Electrospray-assisted modification of proteins: a radical probe of protein structure

A new approach is described to probe the structure of proteins through their reactivity with oxygen-containing radicals. Radical-induced oxidative modification of proteins is achieved within an electrospray ion source using oxygen as a reactive nebulizer gas at high needle voltages. This method facilitates the rapid oxidation of proteins as the molecules emerge from the electrospray needle tip. Electrospray mass spectra of both ubiquitin and lysozyme reveal that over 50% of the protein can be modified under these conditions. The radical-induced oxidative modification of amino acid side chains is correlated with their solvent accessibility to obtain information on a protein's higher-order structure. The oxidation sites in hen lysozyme have been identified by proteolysis of the condensed protein solution and tandem mass spectrometry (MS/MS). Oxidation of tryptophan at positions 62 and 123 occurs exclusively over all other tryptophan residues, consistent with the relative solvent accessibilities of the residue side chains based on the NMR structure of the protein. Radical-induced oxidative modification of cysteine (Cys), methionine (Met), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), histidine (His), and leucine (Leu) residues is also reported, providing sufficient reactive markers to span a protein sequence. This facile oxidation process could be applied to investigate the molecular mechanism by which reactive oxygen species interact with a particular protein domain as a means to investigate the onset of certain diseases.     

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.     

Chemical modification of the arginine residues of proteins and peptides

Methods for the chemical modification of arginine residues with the aid of butan-2,3-dione and its oligomers, cyclohexane-1,2-dione, phenylglyoxal, tetraethoxy-propane, nitromalondialdehyde, and pentane-2,4-dione are considered. The conditions for performing the reactions and their mechanism are given and the stability of the products formed and methods for their identification are discussed.Pacific Ocean Institute of Bioorganic Chemistry, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 591–606, September–October, 1985.     

Chemical modification of the arginine residues of proteins and peptides

Methods for the chemical modification of arginine residues with the aid of butan-2,3-dione and its oligomers, cyclohexane-1,2-dione, phenylglyoxal, tetraethoxy-propane, nitromalondialdehyde, and pentane-2,4-dione are considered. The conditions for performing the reactions and their mechanism are given and the stability of the products formed and methods for their identification are discussed.     

Use of the plastein reaction for the modification of cotton-plant proteins

Pronase hydrolysates have been obtained from cottonseed meal which were then used as a substrate for the performance of the plastein reaction. The possibility has been shown of introducing deficient amino acids — lysine methionine, threonine, and tryptophan — by means of this reaction.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 92–96, January–February, 1986.     

Chemoselective reduction of ketones: trifluoromethylketones versus methylketones

Treatment of an equimolar mixture of trifluoromethylketones (TFMKs) and methylketones (MKs) with Et₂Zn resulted in selective reduction of the TFMKs in good yield. In contrast, treatment of an equimolar mixture of TFMKs and MKs with NaBH₄ in the presence of CeCl₃ in EtOH/H₂O (10:1) at −10 °C reduced only the MKs.     

Chemoselective Acylation of Nucleosides

Acylated nucleoside analogues play an important role in medicinal chemistry and are extremely useful precursors to various other nucleoside analogues. However, chemoselective acylation of nucleosides usually requires several protection and deprotection steps due to the competing nucleophilicity of hydroxy and amino groups. In contrast, direct protecting-group-free chemoselective acylation of nucleosides is a preferred strategy due to lower cost and fewer overall synthetic steps. Herein, a simple and efficient chemoselective acylation of nucleosides and nucleotides under mild reaction conditions, giving either O- or N-acylated products respectively with excellent chemoselectivity is reported.     

Chemoselective aminomethylation of harmol

Chemistry of Heterocyclic Compounds - Aminomethylation of harmol (7-hydroxy-1-methyl-β-carboline) was investigated. It was shown that the reaction proceeds chemoselectively to form...     

Selective N-terminal modification of peptides and proteins: Recent progresses and applications

Numerous strategies for linking desired chemical probes with target peptides and proteins have been developed and applied in the field of biological chemistry. Approaches for site-specific modification of native amino acid residues in test tubes and biological contexts represent novel biological tools for understanding the role of peptides and proteins. Selective N-terminal modification strategies have been broadly studied especially in the last 10 years, as N-terminal positions are typically so...     

Chemoselective hydrosilylation of hydroxyketones

A chemoselective method for the hydrosilylation of ketones has been developed, using the combination of triphenylsilane and a catalyst prepared from Ni(COD)₂ and the simple N-heterocyclic carbene IMes. The most notable feature of this method is that free hydroxyls are largely unaffected, thus providing a simple one-step procedure for the conversion of hydroxyketones to mono-protected diols, wherein the protecting group is exclusively installed on the ketone-derived hydroxyl. The process is typically high yielding with both simple ketones and more complex hydroxyketone substrates.     

Similarity metrics for ligands reflecting the similarity of the target proteins

In this study we evaluate how far the scope of similarity searching can be extended to identify not only ligands binding to the same target as the reference ligand(s) but also ligands of other homologous targets without initially known ligands. This "homology-based similarity searching" requires molecular representations reflecting the ability of a molecule to interact with target proteins. The Similog keys, which are introduced here as a new molecular representation, were designed to fulfill such requirements. They are based only on the molecular constitution and are counts of atom triplets. Each triplet is characterized by the graph distances and the types of its atoms. The atom-typing scheme classifies each atom by its function as H-bond donor or acceptor and by its electronegativity and bulkiness. In this study the Similog keys are investigated in retrospective in silico screening experiments and compared with other conformation independent molecular representations. Studied were molecules of the MDDR database for which the activity data was augmented by standardized target classification information from public protein classification databases. The MDDR molecule set was split randomly into two halves. The first half formed the candidate set. Ligands of four targets (dopamine D2 receptor, opioid delta-receptor, factor Xa serine protease, and progesterone receptor) were taken from the second half to form the respective reference sets. Different similarity calculation methods are used to rank the molecules of the candidate set by their similarity to each of the four reference sets. The accumulated counts of molecules binding to the reference target and groups of targets with decreasing homology to it were examined as a function of the similarity rank for each reference set and similarity method. In summary, similarity searching based on Unity 2D-fingerprints or Similog keys are found to be equally effective in the identification of molecules binding to the same target as the reference set. However, the application of the Similog keys is more effective in comparison with the other investigated methods in the identification of ligands binding to any target belonging to the same family as the reference target. We attribute this superiority to the fact that the Similog keys provide a generalization of the chemical elements and that the keys are counted instead of merely noting their presence or absence in a binary form. The second most effective molecular representation are the occurrence counts of the public ISIS key fragments, which like the Similog method, incorporates key counting as well as a generalization of the chemical elements. The results obtained suggest that ligands for a new target can be identified by the following three-step procedure: 1. Select at least one target with known ligands which is homologous to the new target. 2. Combine the known ligands of the selected target(s) to a reference set. 3. Search candidate ligands for the new targets by their similarity to the reference set using the Similog method. This clearly enlarges the scope of similarity searching from the classical application for a single target to the identification of candidate ligands for whole target families and is expected to be of key utility for further systematic chemogenomics exploration of previously well explored target families.     

Exogenous agents that target transmembrane domains of proteins

Although membrane proteins account for approximately one third of all proteins encoded in the human genome, the functions and structures of their transmembrane domains are much less understood than the water-soluble regions. A major hurdle in studying these transmembrane domains is the lack of appropriate exogenous agents that can be used as specific probes. Despite the daunting challenges, major strides have recently been made in targeting the transmembrane domains of a variety of membrane proteins. High affinity and selectivity have been achieved in model biophysical systems, membranes of bacteria, and mammalian cells.     

Chemoselective ring opening of benzoxazaphosphinines

Cyclization of 2-[(4-chloroanilino)methyl]phenol (1) with thiophosphoryl chloride afforded 2-chloro-3-(4-chlorophenyl)-3,4-dihydro-2H-1,3,2λ⁵-benzoxazaphosphinine-2-thione (2). Reaction of 2 with various heterocyclic amines (3) in the presence of Et₃N/NaH gave 3-(4-chlorophenyl)-2-nitrogen heterocyclic substituted-3,4-dihydro-2H-1,3,2λ⁵-benzoxaza-phosphinine-2-thiones (4). Further reaction of 4 with the N-sodium salt of amino heterocyclics in the presence of HCl at 50-60 °C opened the benzoxazaphosphinine ring chemoselectively at the endocyclic P-O bond and yielded 2-[4-chloro(heterocyclic substituted-phosphorothioyl)anilino]methylphenols 5-13.     

Site-selective modification of proteins for the synthesis of structurally defined multivalent scaffolds

A combination of classical site-directed mutagenesis, genetic code engineering and bioorthogonal reactions delivered a chemically modified barstar protein with one or four carbohydrates installed at specific residues. These protein conjugates were employed in multivalent binding studies, which support the use of proteins as structurally defined scaffolds for the presentation of multivalent ligands.     

Chemoselective alkylation of N-alkylaminooxy-containing peptides

Peptides containing N-alkylaminooxy amino acids were chemoselectively alkylated with allylic, benzylic, and alpha-carbonyl bromides, N-ethylmaleimide, and hexyl acrylate in mildly acidic aqueous/organic solutions. Alkylation at the aminooxy nitrogen proceeds in good yields with excellent to complete chemoselectivity in the presence of all common amino acids except cysteine. This reaction complements the selective glycosylation and acylation of N-alkylaminooxy groups and provides an avenue for the synthesis of peptide arrays comprising a wide variety of neoglycopeptides and neolipopeptides.     

Additive-Free Chemoselective Acylation of Amines

Aliphatic and aromatic amines are efficiently acylated by acetic, pivalic, benzoic, phthalic, or maleic anhydrides in ethyl acetate at room temperature. Under the same experimental conditions, amino alcohols are chemoselectively acylated at the amino group.     

Chemoselective Hydrogenation of Unsaturated Peroxides

Chemoselective hydrogenation of unsaturated monoperoxyketals provides new methodology for the synthesis of saturated and allyl hydroperoxides.