Tryptophan Residues In Peptides Research Articles

Palladium-Catalyzed Olefination of Tryptophan Residues in Peptides

Key words Fujiwara–Moritani reaction - tryptophan - palladium catalysis - late-stage modification

Chemical modifications of tryptophan residues in peptides and proteins.

Chemical protein modifications facilitate the investigation of natural posttranslational protein modifications and allow the design of proteins with new functions. Proteins can be modified at a late stage on amino acid side chains by chemical methods. The indole moiety of tryptophan residues is an emerging target of such chemical modification strategies because of its unique reactivity and low abundance. This review provides an overview of the recently developed methods of tryptophan modification at the peptide and protein levels.

Selective Modification of Tryptophan Residues in Peptides and Proteins Using a Biomimetic Electron Transfer Process.

We report here a photochemical process for the selective modification of tryptophan (Trp) residues in peptides and small proteins using electron-responsive N-carbamoylpyridinium salts and UV-B light. Preliminary mechanistic experiments suggest that the photoconjugation process proceeds through photoinduced electron transfer (PET) between Trp and the pyridinium salt, followed by fragmentation of the pyridinium N-N bond and concomitant transfer of this group to Trp. The reaction displays excellent site selectivity for Trp and is tolerant to other, redox-active amino-acid residues. Moreover, the reaction proceeds in pure aqueous conditions without the requirement of organic cosolvents or photocatalysts, is enhanced by glutathione, and operates efficiently over a wide range of peptide concentrations (10-700 μM). The scope of the process was explored through the labeling of 6-Trp-containing peptides and proteins ranging from 1 to 14 kDa. We demonstrate the versatility of the N-carbamoylpyridinium salt both by tuning the electrochemical and photochemical properties of the pyridinium scaffold to enable challenging photoconjugation reactions and by using the carbamoyl moiety to tether a plethora of productive functional groups, including reactive handles, purification tags, and removable protecting groups.

Palladium-Catalyzed Selective C–H Olefination of Tryptophan Residues in Peptides

Palladium-Catalyzed Selective C–H Olefination of Tryptophan Residues in Peptides

C-H Olefination of Tryptophan Residues in Peptides: Control of Residue Selectivity and Peptide-Amino Acid Cross-linking.

There is high demand for new methods to modify peptides, for application in drug discovery and biomedicine. A C-H functionalization protocol for the olefination of tryptophan residues in peptides is described. The modification is successful for Trp residues at any position in the peptide, has broad scope in the styrene coupling partner, and offers opportunities for conjugating peptides with other biomolecules. For peptides containing both Trp and Phe, directing group manipulation enables full control of residue selectivity.

97 - Use of pyrogallol red to assess carbonate anion radical formation from the bicarbonate-dependent peroxidase activity of superoxide dismutase 1

Background Carbonate radical anion (CO3•–) is a highly reactive free radical generated by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 (SOD-1). CO3•– has been implicated in the crosslinking of human SOD-1, and neurodegenerative disorders as amyotrophic lateral sclerosis. Previous studies have used Pyrogallol Red (PGR) to probe the scavenging activity of polyphenols towards reactive species such as peroxyl radicals, peroxynitrite, superoxide, hypochlorous acid, and nitrogen dioxide. The rate and extent of CO3l-formation by SOD-1 is not however well characterized. Aim To examine the potential of PGR to determine the peroxidase activity of SOD-1, and quantify CO3•– formation. Results PGR solutions (1-100 µM) in bicarbonate buffer (200 mM, pH 7.4) with diethylenetriaminepenta-acetic acid (0.1 mM) were incubated in the presence hydrogen peroxide (1-10 mM), and bovine SOD-1 (0.3-30 µM as dimers) at 37°C. Oxidative consumption of PGR is associated with a progressive decrease in its absorbance at 540 nm, and formation of a new band at 390 nm related to the production of a PGR-derived quinone. The kinetic profiles (followed at 540 nm) showed an efficient PGR oxidation with initial consumption rates in the order of 6-15 µM/min. After a total PGR consumption, successive additions of PGR aliquots showed PGR consumptions with a significantly lower rate. This behavior was observed at different PGR and SOD-1 concentrations and was explained by inactivation of SOD-1 in the first minutes incubation. We observed a similar kinetic behavior when free and peptide tryptophan residues were exposed to the bicarbonate/hydrogen peroxide/SOD-1 system, the experimental conditions affecting the formation of oxygenated products, as well as, tryptophan crosslinking (studied by liquid chromatography with mass detection). Conclusions PGR can be used to detect and quantify oxidant formation by the peroxidase activity of SOD-1.

Degradation of tyrosine and tryptophan residues of peptides by type I photosensitized oxidation

Pterin derivatives are involved in various biological functions, including enzymatic processes that take place in human skin. Unconjugated oxidized pterins are efficient photosensitizers under UV-A irradiation and accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder. These compounds are able to photoinduce the oxidation of the peptide α-melanocyte-stimulating hormone (α-MSH), which stimulates the production and release of melanin by melanocytes in skin and hair. In the present work we have used two peptides in which the amino acid sequence of α-MSH was mutated to specifically investigate the reactivity of tryptophan (Trp) and tyrosine residues (Tyr). The parent compound of oxidized pterins (Ptr) was used as a model photosensitizer in aqueous solution at pH5.5 and was exposed to UV-A radiation, a wavelength range where the peptides do not absorb. Trp residue yields N-formylkynurenine and hydroxytryptophan as oxidized products, whereas the Tyr undergoes dimerization and incorporation of oxygen atoms. In both cases, the first step of the mechanism involves an electron transfer from the amino acid to the photosensitizer triplet excited state, Ptr is not consumed and hydrogen peroxide (H2O2) is released. The role of singlet oxygen produced by energy transfer from 3Ptr⁎ to dissolved O2 was negligible or minor. Other amino acid residues, such as histidine, might be also affected.

Second harmonic generation from tryptophan-rich short peptides: W(n)K(m) and gramicidin A.

We report the first hyperpolarizability of a series of tryptophan-rich short peptides with the respective sequence KWK, KWWK, KWWWK, KWWKWWK, where W and K stand for tryptophan and lysine. The measurements were performed with the technique of hyper-Rayleigh scattering in the bulk of an aqueous Tris buffer solution at a pH of 8.5 and a salt concentration of 150 mM at the non-resonant fundamental wavelength of 784 nm. The first hyperpolarizability of the different peptides follows a simple additive model scaling with the number of tryptophan residues contained in the peptide. However, it appears that the first hyperpolarizability response of a single tryptophan residue in the peptide strongly differs from that of an isolated tryptophan. Hence, it is therefore demonstrated that the local environment of the tryptophan residues within the peptide strongly influences its nonlinear optical response. A comparison with the first hyperpolarizability of the natural peptide gramicidin A measured in trifluoroethanol (TFE) further confirms the key role of the local environment on the first hyperpolarizability of tryptophan residues in peptides.

Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system

ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.

Reaction of the Indole Group with Malondialdehyde: Application for the Derivatization of Tryptophan Residues in Peptides

A method for the selective modification of tryptophan residues based on the reaction of malondialdehyde with the indole nitrogen of the tryptophan side chain at acidic conditions is presented. The condensation reaction is quantitative and leads to a substituted acrolein moiety with a remaining reactive aldehyde group. As is shown, this group can be further converted to a hydrazone using hydrazide compounds, but if hydrazine or phenylhydrazine are used, release of the free indole group is observed upon cleavage of the substitution. Alternatively, secondary amines such as pyrrolidine may also act as cleavage reagents. This general reaction scheme has been adapted and optimized for the derivatization of tryptophan-containing peptides and small N-heterocyclic compounds. It serves as the basis of a reversible tagging scheme for Trp-peptides or molecules of interest carrying indole structures as it allows the specific attachment and removal of a reactive group that may be used for a variety of purposes such as affinity tagging.

Generation, basic chemistry, and detection of N-nitrosotryptophan derivatives

N-Terminal blocked tryptophan derivatives like melatonin or tryptophan residues in peptides are easily nitrosated at the nitrogen atom of the indole ring to give the corresponding N-nitrosotryptophan derivatives. This article provides a comprehensive view of the synthesis, chemical properties, and detection methods of this class of N-nitroso compounds of potential importance in biological systems.

POST-CHROMATOGRAPHIC DETECTION OF OLIGOPEPTIDES USING TERBIUM-SENSITIZED LUMINESCENCE

This report investigated the potential use of oligopeptide complexation with terbium III as a high-performance liquid chromatography method with time-resolved spectrofluorimetric detection for analysis of milk-derived products. The study focused on glycyl-leucyl-phenylalanine, a compound with immunostimulatory properties isolated from milk casein, and also considered tyrosine and tryptophan residues in peptides. As the water content of the mobile phase was highly restrictive for detection, acetate ions were introduced as a potentiation agent. Only post-chromatographic complexation proved feasible, because passage through a column destroyed oligopeptide-terbium chelation. Validation of glycyl-leucylphenylalanine, glycyl-phenylalanine, and leucyl-phenylalanine assays showed linearity in the 8 × 10−5 to 5 × 10−4mol l−1 range (correlation coefficients of 0.96, 0.99 and 0.99 respectively). Results for the assay at 10−5 mol l−1 showed a 7% intra-day (repeatability) coefficient of variation (CV) and a 15% inter-day (reproducibility) CV. No interferences were observed in the presence of various water-soluble vitamins present in whey. This method, when tested in the bromatology field on yoghurts with different milk origins, showed that goat wheys had phenylalanine, tyrosine, and tryptophan residues twice those of cow wheys and three times those of sheep or soja bean wheys.

Specificity in substrate binding by protein folding catalysts: tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas-specific protein disulfide isomerase PDIp.

Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding.

Oxidation of Free Tryptophan and Tryptophan Residues in Peptides and Proteins.

The oxidation of free tryptophan (Trp) and Trp residues in peptides and proteins by hydrogen peroxide as oxidative agent has been used to evaluate Trp losses and the pattern of degradation products formed. Besides free Trp, four Trp-containing peptides and lysozyme were used as substrates in the aqueous model system. The oxidation rate of Trp and the formation of 16 possible degradation compounds were examined using RP-HPLC and UV, fluorescence, and photodiode array detection. The rate of Trp degradation increased from lysozyme to short-chain peptides to unbound Trp. Only approximately 20% of the total Trp loss could be elucidated by the determined Trp degradation compounds. Oxindolylalanine (Oia), 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (PIC), N-formylkynurenine (NFK), dioxindolylalanine (DiOia), kynurenine (Kyn), and 5-hydroxytryptophan (5-OH-Trp) were identified in this order of quantity as degradation compounds, showing the Trp pyrrole moiety to be most susceptible to oxidation. As short peptides such as H-Ala-Trp-Ala-OH were completely hydrolyzed with immobilized Pronase E, Oia and NFK could be identified as main degradation compounds, as could minor amounts of Kyn, DiOia, PIC, and 5-OH-Trp. Acid (6 M HCl), alkaline (4.2 M NaOH), and enzymatic hydrolyses were compared for the determination of Trp degradation compounds in lysozyme. Kyn, Oia, and DiOia could be detected in the hydrogen peroxide treated protein.

ChemInform Abstract: Protein Analysis: Development and Application of Reagents

Reagents and techniques for the analysis of primary structures of proteins have been reviewed with special emphasis on those which had been developed by the present author and his coworkers : Fluorescent Koshland reagent (FKR, 2-carboxy-1-hydroxy-4-naphtylmethyldimethylsulfonium chloride) for tryptophan residues and fluorescent Edman reagent (4-N, N-dimethylainino-l-naphthyl isothiocyanate, DNTC). FKR reacts with tryptophan residues at pH 03 and room temperature to produce fluorescent adduct (λex255 nm, λem 416 nm), the reaction being completed within 2 min. Owing to its high specificity, FKR was applicable to the determination of tryptophan residues in peptides and proteins. Methods for the selective isolation of tryptophan-containing fragments in peptides and proteins, based upon chelating ability of FKR derivative and reversed-phase high performance liquid chromatography, are described. Scope and limitation of DNTC in microsequence analysis of proteins are discussed, together with other Edman-type reagents. Furthermore, the reagents for protein assay and amino acid analysis are briefly reviewed.

タンパク質分析 試薬の開発と応用

Reagents and techniques for the analysis of primary structures of proteins have been reviewed with special emphasis on those which had been developed by the present author and his coworkers : Fluorescent Koshland reagent (FKR, 2-carboxy-1-hydroxy-4-naphtylmethyldimethylsulfonium chloride) for tryptophan residues and fluorescent Edman reagent (4-N, N-dimethylainino-l-naphthyl isothiocyanate, DNTC). FKR reacts with tryptophan residues at pH 03 and room temperature to produce fluorescent adduct (λex255 nm, λem 416 nm), the reaction being completed within 2 min. Owing to its high specificity, FKR was applicable to the determination of tryptophan residues in peptides and proteins. Methods for the selective isolation of tryptophan-containing fragments in peptides and proteins, based upon chelating ability of FKR derivative and reversed-phase high performance liquid chromatography, are described. Scope and limitation of DNTC in microsequence analysis of proteins are discussed, together with other Edman-type reagents. Furthermore, the reagents for protein assay and amino acid analysis are briefly reviewed.

The stacking interaction in 9- (indole-3-propyl) -1-methyl adeninium iodide crystal, a model study on the interaction between tryptophan residue and adenine base in protein-nucleic acid interactions

The crystal structure of 9- [indole-3-propyl] -1-methyl adeninium iodide has been determined by X-ray diffraction method as a model for possible interaction between the tryptophan residue of peptide and the N(1)-protonated or methylated adenine base of nucleic acid. The indole ring stacks intermolecularly with the adeninium ring, which might be caused by their dipoledipole and/or charge-transfer interaction between both the aromatic rings as suggested from the absorption spectra.

An enzymatic approach to the labeling of tryptophan residues in peptides and proteins

An enzymatic approach to the labeling of tryptophan residues in peptides and proteins

New procedure for oxidation of 3-substituted indoles to oxindoles: modification of tryptophan residues in peptides and proteins

A new high-yield procedure for the oxidation to oxindoles of 3-substituted indoles, including tryptophan-containing peptides and proteins, involving the use of a mixture of dimethyl sulphoxide and concentrated HCl, is described.

Formaldehyde-hydrochloric acid treatment. A fluorescence histochemical method for the demonstration of tryptophan residues in peptides and proteins.

Treatment with formaldehyde gas and HCl vapor, simultaneously or in sequence, induces fluorescence with indoles, including tryptophan residues of peptides, as is evident from studies on protein droplet models. Among cells that display intense formaldehyde-HCl-induced fluorescence are pancreatic exocrine cells, gastric chief cells, Paneth cells and enterochromaffin cells. Peptide hormone-producing cells that can be visualized by the formaldehyde-HCl treatment include gastrin cells and glucagon cells. The simultaneous procedure has proved superior to the sequential procedure. Simultaneous formaldehyde-HCl treatment appears to be a useful method for the demonstration of tryptophan residues of peptides and proteins. It seems more sensitive than previously described indole methods.