synthesis-structure-determination-conotoxin-piiia-isomers-with

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

University of Bonn

This review aims to provide a comprehensive overview of the recent advances made in the field of ionic liquids in peptide chemistry and peptide analytics.

Ionic liquid applications in peptide chemistry: synthesis, purification and analytical characterization processes.

The oxidation of the conotoxin μ-SIIIA in different ionic liquids was investigated, and the results were compared with those obtained in [C2 mim][OAc]. Conversion of the reduced precursor into the oxidized product was observed in the protic ILs methyl- and ethylammonium formate (MAF and EAf, respectively), whereas choline dihydrogenphosphate and Ammoeng 110 failed to yield folded peptide. However, the quality and yield of the peptide obtained in MAF and EAF were lower than in the case of the product from [C2 mim][OAc]. Reaction conditions (temperature, water content) also had an impact on peptide conversion. A closer look at the activities of μ-SIIIA versions derived from an up-scaled synthesis in [C2 mim][OAc] revealed a significant loss of the effect on ion channel NaV 1.4 relative to the buffer-oxidized peptide, whereas digestion of either μ-SIIIA product by trypsin was unaffected. This was attributed to adherence of ions from the IL to the peptide, because the disulfide connectivity is basically the same for the differentially oxidized μ-SIIIA versions. 

Application of room-temperature aprotic and protic ionic liquids for oxidative folding of cysteine-rich peptides.

This review focuses on chemical ligation methods for the preparation of oligopeptides and proteins. Chemical ligation is a practical and convenient methodology in peptide and protein synthesis. Longer peptides and proteins can be obtained with high yield in aqueous buffer solutions by coupling unprotected peptide segments even without activation by enzymes or further chemical agents. Several methods and protocols were developed in the past. The potential of the most important approaches of the thioester- and imine-ligation techniques is demonstrated by a broad spectrum of applications. In addition, special features and protocols such as the template-directed ligation, ligation with novel additives or solvent media, microwave-assisted ligation, and the achievements obtained with those are also highlighted herein. 

Synthetic strategies for polypeptides and proteins by chemical ligation.

Propylene carbonate (PC) is a non-toxic solvent currently used in various pharmaceutical formulations. Consequently, a simple, cost-effective and most accurate analytical method for the quantification of this optical inert solvent is of major interest. Based on a consecutive three-step reaction 3,5-diacetyl-1,4-dihydro-2,6-lutidine was obtained from PC and used for quantification by either UV and fluorescent detection. Data were compared with results from LC-ESI-MS as a reference method. After using Mandel's test for linearity assessment of the calibration curves, linear fitting was used for LC-ESI-MS and spectrofluorimetry, while a polynomial 3rd order curve fitting was used for spectrophotometry. High intra- and inter-day precision as well as high accuracy were confirmed for all three analytical methods (spectrophotometry, spectrofluorimetry and LC-ESI-MS). The comparison of all three methods was assessed using correlation coefficients and Bland-Altman plots, both showing satisfying results with a high degree of agreement. The new method confirmed its applicability for PC quantification in two formulations, namely a PC-enriched cream and polyester microimplants. This new quantification method for PC is a reliable alternative to highly sophisticated chromatographic methods. 

Propylene carbonate quantification by its derivative 3,5-diacetyl-1,4-dihydro-2,6-lutidine.

Molecular interaction of δ-conopeptide EVIA with voltage-gated Na(+) channels.

Although toxic when inhaled in high concentrations, the gas carbon monoxide (CO) is endogenously produced in mammals, and various beneficial effects are reported. For potential medicinal applications and studying the molecular processes underlying the pharmacological action of CO, so-called CO-releasing molecules (CORMs), such as tricabonyldichlororuthenium(II) dimer (CORM-2), have been developed and widely used. Yet, it is not readily discriminated whether an observed effect of a CORM is caused by the released CO gas, the CORM itself, or any of its intermediate or final breakdown products. Focusing on Ca- and voltage-dependent K channels (K1.1) and voltage-gated K channels (Kv1.5, Kv11.1) relevant for cardiac safety pharmacology, we demonstrate that, in most cases, the functional impacts of CORM-2 on these channels are not mediated by CO. Instead, when dissolved in aqueous solutions, CORM-2 has the propensity of forming Ru(CO) adducts, preferentially to histidine residues, as demonstrated with synthetic peptides using mass-spectrometry analysis. For K1.1 channels we show that H365 and H394 in the cytosolic gating ring structure are affected by CORM-2. For Kv11.1 channels (hERG1) the extracellularly accessible histidines H578 and H587 are CORM-2 targets. The strong CO-independent action of CORM-2 on Kv11.1 and Kv1.5 channels can be completely abolished when CORM-2 is applied in the presence of an excess of free histidine or human serum albumin; cysteine and methionine are further potential targets. Off-site effects similar to those reported here for CORM-2 are found for CORM-3, another ruthenium-based CORM, but are diminished when using iron-based CORM-S1 and absent for manganese-based CORM-EDE1.

CO-independent modification of K channels by tricarbonyldichlororuthenium(II) dimer (CORM-2).

A series of small, catalytically active metallopeptides, which were derived from the nickel superoxide dismutase (NiSOD) active site were employed to study the mechanism of superoxide degradation especially focusing on the role of the axial imidazole ligand. In the literature, there are contradicting propositions about the catalytic importance of the N-terminal histidine. Therefore, we studied the stability and activity of a set of eight NiSOD model peptides, which represent the major model systems discussed in the literature to date, yet differing in their length and their Ni-coordination. UV-Vis-coupled stopped-flow kinetic measurements and mass spectrometry analysis unveiled their high oxidation sensitivity in the presence of oxygen and superoxide resulting into a much faster Ni(II)-peptide degradation for the amine/amide Ni(II) coordination than for the catalytically inactive bis-amidate Ni(II) coordination. With respect to these results we determined the catalytic activities for all NiSOD mimics studied herein, which turned out to be in almost the same range of about 2 × 10 M s. From these experiments, we concluded that the amine/amide Ni(II) coordination is clearly the key factor for catalytic activity. Finally, we were able to clarify the role of the N-terminal histidine and to resolve the contradictory literature propositions, reported in previous studies.

New insights into the mechanism of nickel superoxide degradation from studies of model peptides.

Peptides and proteins carrying high numbers of cysteines can adopt various 3D structures depending on their disulfide connectivities. The unambiguous verification of such conformational isomers with more than two disulfide bonds is extremely challenging, and experimental strategies for their unequivocal structural analysis are largely lacking. We synthesized all 15 possible isomers of the 22mer conopeptide μ-PIIIA and applied 2D NMR spectroscopy and MS/MS for the elucidation of its structure. This study provides intriguing insights in how the disulfide connectivity alters the global fold of a toxin. We also show that analysis procedures involving comprehensive combinations of conventional methods are required for the unambiguous assignment of disulfides in cysteine-rich peptides and proteins and that standard compounds are crucially needed for the structural analysis of such complex molecules.

Conformational μ-Conotoxin PIIIA Isomers Revisited: Impact of Cysteine Pairing on Disulfide-Bond Assignment and Structure Elucidation.

Direct targeting of intracellular Gα subunits of G protein-coupled receptors by chemical tools is a challenging task in current pharmacological studies and in the development of novel therapeutic approaches. In this study we analyzed novel FR900359-based analogs from natural sources, synthetic cyclic peptides, as well as all so-far known G α inhibitors in a comprehensive study to devise a strategy for the elucidation of characteristics that determine interactions with and inhibition of G in the specific FR/YM-binding pocket. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocyclic structures responsible for binding to the target protein correlating with inhibitory activity. While all novel compounds were devoid of effects on G and G proteins, no inhibitor surpassed the biological activity of FR. This raises the question of whether depsipeptides such as FR already represent valuable chemical tools for specific inhibition of G and, at the same time, are suitable natural lead structures for the development of novel compounds to target Gα subunits other than G .

Article	Year
Ionic liquid applications in peptide chemistry: synthesis, purification and analytical characterization processes. Molecules (Basel, Switzerland)\| PubMed ID: 22481538	2012
Application of room-temperature aprotic and protic ionic liquids for oxidative folding of cysteine-rich peptides. Chembiochem : a European journal of chemical biology\| PubMed ID: 25376613	2014
Synthetic strategies for polypeptides and proteins by chemical ligation. Amino acids\| PubMed ID: 25894893	2015
Propylene carbonate quantification by its derivative 3,5-diacetyl-1,4-dihydro-2,6-lutidine. Talanta\| PubMed ID: 26946012	2016
Molecular interaction of δ-conopeptide EVIA with voltage-gated Na(+) channels. Biochimica et biophysica acta\| PubMed ID: 27316322	2016
CO-independent modification of K channels by tricarbonyldichlororuthenium(II) dimer (CORM-2). European journal of pharmacology\| PubMed ID: 28987271	2017
New insights into the mechanism of nickel superoxide degradation from studies of model peptides. Scientific reports\| PubMed ID: 29222438	2017
Conformational μ-Conotoxin PIIIA Isomers Revisited: Impact of Cysteine Pairing on Disulfide-Bond Assignment and Structure Elucidation. Analytical chemistry\| PubMed ID: 29397705	2018
Deciphering Specificity Determinants for FR900359-Derived G α Inhibitors Based on Computational and Structure-Activity Studies. ChemMedChem\| PubMed ID: 29873888	2018

Pascal Heimer

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