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.
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.
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 Nα-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.