A three-component Mannich-type reaction for selective tyrosine bioconjugation

A new selective bioconjugation reaction is described for the modification of tyrosine residues on protein substrates. The reaction uses imines formed in situ from aldehydes and electron-rich anilines to modify phenolic side chains through a Mannich-type electrophilic aromatic substitution pathway. The reaction takes place under mild pH and temperature conditions and can modify protein substrates at concentrations as low as 20 muM. Using an efficient fluorescence-based assay, we demonstrated the reaction using a number of aldehydes and protein targets. Importantly, proteins lacking surface-accessible tyrosines remained unmodified. It was also demonstrated that enzymatic activity is preserved under the mild reaction conditions. This strategy represents one of the first carbon-carbon bond-forming reactions for protein modification and provides an important complement to more commonly used lysine- and cysteine-based methods.     

Direct, metal-free amination of heterocyclic amides/ureas with NH-heterocycles and N-substituted anilines in POCl3

A POCl(3)-mediated, direct amination reaction of heterocyclic amides/ureas with NH-heterocycles or N-substituted anilines is described. Compared to the existing methods, this operationally simple protocol provides unique reactivity and functional group compatibility because of the metal-free, acidic reaction conditions. The yields are generally excellent.     

Tagless extraction-retentate chromatography: a new global protein digestion strategy for monitoring differential protein expression

A new global protein digestion and selective peptide extraction strategy for the purpose of monitoring differential protein expression, coined as tagless extraction-retentate chromatography, is introduced. Target protein populations are firstly digested under reduced and alkylated conditions, and resultant peptides selectively extracted via covalent attachment to methionine residues by bromoacetyl reactive groups tethered to the surface of glass beads packed in small reaction vessels. After conjugation, reactive beads are stringently washed to remove nonspecifically bound peptides and then later treated with beta-mercaptoethanol to release captured methionine peptides in their nascent state, without complicating affinity tags. Recovered methionine containing peptides are profiled using the surface-enhanced laser desorption/ionization (SELDI) retentate chromatography mass spectrometry (RCMS) method. Selected peptides are further studied employing ProteinChip tandem mass spectrometry (MS/MS) analysis to identify their parent proteins. This approach has been applied to an Escherichia coli lysate model system and has demonstrated facility in reducing global digest complexity, sensitivity to low protein expression levels, and significant quantitative capability. It is envisioned that tagless extraction-RCMS will evolve to be a valuable approach for both basic research and clinical proteomics endeavors.     

Chemoselective attachment of biologically active proteins to surfaces by expressed protein ligation and its application for "protein chip" fabrication

The present work describes a general method for the selective attachment of proteins to solid surfaces through their C-termini that can be used for the efficient creation of protein chips. Our method is based in the chemoselective reaction between a protein C-terminal alpha-thioester and a modified surface containing N-terminal Cys residues. alpha-Thioester proteins can be obtained using standard recombinant techniques by using expression vectors containing engineered inteins. This new method was used to immobilize two fluorescent proteins and a functional SH3 domain using a protein microarrayer.     

Amyloid-beta detection with saccharide immobilized gold nanoparticle on carbon electrode

The electrochemical sensing of saccharide-protein interactions using a couple of sialic acid derivatives and Alzheimer's amyloid-beta (Abeta) is described. The densely-packed saccharide area for recognition of protein was fabricated onto a carbon electrode by three steps, which were electrochemical deposition of Au nanoparticles on a screen printed strip, self-assembled monolayer (SAM) formation of the acetylenyl group on Au nanoparticles, and the cycloaddition reaction of an azide-terminated sialic acid to the acetylenyl group. The attachment of Abeta peptides to the sialic acid layer was confirmed by electrochemistry and atomic force microscopy imaging. The intrinsic oxidation signal of the captured Abeta(1-40) and (1-42) peptides, containing a single tyrosine (Tyr) residues, was monitored at a peak potential of 0.6 V (vs Ag/AgCl within this sensor) in connection with differential pulse voltammetry. The peak current intensities were concentration dependent. The proposed process provides new routes for analysis of saccharide-protein interactions and electrochemical biosensor development.     

A highly efficient synthesis of 1,2,3,4-tetrahydroquinolines by molecular iodine-catalyzed domino reaction of anilines with cyclic enol ethers

A highly efficient method for the synthesis of 1,2,3,4-tetrahydroquinoline derivatives via a molecular iodine catalyzed domino reaction of anilines with cyclic enol ethers, such as 2,3-dihydrofuran and 3,4-dihydro-2H-pyran, is described. The reaction may proceed through an aza-Diels-Alder process between an in situ generated 2-azadiene and another equivalent of cyclic enol.     

Selective Iododeamination of Electron-Deficient Anilines Using Task-Specific Nitrite- and Iodide-Based Ionic Liquids

A convenient and rapid method for the synthesis of aryliodides via the sequential diazotization–iodination of electron-deficient anilines using IL-ONO/[bmim]I is described. IL-ONO and [bmim]I were used as a nitrosonium source for diazotization of anilines and as a iodide ion source for subsequent iodination respectively. Electron-deficient anilines were efficiently and selectively iododeaminated in the presence of other anilines in good to excellent yields. This reaction can be regarded as a new method for the preparation of synthetically and pharmaceutically important iodoarens. Selectively iododeamination of electron-poor anilines in the presence of electron-rich anilines is one of the synthetically important advantages of this new method.     

General method for the synthesis of caged phosphopeptides: tools for the exploration of signal transduction pathways

[reaction: see text] An interassembly approach for the synthesis of peptides containing 1-(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and -tyrosine has been developed. Photochemical uncaging of these peptides releases the 2-nitrophenylethyl protecting group to afford the corresponding phosphopeptide. The peptides described herein are based on phosphorylation sites of kinases involved in cell movement or cell cycle regulation and demonstrate the versatility of the method and compatibility with the synthesis of polypeptides, including a variety of encoded amino acids.     

Visible-light-mediated direct perfluoroalkylation and trifluoromethylation of free anilines

A mild, operationally simple method for direct perfluoroalkylation and trifluoromethylation of anilines through visible-light-mediated photoredox catalysis from broadly available perfluoroalkyl iodides and free anilines is described. The method provides a facile route for application in drug discovery and development.     

非蛋白氨基酸对生物活性多肽的修饰及其构效关系的研究进展

简述了非蛋白氨基酸的结构特征和生理活性。介绍了某些天然存在的含非蛋白氨基酸的生物活性肽及非蛋白氨基酸残基在相应活性肽中的重要作用。着重总结了非蛋白氨基酸对生物活性肽进行修饰的方式和构效关系研究的进展。     

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.     

Lipid modification of proteins through sortase-catalyzed transpeptidation

A general chemoenzymatic method for the site-specific attachment of lipids to protein substrates is described. Sortase A is used to append short lipid-modified oligoglycine peptides to the C terminus of protein substrates bearing a five amino acid sortase A recognition sequence (LPETG). We demonstrate the attachment of a range of hydrophobic modifications in excellent yield (60-90%), including a simple step for removing the sortase enzyme postreaction. Lipoproteins prepared using these procedures were subsequently shown to associate with mammalian cells in a lipid tail-dependent fashion and localized to the plasma membrane and endosomes.     

Modification of aniline containing proteins using an oxidative coupling strategy

A new bioconjugation reaction has been developed based on the chemoselective modification of anilines through an oxidative coupling pathway. Aryl amines were installed on the surface of protein substrates through lysine acylation reactions or through the use of native chemical ligation techniques. Upon exposure to NaIO4 in aqueous buffer, the anilines coupled rapidly to the aromatic rings of N,N-dialkyl-N'-acyl-p-phenylenediamines. The identities of the reaction products were confirmed using ESI-MS and through comparison to small molecule analogs. Control experiments indicated that none of the native amino acids participated in the reaction. The resulting bioconjugates were found to be stable toward hydrolysis from pH 4 to pH 11 and in the presence of many commonly used oxidants, reductants, and nucleophiles. A fluorescent phenylenediamine reagent was synthesized for the selective detection of aniline labeled proteins in mixtures, and the reaction was used to append the C-terminus of the green fluorescent protein with a single PEG chain. When combined with techniques for the incorporation of unnatural amino acids into proteins, this bioorthogonal coupling method should prove useful for a number of applications requiring a high degree of labeling specificity.     

A structural study of model peptides derived from HIV-1 integrase central domain

The HIV-1 integrase (IN) catalyzes the integration of viral DNA in the human genome. In vitro the enzyme displays an equilibrium of monomers, dimers, tetramers and larger oligomers. However, its functional oligomeric form in vivo is not known. We report a study of the auto-associative properties of three peptides denoted K156, E156 and E159. These derive from the alpha4 helix of the IN catalytic core. The alpha4 helix is an amphipatic helix exposed at the surface of the protein and could be involved in the oligomerization process through its hydrophobic face. The peptides were obtained from the replacement of several amino acid residues by more helicogenic ones in the alpha4 helix peptide. K156 carries the basic residues Lys156 and Lys159, which have been shown important for the binding of IN to viral DNA. In E156 and E159 they are replaced with the acidic residue Glu. A fourth peptide K(E)156 obtained from the replacement of hydrophobic residues with Glu in K156 in order to abolish the auto-associative properties is used as a negative control. The capacity shown by peptides for alpha-helical formation is demonstrated by circular dichroism (CD) analysis performed in aqueous solution and in aqueous trifluoroethanol (TFE) mixtures. Both electrospray ionization mass spectrometry (ESI-MS) and glutaraldehyde chemical cross-linking show that peptides adopt different solvent-dependent equilibriums of monomers, dimers, trimers and tetramers. Oligomerization of peptides in aqueous solution is related to their ability to form helical structures. Addition of a small amount of TFE (<10%) stimulates helix stabilization and the interhelical hydrophobic contacts. Higher amounts of TFE alter the hydrophobic contacts and disrupt the oligomeric species. In addition to hydrophobic interactions, the patterns indicate that the biologically important Lys156 and Lys159 residues also participate in helix association. K(E)156 despite its ability to adopt a helical structure is unable to associate into oligomers, demonstrating the importance of hydrophobic contacts for oligomerization. Thus, the designed peptides provide us information on the functional properties of the alpha4 IN that seems to hold a dual role in DNA recognition and protein oligomerization.     

Charge stripping and the site of cationization of substituted aromatic compounds

The site of protonation, methylation and ethylation of anilines, phenols and thiophenols can be determined by tandem mass spectrometry (MS/MS). The extent to which the cation attachment products formed from these compounds undergo charge stripping is related to the site of cation attachment. The formation of doubly charged ions for phenols and anilines is favored when cation attachment occurs on the ring. For thiophenols alkylation occurs almost exclusively on the substituent, and yet an abundant stripping peak is recorded. This behavior is opposite to that seen for anilines and phenols and is accounted for in terms of orbital energies in the three systems. The site of cation attachment, as deduced by charge stripping, agrees with conclusions drawn from the fragmentation behavior of these ions upon collision-induced dissociation.     

Sensitive method of detection, quantitation and purification of peptides using pre-column derivatization with phenyl isothiocyanate

A sensitive method for the detection, quantitation and purification of peptides is described. The method is based on pre-column derivatization of peptides with phenyl isothiocyanate to form phenylthiocarbamoyl derivatives (PTC peptides). The derivatized peptides are analysed by reversed-phase high-performance liquid chromatography on a Zorbax ODS column (5 micron) and detected at 269 nm with a sensitivity limit of 1-5 pmol. The technique was utilized for the separation of a mixture of closely related synthetic peptides. The eluted PTC peptides were collected with an average recovery yield of 75% as determined by amino acid analysis. This method of separation of PTC peptides was also combined with the determination of the complete structure of recovered PTC-dynorphin A-(1-13) using the solid-phase sequenator (Sequemat). The advantages of the derivatization method are the rapidity and completeness of the reaction, the stability of the product, the sensitivity and specificity of the detection of derivatized peptides and the compatibility of the technique with subsequent analytical procedures. A particular application of this method was exemplified by the dosage of enkephalins secreted from perfused bovine adrenal glands.     

Precolumn derivatization of cysteine residues for quantitative analysis of five major cytochrome P450 isoenzymes by liquid chromatography/tandem mass spectrometry

The development of a novel method for absolute quantification of the five most clinically relevant CYP450 isoenzymes is described based on chemical derivatization of cysteine residues. The sulfhydryl-reactive reagents, 2-bromo-4'-chloroacetophenone (p-CPB) and 2-bromo-4'-bromoacetophenone (p-BPB), are proposed for use in quantitative proteomics. After reducing and denaturing, the P450s are derivatized with p-CPB for sulfhydryl alkylation then subjected to trypsin digestion. The resulting p-CPB-attached peptides are enriched using a phenyl resin solid-phase cartridge, then separated on a Zorbax 300SB reversed-phase column, and detected under positive electrospray ionization in the multiple reaction monitoring mode. Quantification is achieved using p-BPB-modified peptides as internal standards. Validation results demonstrated that this method showed good linearity between the concentration range of 10 fmol/microg to 5 pmol/microg for the six selected peptides in a complex matrix (rat liver microsomal protein). Intra-day and inter-day precision, expressed by relative standard deviation, were all less than 18%. Assay accuracy was within +/- 20% in terms of relative error. The quantitative derivatization approach proved to be reproducible, cost-effective and readily suitable for high-throughput assays. The reliability of this method for quantification of intact P450s was demonstrated through comparing with the well-applied isotope-coded affinity tag (ICAT) method.     

Coulometric titration of anilines, mainly containing deactivating functional groups, with bromine

A general method is described for the coulometric titration of various substituted anilines with anodically generated bromine. The reaction is carried out in water-acetic acid medium and the reactivity is governed by varying the water content and the bromide concentration, and by the addition of pyridine. In this way the majority of the substituted anilines tested can be titrated quantitatively. There is no great difference in the reactivity-promoting properties of pyridine-free and pyridine-containing media, but the latter generally give the better results and are therefore to be preferred. The mean relative error, for the best medium for each compound, is +/-0.5% for the haloanilines and +/-1.0% for anilines with oxygenated functional groups.     

α‐Peptide–Oligourea Chimeras: Stabilization of Short α‐Helices by Non‐Peptide Helical Foldamers

Short α‐peptides with less than 10 residues generally display a low propensity to nucleate stable helical conformations. While various strategies to stabilize peptide helices have been previously reported, the ability of non‐peptide helical foldamers to stabilize α‐helices when fused to short α‐peptide segments has not been investigated. Towards this end, structural investigations into a series of chimeric oligomers obtained by joining aliphatic oligoureas to the C‐ or N‐termini of α‐peptides are described. All chimeras were found to be fully helical, with as few as 2 (or 3) urea units sufficient to propagate an α‐helical conformation in the fused peptide segment. The remarkable compatibility of α‐peptides with oligoureas described here, along with the simplicity of the approach, highlights the potential of interfacing natural and non‐peptide backbones as a means to further control the behavior of α‐peptides.     

Electrochemical oxidation and cleavage of peptides analyzed with on-line mass spectrometric detection

An on-line electrochemistry/electrospray mass spectrometry system (EC/MS) is described that allows fast analysis of the oxidation products of peptides. A range of peptides was oxidized in an electrochemical cell by application of a potential ramp from 0 to 1.5 V during passage of the sample. Electrochemical oxidation of peptides was found to occur readily when tyrosine was present. Tyrosine was found to be oxidized between 0.5 and 1.0 V to various oxidation products, including peptide fragments formed by hydrolysis at the C-terminal side of tyrosine. The results confirm earlier knowledge on the mechanisms and reaction products of chemical and electrochemical peptide oxidation. Methionine residues are also readily oxidized, but do not induce peptide cleavage. At potentials higher than about 1.1 V, additional oxidation products were observed in some peptides, including loss of 28 Da from the C-terminus and dimerization. The tyrosine-specific cleavage reaction suggests a possible use of the EC/MS system as an on-line protein digestion and peptide mapping system. In addition, the system can be used to distinguish phosphorylated from unphosphorylated tyrosine residues. Four forms of the ZAP-70 peptide ALGADDSYYTAR with both, either or neither tyrosine phosphorylated were subjected to a 0-1.5 V potential ramp. Oxidation of, and cleavage adjacent to, tyrosine was observed exclusively at unphosphorylated tyrosine residues.