Cysteine Residues In Peptides Research Articles

Synthesis and stability studies of constrained peptide–antimony bicycles

Peptide therapeutics play an increasingly important role in modern drug discovery. Improving the pharmacokinetic profile of bioactive peptides has been effectively achieved with chemical modifications, especially macrocyclisation reactions. Consequently, there is a great demand for highly constrained compounds such as bicyclic peptides. In our previous research, we introduced peptide–bismuth bicycles and peptide–arsenic bicycles as new classes of constrained peptides. In this work, we extend our peptide bicyclisation strategy towards antimony. Similar to arsenic and bismuth, antimony(III) selectively binds to three cysteine residues in peptides, enabling the in situ formation of stable bicycles. The bicyclisation reaction occurs instantaneously under biocompatible conditions at physiological pH. Antimony–peptide bicycles remain largely intact in the presence of the common metal chelator ethylenediaminetetraacetic acid (EDTA) and the main endogenous thiol competitor glutathione (GSH). Furthermore, when challenged with bismuth(III) from water-soluble gastrodenol (bismuth tripotassium dicitrate), antimony–peptide bicycles convert into the corresponding bismuth–peptide bicycle, highlighting the superior thiophilicity of bismuth over other pnictogens. Our study further expands the toolbox of peptide multicyclisation with main group elements previously underexplored in chemical biology.

Enzymatic Generation of Thioaldehyde Motifs by Flavin-Dependent Cysteine Decarboxylases for Peptide Bioconjugation and Macrocyclization.

Thioaldehyde is a highly electrophilic group under aqueous conditions and can be generated via oxidative enzymatic modifications of cysteine residues in peptides and proteins. Herein, we report the installation of thioaldehyde and aldehyde groups at the C-terminus of peptides by flavin-dependent cysteine decarboxylases from the biosynthesis of ribosomally synthesized and post-translationally modified peptides. The in situ generated (thio)aldehyde is utilized as a reactive handle for peptide bioconjugation and macrocyclization.

A 2-formylphenylboronic acid (2FPBA)-maleimide crosslinker: a versatile platform for Cys-peptide-hydrazine conjugation and interplay.

In this work, we describe the preparation of a heterobifunctional 2-formylphenylboronic acid (2-FPBA)-maleimide crosslinker and explore its versatility in the preparation of various bioconjugates. We demonstrate the straightforward attachment of hydrazine payloads to cysteine residues in peptides, as well as the crosslinking of different thiol-bearing peptides or payloads with N-terminal cysteine peptides. Importantly, the dynamic nature of the 2-FPBA handle enables an interplay between the thiazolidine and diazaborine forms, which allows obtaining various products controlled by (and in some cases independent of) the order of addition of the components.

Directed Disulfide Pairing and Folding of Peptides for the De Novo Development of Multicyclic Peptide Libraries.

Disulfide-rich peptides (DRPs) have been an emerging frontier for drug discovery. There have been two DRPs approved as drugs (i.e., Ziconotide and Linaclotide), and many others are undergoing preclinical studies or in clinical trials. All of these DRPs are of nature origin or derived from natural peptides. It is still a challenge to design new DRPs without recourse to natural scaffolds due to the difficulty in handling the disulfide pairing. Here we developed a simple and robust strategy for directing the disulfide pairing and folding of peptides with up to six cysteine residues. Our strategy exploits the dimeric pairing of CPPC (cysteine-proline-proline-cysteine) motifs for directing disulfide formation, and DRPs with different multicyclic topologies were designed and synthesized by regulating the patterns of CPPC motifs and cysteine residues in peptides. As neither sequence manipulations nor unnatural amino acids are involved, the designed DRPs can be used as templates for the de novo development of biosynthetic multicyclic peptide libraries, enabling selection of DRPs with new functions directly from fully randomized sequences. We believe that this work represents as an important step toward the discovery and design of new multicyclic peptide ligands and therapeutics with structures not derived from natural scaffolds.

Vinylphosphonites for Staudinger-induced chemoselective peptide cyclization and functionalization.

In this paper, we introduce vinylphosphonites for chemoselective Staudinger-phosphonite reactions (SPhR) with azides to form vinylphosphonamidates for the subsequent modification of cysteine residues in peptides and proteins. An electron-rich alkene is turned into an electron-deficient vinylphosphonamidate, thereby inducing electrophilic reactivity for a following thiol addition. We show that by varying the phosphonamidate ester substituent we can fine-tune the reactivity of the thiol addition and even control the functional properties of the final conjugate. Furthermore, we observed a drastic increase in thiol addition efficiency when the SPhR is carried out in the presence of a thiol substrate in a one-pot reaction. Hence, we utilize vinylphosphonites for the chemoselective intramolecular cyclization of peptides carrying an azide-containing amino acid and a cysteine in high yields. Our concept was demonstrated for the stapling of a cell-permeable peptidic inhibitor for protein-protein interaction (PPI) between BCL9 and beta-catenin, which is known to create a transcription factor complex playing a role in embryonic development and cancer origin, and for macrocyclization of cell-penetrating peptides (CPPs) to enhance the cellular uptake of proteins.

Stereoretentive C( sp3)-S Cross-Coupling.

We report a stereoretentive cross-coupling reaction of configurationally stable nucleophiles with disulfide and N-sulfenylsuccinimide donors promoted by Cu(I). We demonstrate the utility of this method in the synthesis of thioglycosides derived from simple alkyl and aryl thiols, thioglycosides, and in the glycodiversification of cysteine residues in peptides. These reactions operate well with carbohydrate substrates containing common protective groups and reagents with free hydroxyl and secondary amide functionalities under standardized conditions. Competition experiments in combination with computational DFT studies established that the putative anomeric intermediate is an organocopper species that is configurationally stable and resistant to epimerization due to its short lifetime. The subsequent reductive elimination from the Cu(III) intermediate is rapid and stereoretentive. Taken together, the glycosyl cross-coupling is ideally suited for late stage glycodiversification and bioconjugation under highly controlled installation of the aliphatic carbon-sulfur bonds.

Posttranslational isoprenylation of tryptophan in bacteria.

Posttranslational isoprenylation is generally recognized as a universal modification of the cysteine residues in peptides and the thiol groups of proteins in eukaryotes. In contrast, the Bacillus quorum sensing peptide pheromone, the ComX pheromone, possesses a posttranslationally modified tryptophan residue, and the tryptophan residue is isoprenylated with either a geranyl or farnesyl group at the gamma position to form a tricyclic skeleton that bears a newly formed pyrrolidine, similar to proline. The post-translational dimethylallylation of two tryptophan residues of a cyclic peptide, kawaguchipeptin A, from cyanobacteria has also been reported. Interestingly, the modified tryptophan residues of kawaguchipeptin A have the same scaffold as that of the ComX pheromones, but with the opposite stereochemistry. This review highlights the biosynthetic pathways and posttranslational isoprenylation of tryptophan. In particular, recent studies on peptide modifying enzymes are discussed.

Thiol-Activated Anticancer Agents: The State of the Art

The thiol or sulfhydryl group, as part of low molecular weight non-peptide biomolecules, as well as part of the cysteine residues in peptides and proteins, is known to play extremely important roles in several aspects of cellular function. Glutathione (γ-Glu-Cys-Gly; GSH) is the most abundant thiol-containing peptide in mammals, being present intracellularly in the low millimolar concentration range, but only in the low micromolar concentration range in the majority of extracellular fluids. Notably, intracellular levels of GSH have been found to be significantly upregulated in a number of human cancers, a phenomenon thought to contribute, in concert with overexpression of some GSHassociated enzymes, to the development of tumor cell chemo- and radioresistance. On the other hand, various natural and synthetic chemical entities of different sizes show significant cytotoxic activity only upon interaction with a thiol, and can therefore exploit the GSH-rich intracellular environment of tumors. This review article attempts to summarize the current structural and pharmacological knowledge in the field of thiol-activated anticancer agents, with a focus on the mechanism(s) of their activation. Even though a great part of the available thiol-activated anticancer compounds is still in the preclinical phase of testing, some of them are undergoing trials in cancer patients.

Selective Gas-Phase Ion/Ion Reactions: Enabling Disulfide Mapping via Oxidation and Cleavage of Disulfide Bonds in Intermolecularly-Linked Polypeptide Ions.

The selective gas-phase oxidation of disulfide bonds to their thiosulfinate form using ion/ion reactions and subsequent cleavage is demonstrated here. Oxidizing reagent anions are observed to attach to all polypeptides, regardless of amino acid composition. Direct proton transfer yielding a charge-reduced peptide is also frequently observed. Activation of the ion/ion complex between an oxidizing reagent anion and a disulfide-containing peptide cation results in oxygen transfer from the reagent anion to the peptide cation to form the [M+H+O](+) species. This thiosulfinate derivative can undergo one of several rearrangements that result in cleavage of the disulfide bond. Species containing an intermolecular disulfide bond undergo separation of the two chains upon activation. Further activation can be used to generate more sequence information from each chain. These oxidation ion/ion reactions have been used to illustrate the identification of S-glutathionylated and S-cysteinylated peptides, in which low molecular weight thiols are attached to cysteine residues in peptides via disulfide bonds. The oxidation chemistry effectively labels peptide ions with readily oxidized groups, such as disulfide bonds. This enables a screening approach for the identification of disulfide-linked peptides in a disulfide mapping application involving enzymatic digestion. The mixtures of ions generated by tryptic and peptic digestions of lysozyme and insulin, respectively, without prior separation or isolation were subjected both to oxidation and proton transfer ion/ion chemistry to illustrate the identification of peptides in the mixtures with readily oxidized groups.

Selective and Reversible Photochemical Derivatization of Cysteine Residues in Peptides and Proteins.

Selective derivatization of solvent-exposed cysteine residues in peptides and proteins is achieved by brief irradiation of an aqueous solution containing 3-(hydroxymethyl)-2-naphthol derivatives (NQMPs) with 350 nm fluorescent lamp. NQMP can be conjugated with various moieties, such as PEG, dyes, carbohydrates, or possess a fragment for further selective derivatization, e.g., biotin, azide, alkyne, etc. Attractive features of this labeling approach include an exceptionally fast rate of the reaction and a requirement for low equivalence of the reagent. The NQMP-thioether linkage is stable under ambient conditions, survives protein digestion and MS analysis. Irradiation of NQMP-labeled protein in a dilute solution (<40 μM) or in the presence of a vinyl ether results in a traceless release of the substrate. The reversible biotinylation of bovine serum albumin, as well as capture and release of this protein using NeutrAvidin Agarose resin beads has been demonstrated.

Direct one-step labeling of cysteine residues on peptides with [11C]methyl triflate for the synthesis of PET radiopharmaceuticals

Radiolabeled peptides have emerged as an attractive platform for the diagnostic and therapeutic oncology. However, the (11)C-radiolabeling of peptides for positron emission tomography (PET) has been poorly explored, owing to the relatively short half-life of carbon-11 (t 1/2 = 20.3 min) and time-consuming multi-step radiochemical reactions. Existing methods have found limited use and are not routinely encountered in the production of radiotracers. Herein, we propose a facile one-step direct (11)C-methylation of cysteine residues in peptides using [(11)C]methyl triflate under ambient temperatures (20 °C) and short reaction times, on the order of seconds. Good regioselectivity of this method was demonstrated by HPLC in a simple peptide (glutathione, GSH) and a more complex test decapeptide (Trp-Tyr-Trp-Ser-Arg-Cys-Lys-Trp-Thr-Gly) bearing multiple nucleophilic sites. In addition, we extend this method towards the synthesis of [(11)C]Cys(Me)-[Tyr(3)-octreotate] as a demonstration of applicability for peptides of biological interest. This octreotate derivative was obtained in non-decay-corrected radiochemical yields of 11 ± 2 % (n = 3) with a synthesis time of approx. 30 min.

An Arsenical–Maleimide for the Generation of New Targeted Biochemical Reagents

The finding that arsenic trioxide is an effective treatment for acute promyelocytic leukemia has renewed interest in the pharmacological uses of inorganic and organic arsenicals. Here we synthesized and characterized the reactivity of an arsenical-maleimide (As-Mal) that can be efficiently conjugated to exposed cysteine residues in peptides and proteins with the ultimate goal of directing these As(III) species to vicinal thiols in susceptible targets within cells and tissues. As-Mal conjugated to a surface cysteine in thioredoxin provides a more potent inhibitor for Escherichia coli thioredoxin reductase than comparable simple inorganic or organic arsenicals. As-Mal can be coupled to all of the eight cysteine residues of reduced unfolded ribonuclease A or to site-specific locations using appropriate cysteine mutations. We observed particularly strong binding to the two CxxC motifs of protein disulfide isomerase using a mutant RNase in which As-Mal was specifically incorporated at residues 26 and 110. As-Mal will serve as a facile reagent for the incorporation of As(III) species into a wide range of thiol-containing proteins, biomaterials, and surfaces.

Direct and selective tagging of cysteine residues in peptides and proteins with 4-nitropyridyl lanthanide complexes

A cysteine-selective tagging method in water is reported, based on rapid displacement of a pyridyl nitro-substituent in simple pyridines and lanthanide complexes. The conjugation reaction creates a short link between the tag and peptide, holding the peptide closer to the Ln(3+) ion and with reduced flexibility compared to existing methods.

Peptide peak intensities enhanced by cysteine modifiers and MALDI TOF MS

Two cysteine-specific modifiers we reported previously, N-ethyl maleimide (NEM) and iodoacetanilide (IAA), have been applied to the labeling of cysteine residues of peptides for the purpose of examining the enhancement of ionization efficiencies in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The peak intensities of the peptides as a result of modification with these modifiers were compared with the peak intensities of peptides modified with a commercially available cysteine-specific modifier, iodoacetamide (IA). Our experiments show significant enhancement in the peak intensities of three cysteine-containing synthetic peptides modified with IAA compared to those modified with IA. The results showed a 4.5-6-fold increase as a result of modification with IAA compared to modification with IA. Furthermore, it was found that IAA modification also significantly enhanced the peak intensities of many peptides of a commercially available proteins, bovine serum albumin (BSA), compared to those modified with IA. This significant enhancement helped identify a greater number of peptides of these proteins, leading to a higher sequence coverage with greater confidence scores in identification of proteins with the use of IAA.

ESI-MS Studies of Silver Ion Competitive Interaction with Cysteine-Containing Peptides and Sulfur-Containing Amino Acids

The paper deals with investigation of silver ion interaction with sulfur-bearing amino acids and cysteine-bearing peptides using an electrospray ionisation orthogonal ion introduction time-of-flight (ESI-o-TOF) mass spectrometer. It has been shown that Cys and Hcy demonstrate the largest affinity for silver, both in relation to methionine and cysteine residues in peptides. The studies have for the first time revealed the effect of predominant forming of ions containing two silver atoms per a sulfhydryl group of cysteine-bearing peptides if the nearest microenvironment does not hinder this.

A Mechanistic Investigation into the Irreversible Protein Binding and Antigenicity of p-Phenylenediamine

Exposure to the skin sensitizer p-phenylenediamine (PPD) is associated with allergic contact dermatitis; however, the ability of PPD to modify protein has not been fully investigated. The aims of this study were to characterize the reactions of PPD and the structurally related chemical 2,5-dimethyl-1,4-benzoquinonediamine with model nucleophiles, a synthetic peptide (DS3) containing each of the naturally occurring amino acids and His-tagged glutathione-S-transferase pi (GSTP), and to explore the effect of dimethyl substitution on PPD-specific T-cell responses using lymphocytes from allergic patients. The reductive soft nucleophiles N-acetyl cysteine and glutathione prevented PPD self-conjugation reactions and Bandrowski's base formation, but no adducts were detected. N-Acetyl lysine, a hard nucleophile, did not alter the rate of PPD degradation or form PPD adducts. With PPD and 2,5-dimethyl-1,4-benzoquinonediamine, only cysteine was targeted in the DS3 peptide. PPD and 2,5-dimethyl-1,4-benzoquinonediamine were also found to selectively modify the reactive Cys 47 residue of GSTP, which has a pK(a) of 3.5-4.2 and therefore exists in a largely protonated form. Glutathione formed mixed disulfides with the DS3 peptide, reducing levels of PPD binding. Lymphocytes from PPD allergic patients proliferated in the presence of PPD but not with 2,5-dimethyl-1,4-benzoquinonediamine. These results reveal that PPD and 2,5-dimethyl-1,4-benzoquinonediamine bind selectively to specific cysteine residues in peptides and proteins. Lymphocytes from PPD allergic patients were capable of discriminating between the different haptenic structures, suggesting that the hapten, but not the peptide moiety associated with MHC, is an important determinant for T-cell recognition.

Predicting the redox state and secondary structure of cysteine residues in proteins using NMR chemical shifts

We report 2D cluster analyses of 1Halpha, 1HN, 13Calpha, and 13C' versus 13Cbeta NMR chemical shifts (CSs) that can be used to predict the redox state and secondary structure of cysteine residues in proteins. A database of cysteine 1Halpha, 1Hbeta2, 1Hbeta3, 1HN, 13Calpha, 13Cbeta, 13C', and 15NH CSs as a function of secondary structure and redox state was constructed from BioMagResBank entries. One-dimensional statistical analysis showed that cysteine 1Halpha, 1HN, 13Calpha, 13C', and 15NH CSs reflected the secondary structure, and that cysteine Cbeta CS is extremely sensitive to the redox state. In contrast, cysteine 1Hbeta CS was not correlated with its redox state or secondary structure. Two-dimensional cluster analysis revealed that 2D Calpha/Cbeta, C'/Cbeta, HNu/Cbeta, and Halpha/Cbeta clusters were helpful in distinguishing both the redox state and secondary structure of cysteine residues. Based on these results, we derived rules using a score matrix to predict the redox state and secondary structure of cysteines using their CSs. The score matrix predicts the redox state and secondary structure of cysteine residues in proteins with approximately 90% accuracy. This suggests that the redox state and secondary structure of cysteine residues in peptides and proteins can be obtained from their CSs without recourse to nuclear Overhauser effect measurements.

Metallic Zinc Reduction of Disulfide Bonds between Cysteine Residues in Peptides and Proteins

The use of powdered metallic zinc in acidic solution for the reduction of disulfide bonds in peptides and proteins has been investigated. The method has several advantages over the traditional mere ...

On-line counting of cysteine residues in peptides during electrospray ionization by electrogenerated tags and their application to protein identification

The electrochemically induced mass spectrometric tagging of cysteines by substituted hydroquinones was studied for peptides in a classical electrospray solvent (i.e., MeOH/H2O/AcOH 50/49/1). The tagging efficiency was tested with different hydroquinone compounds on an undecapeptide containing one cysteine residue. 2-carboxymethylhydroquinone was the most reactive probe and revealed to be suitable for cysteine quantification in peptides containing up to three cysteine residues, even in the case of two consecutive cysteines in the sequence. We demonstrate the compatibility of the on-line electrochemical tagging method for the cysteine content analysis of peptides coming from gel-free protein digestion procedures. The identification of bovine serum albumin and human alpha-lactalbumin digest samples in a peptide mapping strategy was greatly improved by the application of the electrotagging technique as post-column treatment. Indeed, the determination of cysteine content in the tryptic peptides provided powerful information in order to enhance the identification score as well as the discrimination against other protein candidates. The tagging method was then applied to the determination of four proteins in a model mixture.

The Sulfinic Acid Switch in Proteins

AbstractFor Abstract see ChemInform Abstract in Full Text.