Cysteine Derivatives Research Articles

Chemical composition, nutritional value and antioxidant properties of Allium caepa L. Var. tropeana (red onion) seeds

Chemical analysis of Allium caepa L. var Tropeana (red onion) seeds showed high amounts of oil (20.4%), fibre (22.4%), crude protein (24.8%), calcium (175.0 mg/100 g), potassium (1010 mg/100 g), low amounts of sodium (11.2 mg/100 g) and six cysteine derivatives, of which the S-propylmercapto-cysteine has never been reported in onion before. The antioxidant capacity of seed extracts containing cysteine derivatives (SECCD), before and after boiling the seeds, and of cooking water extracts containing cysteine derivatives (CWECCD), was also evaluated, by the ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picrilhydrazyl (DPPH) assays. The extracts showed discrete antioxidant capacity which increased after boiling, although cooking methods caused significant losses of the cysteine derivatives in water.

Influence of various combinations of mucolytic agent and non-ionic surfactant on intestinal absorption of poorly absorbed hydrophilic compounds

The absorption enhancing effects of various combinations of a mucolytic agent and a non-ionic surfactant on the intestinal absorption of poorly absorbed hydrophilic compounds were examined. Fluorescein isothiocyanate-labeled dextran with an average molecular weight of ca. 4.4 kDa (FD-4) was used as a model compound. Cysteine derivatives such as N-acetylcysteine (NAC), S-carboxymethylcysteine (SCMC), S-ethylcysteine (SEC), and S-methylcysteine (SMC) were selected as mucolytic agents. A homogeneous series of single chain polyoxyethylene alkyl ethers were employed as non-ionic surfactants. Various dosing solutions were administered into rat jejunum, and the bioavailability of FD-4 was determined. Unlike NAC, the agents such as SCMC, SEC, and SMC, which do not possess a free thiol group, did not show any apparent enhancement of intestinal FD-4 absorption, when they were co-administered with p- t-octyl phenol polyoxyethylene-9.5 (Triton ® X-100, TX-100). In addition, the absorption enhancement was dependent on the kinds of polyoxyethylene alkyl ethers used, when used in combination with NAC. For a constant alkyl chain of 12 with a varying polyoxyethylene (POE) chain length, the surfactant with a short to medium POE chain length such as lauryl poly (4.2) oxyethylene ether (BL-4.2) and lauryl poly (9) oxyethylene ether (BL-9) were effective. In addition, for a constant alkyl chain of 18 with a varying POE chain length, the surfactants with a longer POE chain length such as oleyl poly (15) oxyethylene ether (BO-15) and stearyl poly (20) oxyethylene ether (BS-20) showed the effective enhancement. All these results suggest that a mucolytic agent not possessing a free thiol group is not effective for enhancing the intestinal absorption of poorly absorbed hydrophilic compounds. Also, they indicate that the combination of a mucolytic agent possessing a free thiol group and a non-ionic surfactant either with a short to medium POE chain length and a medium alkyl chain length, or with a longer POE chain length and a longer alkyl chain length shows the effective enhancement. This fundamental information might be useful for finding the optimal combination.

Effect of Antioxidant Treatments on the Gut–Liver Axis Oxidative Status and Function in Bile Duct‐Ligated Rats

Experimental and clinical studies have demonstrated the pivotal role of oxidative stress in the promotion of hepatic and intestinal injury in obstructive jaundice. The present study was undertaken to investigate the effect of well known antioxidant treatments on the gut-liver axis oxidative status and function in bile duct-ligated rats. A total of 60 male Wistar rats were randomly divided into six groups of 10 animals each: controls, sham operated, bile duct ligated (BDL), and BDL treated with either N-acetylcysteine (NAC), allopurinol, or alpha-tocopherol (alpha-TC). Ten days after treatment, the hepatic and intestinal oxidative status was estimated by measuring lipid peroxidation and a battery of biochemical markers comprising the organ's thiol redox state (i.e., glutathione, cysteine, protein thiols, oxidized glutathione, nonprotein mixed disulfides, oxidized cysteine derivatives, protein symmetrical disulfides, and protein mixed disulfides). Portal and aortic endotoxin concentrations and alanine aminotransferase (ALT) levels were also determined. All antioxidant treatments significantly improved intestinal barrier function and protected from cholestatic liver injury, as evidenced by reduction of the portal and aortic endotoxin concentration and ALT levels, respectively. This effect accompanied their significant antioxidant action in both organs, mediated by a certain influence profile on the thiol redox state by each treatment. NAC, allopurinol, and alpha-TC, exerting a potent combined antioxidant effect on the intestine and liver in experimental obstructive jaundice, significantly prevented intestinal barrier dysfunction and liver injury. The variety of results depending on the antioxidant agent that was administered and the marker of oxidative stress that was estimated, indicates that a battery of biomarkers would be more appropriate in assessing pharmacologic responses to therapeutic interventions.

Convenient Solid‐Phase Synthesis of Ureido‐Pyrimidinone Modified Peptides

AbstractPeptides have been modified with quadruple hydrogen bonding ureido‐pyrimidinone moieties to be applied in supramolecular architectures for biomedical applications. A convenient solid‐phase synthesis method was developed to functionalize peptide sequences with ureido‐pyrimidinone units. Two different ureido‐pyrimidinone synthons were used: based on a carbonyldiimidazole‐activated amine or on an isocyanate functionality. Oligopeptides were functionalized on the solid support using two coupling strategies: on the N‐terminus or selectively on the ϵ‐position of a C‐terminal lysine. Several peptides were modified to show the generality of the approach, varying from cell adhesion sequences, to collagen binding peptides and cysteine derivatives which can be used for native chemical ligation. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Topically Applied Vitamin C and Cysteine Derivatives Protect Against UVA-induced Photodegradation of Suprofen in Ex Vivo Pigskin¶

Topically Applied Vitamin C and Cysteine Derivatives Protect Against UVA-induced Photodegradation of Suprofen in Ex Vivo Pigskin¶

Electrospray Ionization Tandem Mass Spectrometry Sequencing of Novel Skin Peptides from Ranid Frogs Containing Disulfide Bridges

Tandem mass spectrometry sequencing, as well as Edman sequencing of peptides belonging to the Rana genus, represents a difficult task due to the presence of a disulfide bridge at the C-terminus and their rather high molecular masses (over 2000 Da). The present study throws light upon the sequence of three rather long peptides (more than 20 amino acid residues each) isolated from the skin secretion of Russian frogs, Rana ridibunda and Rana arvalis. This novel aspect involves the fact that the sequences (including two sequences established de novo) were determined exclusively by means of mass spectrometry. A combination of electron capture dissociation (ECD) and collision-induced dissociaiton (CID) data accompanied by exact mass measurements (LTQ Fourier transform ion cyclotron resonance mass spectrometer) facilitated reaching the goal. To overcome the difficulty dealing with disulphide bridges ("Rana box"), reduction of the S-S bond with dithiotreitol followed by derivatization of Cys residues with iodoacetamide was used. The sequence was determined using combined spectral data on y and b series of fragment ions. A multiple mass spectrometry (MS(3)) experiment was also used to elucidate the sequence inside the "Rana box" after cysteine derivatization. Exact mass measurements were used to differentiate between Lys and Gln residues, while characteristic losses of 29 and 43 Da (d and w fragment ions) in CID and ECD experiments allowed us to distinguish between Ile and Leu isomeric acids.

Inhibition of polyphenoloxidase activity by mixtures of heated cysteine derivatives with carbonyl compounds

It had previously been shown that soluble Maillard reaction products (MRP) made from thiol compounds and glucose or fructose contained powerful inhibitors of various fruit and vegetable polyphenoloxidase (PPO) activity. In MRP from cysteine and glucose, the amount of hydroxymethylfurfural (HMF) formed increased with the increase in glucose concentration (0-1 M), particularly under acidic (pH 2) conditions. Using model mixtures containing a preheated cysteine-derived compound and a carbonyl component, especially HMF, furfural and benzaldehyde, we showed that the neoformed compounds produced exhibited a stronger inhibitory potency toward PPO activity of eggplant, apple, and mushroom than former MRP. Optimal reaction conditions for the formation of inhibitory compounds when HMF reacted with preheated cysteine were investigated. It was found that a reactants molar ratio of 1:1 and a reaction time exceeding 1 h were the most efficient reaction conditions to generate inhibitory compounds. The stability of the newly formed products, evaluated during storage, showed that their inhibitory potency was globally kept at 4, 21, and 37 degrees C for 72 h but was unstable when stored at -20 degrees C and lost when exposed to UV radiations for 24 h.

A Novel Salt Bridge Mechanism Highlights the Need for Nonmobile Proton Conditions to Promote Disulfide Bond Cleavage in Protonated Peptides Under Low-Energy Collisional Activation

The gas-phase fragmentation mechanisms of small models for peptides containing intermolecular disulfide links have been studied using a combination of tandem mass spectrometry experiments, isotopic labeling, structural labeling, accurate mass measurements of product ions, and theoretical calculations (at the MP2/6-311 + G(2d,p)//B3LYP/3-21G(d) level of theory). Cystine and its C-terminal derivatives were observed to fragment via a range of pathways, including loss of neutral molecules, amide bond cleavage, and S-S and C-S bond cleavages. Various mechanisms were considered to rationalize S-S and C-S bond cleavage processes, including charge directed neighboring group processes and nonmobile proton salt bridge mechanism. Three low-energy fragmentation pathways were identified from theoretical calculations on cystine N-methyl amide: (1) S-S bond cleavage dominated by a neighboring group process involving the C-terminal amide N to form either a protonated cysteine derivative or protonated sulfenyl amide product ion (44.3 kcal mol(-1)); (2) C-S bond cleavage via a salt bridge mechanism, involving abstraction of the alpha-hydrogen by the N-terminal amino group to form a protonated thiocysteine derivative (35.0 kcal mol(-1)); and (3) C-S bond cleavage via a Grob-like fragmentation process in which the nucleophilic N-terminal amino group forms a protonated dithiazolidine (57.9 kcal mol(-1)). Interestingly, C-S bond cleavage by neighboring group processes have high activation barriers (63.1 kcal mol(-1)) and are thus not expected to be accessible during low-energy CID experiments. In comparison to the energetics of simple amide bond cleavage, these S-S and C-S bond cleavage reactions are higher in energy, which helps rationalize why bond cleavage processes involving the disulfide bond are rarely observed for low-energy CID of peptides with mobile proton(s) containing intermolecular disulfide bonds. On the other hand, the absence of a mobile proton appears to "switch on" disulfide bond cleavage reactions, which can be rationalized by the salt bridge mechanism. This potentially has important ramifications in explaining the prevalence of disulfide bond cleavage in singly protonated peptides under MALDI conditions.

Enantioselective Rauhut-Currier Reactions Promoted by Protected Cysteine

We report highly enantioselective examples of the Rauhut−Currier cycloisomerization reaction (the “vinylogous Morita−Baylis−Hillman reaction”). The reaction is highly practical and is catalyzed by a commercially available derivative of the proteinogenic amino acid cysteine. Reactions are conducted in the presence of potassium tert-butoxide and a critical concentration of water in bulk acetonitrile. A mechanistic model is advanced that may account for reaction selectivity that is predicated on organizational chelation of K ion in the product-determining step in which the Cys derivative undergoes elimination.

Reduction and S-Carboxymethylation of Proteins: Large-Scale Method

INTRODUCTIONIntact interchain and/or intrachain disulfide linkage in a protein can present problems during proteolytic or chemical fragmentation procedures. Disulfide bonds are commonly cleaved by reducing cystine to yield cysteine residues. However, cysteine residues are highly reactive, which can complicate sequence work. In this protocol, the intact protein (>1 mg) is reduced and then S-alkylated with iodoacetic acid (or iodoacetamide). The resulting cysteine derivative S-carboxymethylcysteine (or S-carboximadomethylcysteine) is easily detectable during chemical sequencing.

Visualization of RecA Filaments and DNA by Fluorescence Microscopy

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.

Effect of Cysteine Derivatives Administration in Healthy Men Exposed to Intense Resistance Exercise by Evaluation of Pro-Antioxidant Ratio

The aim of this study was to ascertain the influence of cysteine derivatives on pro-antioxidant equilibrium and to compare the antioxidant effectiveness of N-acetylcysteine, alpha-lipoic acid, and taurine by using Loverro's coefficient (pro-antioxidant ratio) in healthy men exposed to intensity-resistance exercise. Fifty-five men were randomly assigned to one of four groups: control (CON, placebo), N-acetylcysteine (NAC 1.8 g.day(-1), 3 days), alpha-lipoic acid (LIP 1.2 g.day(-1), 3 days), or taurine (TAU 3 g.day(-1), 3 days). The erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, lipid peroxidation products (TBARS), and plasma protein thiol concentrations were evaluated. The P/A ratio was determined from the mean values of TBARS, SOD, GPx, and CAT. The applied exercise at maximal intensity induced the significant changes in pro-antioxidant equilibrium toward peroxidation, which was proved by a 25% increase in TBARS concentration in the CON group. The peroxidation was significantly diminished by NAC (-14%) and LIP (-16%), whereas TAU had no effect on the TBARS concentration. Cysteine derivatives administration prevented exercise-induced decline in SOD activity and increased in GPx activity during exercise. CAT activity changed only in the LIP group. The estimation of P/A ratio showed the lowest level of pro-antioxidant equilibrium after LIP administration. In the CON group, P/A ratio was directly correlated with the protein thiols level (r = 0.495, p < 0.001). These data confirm the antioxidant action of tested cysteine derivatives, particularly lipoic acid, and demonstrate the practical application of P/A ratio to evaluate the effectiveness of antioxidants in athletes.

Solid-Phase Synthesis of a Peptide-Based P,S-Ligand System Designed for Generation of Combinatorial Catalyst Libraries

An efficient methodology for the solid-phase synthesis of diverse combinatorial peptide-based P,S-ligand libraries based on a modular approach was developed. Chiral thioethers were introduced into a series of peptide scaffolds using commercially available Fmoc-protected cysteine derivatives, and secondary amines were incorporated into the peptide backbones by reductive alkylation using readily available Fmoc-protected amino aldehydes. Phosphinylation of the secondary amines of the scaffolds, applying two different reagents, yielded two different types of ligands. Subsequent complexation with palladium afforded six- or seven-membered chelates, respectively. The selectivity, in an asymmetric allylic substitution reaction, of the two different types of chelates, derived from the same peptide scaffold, was complementary in all cases studied, affording the product as opposite stereoisomers with up to 60% ee. These results hold great promise for the identification of highly selective catalysts upon screening of larger P,S-based catalyst libraries.

Enantioselective Rauhut−Currier Reactions Promoted by Protected Cysteine

We report highly enantioselective examples of the Rauhut−Currier cycloisomerization reaction (the “vinylogous Morita−Baylis−Hillman reaction”). The reaction is highly practical and is catalyzed by a commercially available derivative of the proteinogenic amino acid cysteine. Reactions are conducted in the presence of potassium tert-butoxide and a critical concentration of water in bulk acetonitrile. A mechanistic model is advanced that may account for reaction selectivity that is predicated on organizational chelation of K ion in the product-determining step in which the Cys derivative undergoes elimination.

Leaving group effects on the selectivity of the gas‐phase fragmentation reactions of side chain fixed‐charge‐containing peptide ions

The effect of trialkylsulfonium versus quaternaryalkylammonium ions on the multistage gas-phase fragmentation reactions of side chain, fixed-charge, cysteine-containing peptides has been examined in a quadrupole linear ion trap. These tandem mass spectrometry experiments reveal that selective loss of dialkylsulfide from fixed-charge sulfonium ion derivatives is the dominant fragmentation pathway regardless of the degree of proton mobility, indicating that it is the most energetically favored fragmentation pathway. In contrast, the loss of trimethylamine from the side chain of fixed-charge ammonium-ion-containing cysteine derivatives appears to be less energetically favored, and as a result extensive charge-remote fragmentation is observed under low proton mobility conditions, while under conditions of high proton mobility, amide bond fragmentation reactions dominate. These findings are supported by molecular orbital calculations at the B3LYP/6-31 + G(d, p) level of theory, which showed that the neutral loss of dimethylsulfide is both thermodynamically and kinetically preferred over the loss of trimethylamine.

N-Alkyl cysteine-assisted thioesterification of peptides

A new method for the preparation of peptide thioester by the post-solid phase peptide synthesis (SPPS) approach was developed. A series of N-alkyl cysteine derivatives were prepared and used as the C-terminus residue of the peptides prepared by the Fmoc SPPS. The synthetic peptides released from resin by TFA were readily converted to the peptide thioester in aqueous 3-mercaptopropionic acid (MPA) without significant side reactions.

Sigmatropic Rearrangements as Tools for Amino Acid and Peptide Modification: Application of the Allylic Sulfur Ylide Rearrangement to the Preparation of Neoglycoconjugates and Other Conjugates

Reaction of S-allyl cysteine derivatives, generated by the selenocysteine ligation, with rhodium carbenoids, stabilized and unstabilized, enables the attachment of diverse functionality onto cysteine residues. The reaction is successfully applied to the introduction of lipid-like residues, a fluorous alkyl chain, and mono- and disaccharides.

Site-Specific Arylation of Rat Glutathione S-Transferase A1 and A2 by Bromobenzene Metabolites in Vivo

The hepatotoxicity of bromobenzene (BB) derives from its reactive metabolites (epoxides and quinones), which arylate cellular proteins. Application of proteomic methods to liver proteins from rats treated with a hepatotoxic dose of [14C]-BB has identified more than 40 target proteins, but no adducted peptides have yet been observed. Because such proteins are known to contain bromophenyl- and bromodihydroxyphenyl derivatives of cysteine, histidine, and lysine, the failure to observe modified peptides has been attributed to the low level of total covalent binding and to the "dilution" effect of multiple metabolites reacting at multiple sites on multiple proteins. In this work glutathione S-transferase (GST), a well-known and abundant BB-target protein, was isolated from liver cytosol of rats treated with 14C-BB by use of a glutathione (GSH)-agarose affinity column and further resolved by reverse-phase high-performance liquid chromatography (HPLC) into subunits M1, M2, A1, A2 and A3. The subunits were identified by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whole-molecule mass spectrometry, and peptide mass mapping and found to contain radioactivity corresponding to 0.01-0.05 adduct per molecule of protein. Examination of tryptic digests of these subunits by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization mass spectrometry (ESI-MS) failed to reveal any apparent adducted peptides despite observed sequence coverages up to 87%. However, use of HPLC-linear ion-trap quadrupole Fourier transform mass spectrometry (LTQ-FTMS) to search for predicted modified tryptic peptides revealed peaks corresponding, with a high degree of mass accuracy, to a bromobenzoquinone adduct of peptide 89-119 in both GSTA1 and A2. The identity of these adducts and their location at Cys-111 was confirmed by tandem mass spectrometry (MS-MS). No evidence for the presence of any putative BB-adducts in GST M1, M2, or A3 was obtained. This work highlights the challenges involved in the unambiguous identification of reactive metabolite adducts formed in vivo.

Synthesis of heterocyclic carbene ligands via 1,2,3-diheterocyclization of allenylidene complexes with dinucleophiles

Heterocyclic carbene complexes are accessible from π-donor-substituted allenylidene complexes, [(CO) 5Cr C C C(NMe 2)Ph] ( 1) and [(CO) 5Cr C C C(O- endo-Bornyl)OEt] ( 4), and various dinucleophiles by 1,2,3-diheterocyclization. The reaction of 1 with 1,2-dimethylhydrazine gives the 1,2-dimethylpyrazolylidene complex ▪ ( 2) in high yield in addition to small amounts of the α,β-unsaturated carbene complex [(CO) 5Cr C(NMe 2)–C(H) C(NMe 2)Ph] ( 3). The analogous reaction of 4 with 1,2-dimethylhydrazine affords the 1,2-dimethylpyrazolylidene complex ▪ ( 5) and, via displacement of the C γ-bound ethoxy substituent, the hydrazinoallenylidene complex [(CO) 5Cr C C C(O- endo-Bornyl){NMe-N(H)Me}] ( 6). Treatment of 6 with catalytic amounts of acids induces cyclization to 5. On addition of 1,1-dimethylhydrazine to 1 the zwitterionic pyrazolium-5-ylidene complex ▪ ( 7) is formed. The reaction of 1 with 1,2-diaminocyclohexane affords a octahydro-benzo[1,4]diazepinylidene complex ( 10) and, via intermolecular substitution, a binuclear bisallenylidene complex ( 11). Thiazepinylidene complexes ( 12– 14), containing 7-membered N/S-heterocyclic carbene ligands, are formed highly selectively in the reaction of 1 with 2-aminoethanethiol or related cysteine derivatives by a substitution/cyclization sequence. The analogous reaction of 1 with homocysteine methylester yields a thiazocanylidene complex ( 15). All new heterocyclic carbene ligands are strong donors exhibiting σ-donor/π-acceptor ratios similar to those of the known imidazolylidene complexes. On photolysis of 2 and 12 in the presence of triphenylphosphine, the corresponding cis-carbene tetracarbonyl triphenylphosphine complexes ( 16 and 17) are formed. The solid state structure of complexes 2, 7, 14, 15, and 16 is established by X-ray structural analysis.

Homocysteine and Cardiovascular Disease

See related article, pages 598–606 An elevated plasma level of homocysteine has long been known as an independent predictor of cardiovascular disease.1,2 However, in the absence of a clear mechanism linking homocysteine to cardiovascular disease, there has been an ongoing debate about whether this relationship is one of cause and effect or whether an elevated level of plasma homocysteine is an epiphenomenon, reflecting the presence of some other proatherogenic factor that is actually responsible for the cardiovascular disease. In the current issue of this journal, Liao et al3 suggest that the mechanistic link may be a homocysteine-induced reduction in the concentration of high density lipoproteins (HDLs). Liao et al3 report that homocysteine reduces the concentration of HDL cholesterol in plasma by inhibiting the hepatic synthesis of apoA-I, the main HDL apolipoprotein. This conclusion supports the findings from another recent study by Mikael et al4 who also reported that homocysteine inhibits apoA-I synthesis. The results of these 2 studies not only explain the documented inverse correlation between the plasma concentrations of HDL cholesterol and homocysteine5,6 but also raise the real possibility that a homocysteine-induced inhibition of apoA-I synthesis is the mechanism linking homocysteine to the development of atherosclerosis. A low concentration of HDL cholesterol has been shown in numerous human population studies to be highly predictive of premature cardiovascular disease.7,8 Furthermore, treatments that increase the level of HDL cholesterol in plasma in both animals9,10 and humans11,12 reduce the progression or even promote …