Photochemical Transformations of Peptides Containing the N-(2-Selenoethyl)glycine Moiety.

The diselenide bond has attracted considerable attention due to its ability to undergo the metathesis reaction in response to visible light. In our previous study, we demonstrated visible-light-induced diselenide metathesis of selenocysteine-containing linear peptides, allowing for the convenient generation of peptide libraries. Here, we investigated the transformation of linear and cyclic peptides containing the N-(2-selenoethyl)glycine moiety. The linear peptides were highly susceptible to the metathesis reaction, whereas the cyclic systems gave only limited conversion yields of the metathesis product. In both cases, side reactions leading to the formation of mono-, di-, and polyselenides were observed upon prolonged irradiation. To confirm the radical mechanism of the reaction, the radical initiator 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) was tested, and it was found to induce diselenide metathesis without photochemical activation. The data were interpreted in the light of quantum-chemical simulations based on density functional theory (DFT). The simulations were performed at the B3LYP-D3BJ/def2-TZVP level of theory using a continuum solvation model (IEF-PCM) and methanol as a solvent.

Stapling of leu-enkephalin analogs with bifunctional reagents for prolonged analgesic activity.

The design and synthesis of leu-enkephalin analogs by replacing the glycine residues with N-(2-thioethyl)glycines and opening the cyclisation potential is presented. The cyclization (stapling) was achieved using bifunctional reagents (hexafluorobenzene and trithiocyanuric acid derivatives). The CD conformational studies of the stapled analogs suggest that the peptides adopt the type I ß-turn conformation, which is in agreement with the theoretical analysis. The analog containing a trithiocyanuric acid derivative with a benzyl substituent shows potent analgesic activity.

Visible Light-Induced Templated Metathesis of Peptide-Nucleic Acid Conjugates with a Diselenide Bridge.

The use of template molecules as chemical scaffolds that significantly influence the course of the reaction has recently been intensively studied. Peptide nucleic acids (PNA) are molecules that mimic natural nucleic acids. They are a promising matrix in such reactions because they possess high affinity and specificity in their interactions. The manner of PNA interaction is predictable based on sequence complementarity. Recently, we report the visible light-induced metathesis reaction in peptides containing a diselenide bond. Herein, we present an efficient and straightforward method of the visible light-driven diselenide-based metathesis of peptide-nucleic acid conjugates. Compared to a similar photochemical transformation in peptides, a significant increase in the metathesis efficiency was obtained due to the template effect.

Alkyl Thiocyanurates as Thioester Mimetics. Transthioesterification and Ligation Reactions with High Potential in Dynamic Covalent Chemistry.

Alkyl thiocyanurates, the compounds formed in the SN reaction of thiocyanuric acid and alkyl halides, are susceptible to transthioesterification and ligation with molecules containing cysteamine, analogous to native chemical ligation of thioesters with peptides with an N-terminal cysteine moiety. The ligation is irreversible and results in the formation of mono- and disubstituted products dominantly. Transthioesterification, in contrast, is fully reversible and may be used in constructing dynamic systems. The application of this reactivity in dynamic covalent chemistry has been exemplified by the preparation of a library of mixed thiocyanurates of glutathione and thioglycolic acid with self-assembly abilities and metathesis between thiocyanurates of tris(carboxymethyl) and tris(carboxamidomethyl) catalyzed by MESNa (sodium 2-mercaptoethylsulphonate) or MPAA (4-mercaptophenylacetic acid). Differences in reactivity of thiocyanurates toward cysteamines and thiols has been explained based on conceptual DFT.

Peptide Stapling with Boronate Esters- A Reversible Folding of (Artificial) Peptide Chain to α-Helix.

Stabilization of a peptide conformation via stapling strategy may be realized by the reversible or more often irreversible connection of side chains being in mutually appropriate geometry. An incorporation of phenylboronic acid and sugar residues (fructonic or galacturonic acid), attached to two lysine side chains via amide bonds and separated by 2, 3, or 6 other residues in the C-terminal fragment of RNase A introduces the intramolecular interaction stabilizing the α-helical organization. The boronate ester stapling is stabilized in mild basic conditions and may be switched off by acidification leading to unfolded organization of the peptide chain. We investigated the possibility of using switchable stapling by mass spectrometry, NMR and UV-CD spectroscopies, and DFT calculations.

Selenium in Peptide Chemistry.

In recent years, researchers have been exploring the potential of incorporating selenium into peptides, as this element possesses unique properties that can enhance the reactivity of these compounds. Selenium is a non-metallic element that has a similar electronic configuration to sulfur. However, due to its larger atomic size and lower electronegativity, it is more nucleophilic than sulfur. This property makes selenium more reactive toward electrophiles. One of the most significant differences between selenium and sulfur is the dissociation of the Se-H bond. The Se-H bond is more easily dissociated than the S-H bond, leading to higher acidity of selenocysteine (Sec) compared to cysteine (Cys). This difference in acidity can be exploited to selectively modify the reactivity of peptides containing Sec. Furthermore, Se-H bonds in selenium-containing peptides are more susceptible to oxidation than their sulfur analogs. This property can be used to selectively modify the peptides by introducing new functional groups, such as disulfide bonds, which are important for protein folding and stability. These unique properties of selenium-containing peptides have found numerous applications in the field of chemical biology. For instance, selenium-containing peptides have been used in native chemical ligation (NCL). In addition, the reactivity of Sec can be harnessed to create cyclic and stapled peptides. Other chemical modifications, such as oxidation, reduction, and photochemical reactions, have also been applied to selenium-containing peptides to create novel molecules with unique biological properties.

High-Temperature, Solid-Phase Reaction of α-Amino Groups in Peptides with Lactose and Glucose: An Alternative Mechanism Leading to an α-Ketoacyl Derivative.

The reaction of proteins with reducing sugars results in the formation of Amadori products, which involves the N-terminal group and/or ε-amino group of the lysine side chain. However, less attention has been given to the reactivity of the N-terminus of a peptide chain under similar conditions. In our work, we focused on the reaction of the α-amino group of peptides in the presence of a reducing sugar, specifically lactose. We optimized the reaction conditions of model peptides with lactose in the solid phase and showed that temperatures above 120 °C lead to the deamination of the N-terminal amino acid moiety, ultimately resulting in α-ketoacids. We carried out detailed studies to confirm the structure of the deaminated product using analytical methods such as ESI-MS and LC-MS/MS, as well as chemical methods that involved the reduction of the carbonyl group combined with isotopic exchange and the reactivity of the carbonyl group with the hydroxylamine derivative. The structure of the reaction product was also confirmed by chemical synthesis. We suggested plausible mechanisms for the formation of the deaminated product and considered the probable path of its formation. Our aim was to determine whether the reaction proceeds according to the Strecker-based mechanism and direct imine isomerization by carrying out reactions of model peptides in the presence of lactose under aerobic and anaerobic conditions and comparing the results obtained.

HPLC-free method of synthesis of isotopically labeled deoxyfructosylated peptides.

The biomarker strategy, based on multiple specific glycation sites in plasma proteins, could essentially increase the efficiency of glycemic control and disease prediction. Besides glycated albumin being a potential biomarker of early states of diabetes mellitus and control of short-term, it has been shown that the glycation of fibrinogen may also impact the formation of the fibrin network, while quantification of glycation of the CD59 protein allows for early detection of glucose intolerance in pregnant women. A different level of glycation of individual lysine residues in proteins has a crucial influence on the stages of the disease. The quantification of new biomarkers of different stages of diabetes requires appropriate isotope-labeled analogs that may improve biomarker search by providing more accurate quantitative data and by more robust detection/quantitation of low-abundance biomarkers. In the presented work, we proposed a fast and simple protocol for the synthesis of isotopically labeled and bi-labeled deoxyfructosylated peptide based on a combination of microwave-assisted synthesis and boronic affinity chromatography using functionalized resin (PhB-Lys(PhB)-ChemMatrix® Rink resin) developed by us. Our method is focused on the synthesis of glycated peptides identified in glycated albumin (GA) after enzymatic hydrolysis catalyzed by trypsin after arginine residues. Thereby, the standard peptides comprised [13C6]-deoxyfructose attached to lysine residue side chain, a dabcyl moiety for determination of standard amounts, and a cleavable linker. Moreover, we applied bi-labeled deoxyfructosylated peptide to determine the concentration of appropriate analog in a sample of human serum albumin glycated in vitro.

Analysis of Fragmentation Pathways of Peptide Modified with Quaternary Ammonium and Phosphonium Group as Ionization Enhancers.

Peptide modification by a quaternary ammonium group containing a permanent positive charge is a promising method of increasing the ionization efficiency of the analyzed compounds, making ultra-sensitive detection even at the attomolar level possible. Charge-derivatized peptides may undergo both charge remote (ChR) and charge-directed (ChD) fragmentation. A series of model peptide conjugates derivatized with N,N,N-triethyloammonium (TEA), 1-azoniabicyclo[2.2.2]octane (ABCO), 2,4,6-triphenylopyridinium (TPP) and tris(2,4,6-trimetoxyphenylo)phosphonium (TMPP) groups were analyzed by their fragmentation pathways both in collision-induced dissociation (CID) and electron-capture dissociation (ECD) mode. The effect of the fixed-charge tag type and peptide sequence on the fragmentation pathways was investigated. We found that the aspartic acid effect plays a crucial role in the CID fragmentation of TPP and TEA peptide conjugates whereas it was not resolved for the peptides derivatized with the phosphonium group. ECD spectra are mostly dominated by cn ions. ECD fragmentation of TMPP-modified peptides results in the formation of intense fragments derived from this fixed-charge tag, which may serve as reporter ion.

One-Pot Cyclization and Cleavage of Peptides with N-Terminal Cysteine via the N,S-Acyl Shift of the N-2-[Thioethyl]glycine Residue.

We developed a one-pot method for peptide cleavage from a solid support via the N,S-acyl shift of N-2-[thioethyl]glycine and transthioesterification using external thiols to produce cyclic peptides through native chemical self-ligation with the N-terminal cysteine. The feasibility of this methodology is validated by the syntheses of model short peptides, including a tetrapeptide, the bicyclic sunflower trypsin inhibitor SFTI-1, and rhesus Θ-defensin RTD-1. Synthesis of the whole peptide precursor can be fully automated and proceeds without epimerization or dimerization.

Selective ESI-MS detection of carbonyl containing compounds by aminooxyacetic acid immobilized on a resin.

There are numerous examples of bioactive compounds containing carbonyl groups including modified proteins with oxidation of side chain of amino acid residues to aldehyde/ketone groups which are frequently considered as markers of oxidative stress. The carbonyl unit can be also distinguished as a substructure in many illegal drugs including anabolic steroids as well as cations derivatives. Based on chemoselective formation of oximes by solid phase immobilized hydroxylamine derivatives we proposed the protocol for derivatization and selective detection of carbonylated compounds in human serum albumin hydrolysate as a complex peptide mixture and of testosterone in urine samples. This allowed for the removal of the matrix and the qualitative and quantitative analysis of the derivatized analyte by LC-MS/MS (or LC-MRM). Herein we report the preparation and chemical characterization of a novel, ChemMatrix - based resin functionalized with aminooxyacetic acid (AOA). The hydroxylamine moiety in this resin is combined with a peptide linker (GRG) containing an arginine residue to enhance the ionization efficiency. Application of an isotopically labeled carbonylated peptide ((H-Leu-Val-Thr(O)-Asp-Leu-Thr-Lys [13C6,15N2]-OH and testosterone-d3 allowed us to carry out quantitative analyses of detected compounds. Our method is general and may be applied for analysis of carbonylated compounds in biological samples. Our method based on application of functionalized resin allowed to quantify the level of free testosterone in small sample (0.5 mL) of urine, while the non-derivatized testosterone from urine sample was not detected during direct LC-MRM analysis.

Isolation, characterization, anti-MRSA evaluation, and in-silico multi-target anti-microbial validations of actinomycin X2 and actinomycin D produced by novel Streptomyces smyrnaeus UKAQ_23.

Streptomyces smyrnaeus UKAQ_23, isolated from the mangrove-sediment, collected from Jubail,Saudi Arabia, exhibited substantial antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), including non-MRSA Gram-positive test bacteria. The novel isolate, under laboratory-scale conditions, produced the highest yield (561.3 ± 0.3 mg/kg fermented agar) of antimicrobial compounds in modified ISP-4 agar at pH 6.5, temperature 35 °C, inoculum 5% v/w, agar 1.5% w/v, and an incubation period of 7 days. The two major compounds, K1 and K2, were isolated from fermented medium and identified as Actinomycin X2 and Actinomycin D, respectively, based on their structural analysis. The antimicrobial screening showed that Actinomycin X2 had the highest antimicrobial activity compared to Actinomycin D, and the actinomycins-mixture (X2:D, 1:1, w/w) against MRSA and non-MRSA Gram-positive test bacteria, at 5 µg/disc concentrations. The MIC of Actinomycin X2 ranged from 1.56-12.5 µg/ml for non-MRSA and 3.125-12.5 µg/ml for MRSA test bacteria. An in-silico molecular docking demonstrated isoleucyl tRNA synthetase as the most-favored antimicrobial protein target for both actinomycins, X2 and D, while the penicillin-binding protein-1a, was the least-favorable target-protein. In conclusion, Streptomyces smyrnaeus UKAQ_23 emerged as a promising source of Actinomycin X2 with the potential to be scaled up for industrial production, which could benefit the pharmaceutical industry.

Lossen Rearrangement of p-Toluenesulfonates of N-Oxyimides in Basic Condition, Theoretical Study, and Molecular Docking.

The sulfonic esters of N-oxyimides are a group of compounds with a wide range of biological activities, as well as a unique reactivity toward amines. They undergo this reaction with primary amines and other nucleophilic reagents according to a Lossen-like rearrangement. The reaction is initiated by nucleophilic attack on a carbonyl group in the succinimide ring followed by isocyanate formation, which next interacts with another nucleophile molecule forming an addition product (e.g., ureido or urethane derivative). However, the secondary amines are also susceptible to other reactions leading to products containing the maleimide ring formed by sulphonic acid elimination. In the case of tertiary amines, this reaction is predominant. To explain the phenomenon of the reactivity of the N- oxyimides toward different types of amines, we employed various spectroscopic and X-ray approaches as well as DFT calculation. Results suggest that the basicity of the amine used for the reaction plays a crucial role in the reaction mechanism that eventually dominates the entire chemical process. Moreover, we applied molecular docking to investigate the ability of the products to act as serine protease inhibitors using human leukocyte elastase (HLE).

Intramolecularly stapled amphipathic peptides via a boron-sugar interaction.

Amadori products (deoxyfructosyllysine derivatives) that can selectively interact with phenylboronic acids and borate ions were synthesized. The intramolecular interactions between the fructosyl moiety and phenylboronic acid incorporated into various positions of the peptide chain were investigated using high-resolution mass spectrometry (HR-MS), circular dichroism (CD), and nuclear magnetic resonance (NMR).

Attempting to synthesize lasso peptides using high pressure.

Lasso peptides are unique in that the tail of the lasso peptide threads through its macrolactam ring. The unusual structure and biological activity of lasso peptides have generated increased interest from the scientific community in recent years. Because of this, many new types of lasso peptides have been discovered. These peptides can be synthesized by microorganisms efficiently, and yet, their chemical assembly is challenging. Herein, we investigated the possibility of high pressure inducing the cyclization of linear precursors of lasso peptides. Unlike other molecules like rotaxanes which mechanically interlock at high pressure, the threaded lasso peptides did not form, even at pressures the high pressure up to 14 000 kbar.

Detection of Podocin in Human Urine Sediment Samples by Charge Derivatization and LC-MS-MRM Method.

Detection of podocytes in urine might serve as a useful diagnostic tool in both primary and secondary glomerular diseases. The utility of podocyturia has been confirmed for both pre-eclampsia and glomerulonephritis. Here, we present a new and sensitive method for qualitative LC-MS-multiple-reaction-monitoring (MRM) analysis of podocin, serving as a podocyturia biomarker in urine sediments. The following podocin tryptic peptides with the 169LQTLEIPFHEIVTK182, 213AVQFLVQTTMK223, 240SIAQDAK246, and 292MIAAEAEK299 sequences were applied as a model. The selective chemical derivatization of the ε amino group of C-terminal lysine residue in tryptic peptides, by 2,4,6-triphenylpyrylium salt (TPP) as a fixed charge tag, was employed to increase the ionization efficiency, in routine ESI-MS analysis. Additionally, the generation of a reporter ion, in the form of a protonated 2,4,6-triphenylpyridinium cation, makes the derivatized peptide analysis in the MRM mode unambiguous. Identification of derivatized and non-derivatized peptides were performed, and the obtained results suggest that the peptide with the 292MIAAEAEK299 sequence may serve as a marker of podocyturia.

An Optimised Di-Boronate-ChemMatrix Affinity Chromatography to Trap Deoxyfructosylated Peptides as Biomarkers of Glycation.

We report herein a novel ChemMatrix® Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N-α and N-ε amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix® Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix® Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics.

Light-Driven Diselenide Metathesis in Peptides.

Peptides containing selenocysteine moieties are susceptible to non-catalytic reactions of diselenide bonds metathesis induced by visible light. In contrast to previously reported radical metathesis of disulfide bridges in cysteine derivatives, this newly developed reaction is fast and clean, and proceeds without decomposition of peptides and without formation of side products. The diselenide bond in peptides was reported in literature to be more stable than the disulfide one and also less susceptible to metathesis induced by thiols and reducing reagents. We demonstrated that visible light induces fast metathesis of Se-Se bonds in peptides. This reaction is important for the folding of peptides containing selenocysteine residues and may find application in designing dynamic combinatorial libraries of peptides responsive to external influence.

Trends in the Design of New Isobaric Labeling Reagents for Quantitative Proteomics.

Modern mass spectrometry is one of the most frequently used methods of quantitative proteomics, enabling determination of the amount of peptides in a sample. Although mass spectrometry is not inherently a quantitative method due to differences in the ionization efficiency of various analytes, the application of isotope-coded labeling allows relative quantification of proteins and proteins. Over the past decade, a new method for derivatization of tryptic peptides using isobaric labels has been proposed. The labels consist of reporter and balanced groups. They have the same molecular weights and chemical properties, but differ in the distribution of stable heavy isotopes. These tags are designed in such a way that during high energy collision induced dissociation (CID) by tandem mass spectrometry, the isobaric tag is fragmented in the specific linker region, yielding reporter ions with different masses. The mass shifts among the reporter groups are compensated by the balancing groups so that the overall mass is the same for all forms of the reagent. Samples of peptides are labeled with the isobaric mass tags in parallel and combined for analysis. Quantification of individual peptides is achieved by comparing the intensity of reporter ions in the tandem mass (MS/MS) spectra. Isobaric markers have found a wide range of potential applications in proteomics. However, the currently available isobaric labeling reagents have some drawbacks, such as high cost of production, insufficient selectivity of the derivatization, and relatively limited enhancement of sensitivity of the analysis. Therefore, efforts have been devoted to the development of new isobaric markers with increased usability. The search for new isobaric markers is focused on developing a more selective method of introducing a tag into a peptide molecule, increasing the multiplexicity of markers, lowering the cost of synthesis, and increasing the sensitivity of measurement by using ionization tags containing quaternary ammonium salts. Here, the trends in the design of new isobaric labeling reagents for quantitative proteomics isobaric derivatization strategies in proteomics are reviewed, with a particular emphasis on isobaric ionization tags. The presented review focused on different types of isobaric reagents used in quantitative proteomics, their chemistry, and advantages offer by their application.

Isobaric duplex based on a combination of 16O/18O enzymatic exchange and labeling with pyrylium salts.

Enzymatic 18O exchange, the well-established approach in comparative proteomics, has some disadvantages such as back exchange of labeled oxygen and overlapping the peak of a labeled peptide with isotopic peaks of an unlabeled one. Herein we demonstrated a simple procedure in which samples digested with a trypsin (with and without H218O) were reacted with unlabeled and quadrupled 13C-labeled pyrylium salt respectively which results in formation of pyridinium cations. Thus, each isobarically labeled peptide containing zero or four 13C atoms in the mass reporter group, during tandem MS/MS forms an unique reporter ion useful for a relative quantitation. Such a sample treatment improves the signal to noise ratio, reduces overlapping of the isotopic peaks and completely eliminates the back exchange problem.