The Experts below are selected from a list of 6432 Experts worldwide ranked by ideXlab platform
Andrij Pich - One of the best experts on this subject based on the ideXlab platform.
-
reactive copolymers based on n vinyl lactams with pyridyl Disulfide side groups via raft polymerization and postmodification via thiol Disulfide Exchange reaction
Macromolecules, 2016Co-Authors: Huan Peng, Xiaobin Huang, Felix Jakob, Marcel Karperien, Ulrich Schwaneberg, Andrij Pich, Kristin RubsamAbstract:Herein, we report the synthesis of a series of novel pyridyl Disulfide (PDS)-functionalized statistical reactive copolymers that enable facile access to complex polymeric architectures through highly selective thiol–Disulfide Exchange reaction with thiol-containing ligands or proteins. Functional reactive poly(N-vinyl lactam)-based copolymers including poly(N-vinylpyrrolidone-co-pyridyl Disulfide ethyl methacrylate) (PVPD), poly(N-vinylpiperidone-co-pyridyl Disulfide ethyl methacrylate) (PVPID), and poly(N-vinylcaprolactam-co-pyridyl Disulfide ethyl methacrylate) (PVD) with PDS side groups were synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization at 60 °C in anisole with methyl 2-(ethoxycarbonothioylthio)propanoate as chain transfer agent. The PDS contents in the synthesized copolymers were varied from 2 to 10 mol % (as confirmed by systematical characterization with FTIR/Raman and 1H NMR spectroscopy) using well-controlled continuous feeding method. The kinetics study su...
-
Reactive Copolymers Based on N‑Vinyl Lactams with Pyridyl Disulfide Side Groups via RAFT Polymerization and Postmodification via Thiol–Disulfide Exchange Reaction
2016Co-Authors: Huan Peng, Kristin Rübsam, Xiaobin Huang, Felix Jakob, Marcel Karperien, Ulrich Schwaneberg, Andrij PichAbstract:Herein, we report the synthesis of a series of novel pyridyl Disulfide (PDS)-functionalized statistical reactive copolymers that enable facile access to complex polymeric architectures through highly selective thiol–Disulfide Exchange reaction with thiol-containing ligands or proteins. Functional reactive poly(N-vinyl lactam)-based copolymers including poly(N-vinylpyrrolidone-co-pyridyl Disulfide ethyl methacrylate) (PVPD), poly(N-vinylpiperidone-co-pyridyl Disulfide ethyl methacrylate) (PVPID), and poly(N-vinylcaprolactam-co-pyridyl Disulfide ethyl methacrylate) (PVD) with PDS side groups were synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization at 60 °C in anisole with methyl 2-(ethoxycarbonothioylthio)propanoate as chain transfer agent. The PDS contents in the synthesized copolymers were varied from 2 to 10 mol % (as confirmed by systematical characterization with FTIR/Raman and 1H NMR spectroscopy) using well-controlled continuous feeding method. The kinetics study suggested that copolymerizations were less favored with the enlargement of the lactam rings, indicated by lower conversions and larger dispersity indexes (Đ). The PDS-functionalized reactive polymers were amenable to functionalization with a variety of thiol-containing molecules, including 3-mercaptopropionic acid (3M), 2-phenylethanethiol (2P), methyl 3-mercaptopropionate (M3), 2-mercaptoethanol (2M), 2-aminoethanethiol (2A), poly(ethylene glycol) methyl ether thiol (PEG-SH), and enhanced green fluorescent protein (EGFP) via thiol–Disulfide Exchange reaction under mild conditions, confirmed by 1H NMR and SDS-PAGE. The conversions in all cases were higher than 95%, displaying that the thiol–Disulfide Exchange reaction to PDS groups with thiol-containing molecules is highly selective and tolerant to different ligands including amine, carboxyl, hydroxyl, phenyl, PEG and even polypeptides, providing a versatile scaffold for facile conjugation of various biological components. The contact angle measurement results and fluorescence microscopy study indicated that the reactive films based on the PDS-functionalized copolymers allowed facile, direct, and environmental-friendly surface engineering of surfaces from aqueous solution suggesting potential application in surface decoration of tissue-engineering scaffolds and medical implants. The initial cell culture experiments with HeLa cells displayed that the unmodified PVPD film was nontoxic and biocompatible while the film modified with PEG (a type of antifouling polymer) showed diminished cell attachment and growth, indicating that elegant engineering of the film surface can meet demands of particular applications
Elizabeth M Topp - One of the best experts on this subject based on the ideXlab platform.
-
thiol Disulfide Exchange in human growth hormone
Pharmaceutical Research, 2016Co-Authors: Saradha Chandrasekhar, Balakrishnan S Moorthy, Ruichao Xie, Elizabeth M ToppAbstract:Thiol-Disulfide Exchange was monitored in recombinant human growth hormone (hGH) and in model tryptic peptides derived from hGH to investigate the effects of higher-order structure on the reaction. Different free thiol-containing peptides, varying in length and amino acid sequence, were used to initiate the reaction at pH 7.0 and 37°C in hGH. Protein samples were digested with trypsin and analyzed for native Disulfides, scrambled Disulfides and free thiols using LC/MS. The loss of native Disulfide and Disulfide Exchange was compared with model peptides derived from hGH. Loss of native Disulfide in cyclic (cT20-T21) and linear peptides (T20-T21pep) derived from the C-terminal hGH Disulfide during the first 60 min of reaction was greater than loss of the C-terminal Disulfide in hGH itself. Of the thiols tested, glutathione (GSH) was the most reactive, forming the highest percentage of mixed Disulfides in intact hGH and in the model peptides. At longer reaction times (>240 min), native Disulfides in both hGH and cT20-T21 were regenerated. The fastest rates of regeneration were observed for Cys and the di- or tripeptide containing an Arg residue adjacent to Cys, suggesting that they may be useful in refolding. Thiol-Disulfide Exchange reactions in hGH and related model peptides were influenced by higher order structure, by the size of the thiol reactant and by an Arg residue adjacent to Cys in the thiol reactant. Reduction of Disulfide bonds in hGH did not affect higher order structure as measured by CD and HDX-MS.
-
thiol Disulfide Exchange in peptides derived from human growth hormone during lyophilization and storage in the solid state
Journal of Pharmaceutical Sciences, 2015Co-Authors: Saradha Chandrasekhar, Elizabeth M ToppAbstract:Lyophilization (freeze-drying) is frequently used to stabilize protein therapeutics. However, covalent modifications such as thiol-Disulfide Exchange and Disulfide scrambling can occur even in the solid state. The effects of lyophilization and storage of lyophilized powders on the mechanism and kinetics of thiol-Disulfide Exchange have not been elucidated and are explored here. Reaction kinetics was monitored in peptides corresponding to tryptic fragments of human growth hormone (T20 + T20-T21 or T20 + cT20-T21) during different stages of lyophilization and during storage of the lyophilized powders at 22°C and ambient RH. The concentrations of reactants and products were determined using RP-HPLC and product identity confirmed using liquid chromatography-mass spectrometry. Loss of native Disulfide was observed for the reaction of T20 with both linear (T20-T21) and cyclic (cT20-T21) peptides during the primary drying step; however, the native Disulfides were regenerated during secondary drying with no further change till the end of lyophilization. Deviations from Arrhenius parameters predicted from solution studies and the absence of buffer effects during lyophilization suggest that factors such as temperature, initial peptide concentration, buffer type, and concentration do not influence thiol-Disulfide Exchange during lyophilization. Results from a "cold finger" method used to study peptide adsorption to ice indicate that there is no preferential adsorption to the ice surface and that its presence may not influence Disulfide reactivity during primary drying. Overall, reaction rates and product distribution differ for the reaction of T20 with T20-T21 or cT20-T21 in the solid state and aqueous solution, whereas the mechanism of thiol-Disulfide remains unchanged. Increased reactivity of the cyclic peptide in the solid state suggests that peptide cyclization does not offer protection against lyophilization and that damage induced by a process stress further affects storage stability at 22°C and ambient RH.
-
thiol Disulfide Exchange in peptides derived from human growth hormone
Journal of Pharmaceutical Sciences, 2014Co-Authors: Saradha Chandrasekhar, Daniel E Epling, Andreas M Sophocleous, Elizabeth M ToppAbstract:Disulfide bonds stabilize proteins by cross-linking distant regions into a compact three-dimensional structure. They can also participate in hydrolytic and oxidative pathways to form nonnative Disulfide bonds and other reactive species. Such covalent modifications can contribute to protein aggregation. Here, we present experimental data for the mechanism of thiol-Disulfide Exchange in tryptic peptides derived from human growth hormone in aqueous solution. Reaction kinetics was monitored to investigate the effect of pH (6.0-10.0), temperature (4-50°C), oxidation suppressants [ethylenediaminetetraacetic acid (EDTA) and N2 sparging], and peptide secondary structure (amide cyclized vs. open form). The concentrations of free thiol containing peptides, scrambled Disulfides, and native Disulfide-linked peptides generated via thiol-Disulfide Exchange and oxidation reactions were determined using reverse-phase HPLC and liquid chromatography-mass spectrometry. Concentration versus time data were fitted to a mathematical model using nonlinear least squares regression analysis. At all pH values, the model was able to fit the data with R(2) ≥ 0.95. Excluding oxidation suppressants (EDTA and N2 sparging) resulted in an increase in the formation of scrambled Disulfides via oxidative pathways but did not influence the intrinsic rate of thiol-Disulfide Exchange. In addition, peptide secondary structure was found to influence the rate of thiol-Disulfide Exchange.
Ricardo L E Furlan - One of the best experts on this subject based on the ideXlab platform.
-
dithioacetal Exchange a new reversible reaction for dynamic combinatorial chemistry
Chemistry: A European Journal, 2016Co-Authors: Gaston A Orrillo, Andrea M Escalante, Ricardo L E FurlanAbstract:Reversibility of dithioacetal bond formation is reported under acidic mild conditions. Its utility for dynamic combinatorial chemistry was explored by combining it with orthogonal Disulfide Exchange. In such a setup, thiols are positioned at the intersection of both chemistries, constituting a connecting node between temporally separated networks.
Brian W Matthews - One of the best experts on this subject based on the ideXlab platform.
-
thiol Disulfide Exchange is involved in the catalytic mechanism of peptide methionine sulfoxide reductase
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Todd W Lowther, Nathan Brot, Herbert Weissbach, John F Honek, Brian W MatthewsAbstract:Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) reverses the inactivation of many proteins due to the oxidation of critical methionine residues by reducing methionine sulfoxide, Met(O), to methionine. MsrA activity is independent of bound metal and cofactors but does require reducing equivalents from either DTT or a thioredoxin-regenerating system. In an effort to understand these observations, the four cysteine residues of bovine MsrA were mutated to serine in a series of permutations. An analysis of the enzymatic activity of the variants and their free sulfhydryl states by mass spectrometry revealed that thiol–Disulfide Exchange occurs during catalysis. In particular, the strictly conserved Cys-72 was found to be essential for activity and could form Disulfide bonds, only upon incubation with substrate, with either Cys-218 or Cys-227, located at the C terminus. The significantly decreased activity of the Cys-218 and Cys-227 variants in the presence of thioredoxin suggested that these residues shuttle reducing equivalents from thioredoxin to the active site. A reaction mechanism based on the known reactivities of thiols with sulfoxides and the available data for MsrA was formulated. In this scheme, Cys-72 acts as a nucleophile and attacks the sulfur atom of the sulfoxide moiety, leading to the formation of a covalent, tetracoordinate intermediate. Collapse of the intermediate is facilitated by proton transfer and the concomitant attack of Cys-218 on Cys-72, leading to the formation of a Disulfide bond. The active site is returned to the reduced state for another round of catalysis by a series of thiol—Disulfide Exchange reactions via Cys-227, DTT, or thioredoxin.
-
thiol Disulfide Exchange is involved in the catalytic mechanism of peptide methionine sulfoxide reductase
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Todd W Lowther, Nathan Brot, Herbert Weissbach, John F Honek, Brian W MatthewsAbstract:Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) reverses the inactivation of many proteins due to the oxidation of critical methionine residues by reducing methionine sulfoxide, Met(O), to methionine. MsrA activity is independent of bound metal and cofactors but does require reducing equivalents from either DTT or a thioredoxin-regenerating system. In an effort to understand these observations, the four cysteine residues of bovine MsrA were mutated to serine in a series of permutations. An analysis of the enzymatic activity of the variants and their free sulfhydryl states by mass spectrometry revealed that thiol–Disulfide Exchange occurs during catalysis. In particular, the strictly conserved Cys-72 was found to be essential for activity and could form Disulfide bonds, only upon incubation with substrate, with either Cys-218 or Cys-227, located at the C terminus. The significantly decreased activity of the Cys-218 and Cys-227 variants in the presence of thioredoxin suggested that these residues shuttle reducing equivalents from thioredoxin to the active site. A reaction mechanism based on the known reactivities of thiols with sulfoxides and the available data for MsrA was formulated. In this scheme, Cys-72 acts as a nucleophile and attacks the sulfur atom of the sulfoxide moiety, leading to the formation of a covalent, tetracoordinate intermediate. Collapse of the intermediate is facilitated by proton transfer and the concomitant attack of Cys-218 on Cys-72, leading to the formation of a Disulfide bond. The active site is returned to the reduced state for another round of catalysis by a series of thiol—Disulfide Exchange reactions via Cys-227, DTT, or thioredoxin.
Artur R Stefankiewicz - One of the best experts on this subject based on the ideXlab platform.
-
Disulfide Exchange exposing supramolecular reactivity through dynamic covalent chemistry
Chemical Society Reviews, 2014Co-Authors: Samuel P Black, Jeremy K M Sanders, Artur R StefankiewiczAbstract:A Tutorial Review of the subtle supramolecular interactions influencing the outcomes of equilibrating systems, focusing on the dynamic covalent chemistry (DCC) of Disulfide Exchange reactions, is presented. We discuss the topics of cation–π interactions (2.1), hydrophobic effects (2.2), hydrogen bonding interactions (2.3) aromatic donor–acceptor interactions (2.4), and metal–ligand interactions (2.5) in the context of dynamic Disulfide chemistry.