum was found to correlate with many disease states, including immunological and inflammatory disorders. Oxytocin (OT) is a neuropeptide, and its activity is modulated by zinc and copper ion binding. Harnessing the intrinsic properties of OT is one of the attractive ways to develop valuable metal ion sensors. Here, we report for the first time an OT-based metal ion sensor prepared by immobilizing the neuropeptide onto a glassy carbon electrode. The developed impedimetric biosensor was ultrasensitive to Zn2+ and Cu2+ ions at physiological pH and not to other biologically relevant ions. Interestingly, the electrochemical impedance signal of two hemicircle systems was recorded after the attachment of OT to the surface. These two semicircles suggest two capacitive regions that result from two different domains in the OT monolayer. Moreover, the change in the charge-transfer resistance of either Zn2+ or Cu2+ was not similar in response to binding. This suggests that the metal-dependent conformational changes of OT can be translated to distinct impedimetric data. Selective masking of Zn2+ and Cu2+ was used to allow for the simultaneous determination of zinc to copper ions ratio by the OT sensor. The OT sensor was able to distinguish between healthy control and multiple sclerosis patients diluted sera samples by determining the Zn/Cu ratio similar to the state-of-the-art techniques. The OT sensor presented herein is likely to have numerous applications in biomedical research and pave the way to other types of neuropeptide-derived sensors.

 

 

Automated glycan assembly (AGA) has advanced from a concept to a commercial technology that rapidly provides access to diverse oligosaccharide chains as long as 30-mers. To date, AGA was mainly employed to incorporate trans-glycosidic linkages, where C2 participating protecting groups ensure stereoselective couplings. Stereocontrol during the installation of cis-glycosidic linkages cannot rely on C2-participation and anomeric mixtures are typically formed. Here, we demonstrate that oligosaccharides containing multiple cis-glycosidic linkages can be prepared efficiently by AGA using monosaccharide building blocks equipped with remote participating protecting groups. The concept is illustrated by the automated syntheses of biologically relevant oligosaccharides bearing various cis-galactosidic and cis-glucosidic linkages. This work provides further proof that AGA facilitates the synthesis of complex oligosaccharides with multiple cis-linkages and other biologically important oligosaccharides.

 

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.

We present a new approach for the covalent inhibition of HIV-1 integrase (IN) by an LEDGF/p75-derived peptide modified with an N-terminal succinimide group. The covalent inhibition is mediated by direct binding of the succinimide to the amine group of a lysine residue in IN. The peptide serves as a specific recognition sequence for the target protein, while the succinimide serves as the binding moiety. The combination of a readily synthesizable peptide precursor with easy and efficient binding to the target protein makes this approach a promising new strategy for designing lead compounds.

 

Chirality is an important aspect in many pharmacological processes including drug transport and metabolism. The current investigation examined the stereospecific transport and entry inhibitory activity of four diastereomers derived from a small (macrocyclic) molecule that has two chiral centers. These molecules were designed to mimic the interaction between CD4 and gp120 site of HIV-1 and thereby to function as entry inhibitor(s). Intestinal permeability was assessed by ex-vivo model using excised rat intestine mounted in side-byside diffusion chambers. The entry inhibitory activity was monitored using indicator HeLa-CD4-LTR-beta-gal cells (MAGI assay). The (S/S) diastereomer, named CG-1, exhibited superiority in both unrelated tested biological processes: (I) high transport through the intestine and (II) entry inhibition activity (in the low mu M range). The permeability screening revealed a unique transporter-mediated absorption pathway of CG-1, suggesting a significant role of the molecule's conformation on the mechanism of intestinal absorption. Here we highlight that only the S, S enantiomer (CG-1) has both (I) promising anti HIV-1 entry inhibitory properties and (II) high transporter mediated intestinal permeability. Hence we suggest preference in pharmacological processes to the S, S conformation. This report augments the knowledge regarding stereoselectivity in receptor mediated and protein-protein interaction processes. (C) 2015 Elsevier B.V. All rights reserved.

 

G-protein coupled receptors (GPCRs) mediate a large number of biological pathways and are major therapeutic targets. One of the most exiting phenomena of GPCRs is their ability to interact with other GPCRs. GPCR-GPCR interactions, also known as GPCR oligomerization, may create various functional entities such as homo- and heterodimers and also form complex multimeric GPCR clusters. In many biological systems, GPCR-GPCR interactions are crucial for signal regulation. The interaction with other receptors results in allosteric modifications of GPCRs through conformational changes. Allosteric inhibition of GPCRs is considered an attractive strategy for drug development and does not involve targeting the orthosteric site. Understanding the nature of GPCR-GPCR interactions is mandatory for developing allosteric inhibitors. Studying GPCR-GPCR interactions is a challenging task and many methods have been developed to analyze these events. This review will highlight some of the methods developed to study GPCR-GPCR interactions and will describe pivotal studies that provided the basic understanding of the importance of GPCR oligomerization. We will also describe the significance of GPCR interaction networks for drug development. Recent studies will be reviewed to illustrate the use of state-of-the-art biophysical and spectroscopic methods for the discovery of GPCR oligomerization modulators.

 

Current strategies for the synthesis of glycopeptides require multiple manual synthetic steps. Here, we describe a synthesis concept that merges solid phase peptide and oligosaccharide syntheses and can be executed automatically using a single instrument.