Intramolecular Hydrogen Transfer Reactions of Thiyl Radicals from Glutathione: Formation of Carbon-Centered Radical at Glu, Cys, and Gly

Mozziconacci, Olivier; Williams, Todd D.; Schöneich, Christian

doi:10.1021/tx3000494

Cited by 30 publications

(13 citation statements)

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“…Theoretical calculations by Rauk and coworkers suggested that HAT reactions occur between thiyl radicals and the α C–H bonds of amino acids located in random and β-sheet conformations, but these do not occur when amino acids are located in α-helices [43]. Experimentally, the inter- and intramolecular HAT reactions of thiyl radicals have been demonstrated for amino acids, amino acid derivatives and peptides, including glutathione, in solution and in the gas phase [19,44,45,46,47,48,49,50]. Importantly, these HAT reactions do not only target α C–H bonds but also C–H bonds of amino acid side chains [51,52].…”

Section: Thiyl Radicals In Reversible Hat Reactionsmentioning

confidence: 99%

Thiyl Radical Reactions in the Chemical Degradation of Pharmaceutical Proteins

Schöneich

2019

Molecules

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Free radical pathways play a major role in the degradation of protein pharmaceuticals. Inspired by biochemical reactions carried out by thiyl radicals in various enzymatic processes, this review focuses on the role of thiyl radicals in pharmaceutical protein degradation through hydrogen atom transfer, electron transfer, and addition reactions. These processes can lead to the epimerization of amino acids, as well as the formation of various cleavage products and cross-links. Examples are presented for human insulin, human and mouse growth hormone, and monoclonal antibodies.

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Section: Thiyl Radicals In Reversible Hat Reactionsmentioning

confidence: 99%

Thiyl Radical Reactions in the Chemical Degradation of Pharmaceutical Proteins

Schöneich

2019

Molecules

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“…24−29 A pair of CysS • can also be generated through the direct photolysis of cystine (reactions 3). 26 −30 Our recent studies on the reactivity of CysS • radicals in peptides and proteins, including IgG1 and IgG2, have shown a pronounced tendency of CysS • to reversibly and selectively abstract hydrogen atoms from adjacent amino acids, from both the α C−H and side chain C−H bonds (equilibrium 4). 3,31 In a model peptide containing the sequence -Ala-CysAla-, such hydrogen transfer reactions resulted in the conversion of L-Ala into D-Ala, i.e., epimerization of the peptide sequence.…”

Section: Introductionmentioning

confidence: 99%

Sequence-Specific Formation of d-Amino Acids in a Monoclonal Antibody during Light Exposure

Mozziconacci

Schöneich

2014

Mol. Pharmaceutics

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The photoirradiation of a monoclonal antibody 1 (mAb1) at λ = 254 nm and λmax = 305 nm resulted in the sequence-specific generation of d-Val, d-Tyr, and potentially d-Ala and d-Arg, in the heavy chain sequence [95-101] YCARVVY. d-Amino acid formation is most likely the product of reversible intermediary carbon-centered radical formation at the (α)C-positions of the respective amino acids ((α)C(•) radicals) through the action of Cys thiyl radicals (CysS(•)). The latter can be generated photochemically either through direct homolysis of cystine or through photoinduced electron transfer from Trp and/or Tyr residues. The potential of mAb1 sequences to undergo epimerization was first evaluated through covalent H/D exchange during photoirradiation in D2O, and proteolytic peptides exhibiting deuterium incorporation were monitored by HPLC-MS/MS analysis. Subsequently, mAb1 was photoirradiated in H2O, and peptides, for which deuterium incorporation in D2O had been documented, were purified by HPLC and subjected to hydrolysis and amino acid analysis. Importantly, not all peptide sequences which incorporated deuterium during photoirradiation in D2O also exhibited photoinduced d-amino acid formation. For example, the heavy chain sequence [12-18] VQPGGSL showed significant deuterium incorporation during photoirradiation in D2O, but no photoinduced formation of d-amino acids was detected. Instead this sequence contained ca. 22% d-Val in both a photoirradiated and a control sample. This observation could indicate that d-Val may have been generated either during production and/or storage or during sample preparation. While sample preparation did not lead to the formation of d-Val or other d-amino acids in the control sample for the heavy chain sequence [95-101] YCARVVY, we may have to consider that during hydrolysis N-terminal residues (such as in VQPGGSL) may be more prone to epimerization. We conclude that the photoinduced, radical-dependent formation of d-amino acids requires not only the intermediary formation of a (α)C(•) radical but also sufficient flexibility of the protein domain to allow both pro-chiral faces of the (α)C(•) radical to accept a hydrogen atom.

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“…Therefore, we generated the biologically relevant thiyl radicals from glutathione and cysteine, abbreviated as and , respectively, to monitor their reaction with the and HE probes. One should note that and exist in equilibrium with their carbon-centered radicals as reported by Schöneich and coworkers 41 . Although this equilibrium is shifted to the side of the sulfur-centered radicals and is rather fast 42 , it may affect the reaction kinetics, and thus the determined rate constants should be treated as the “apparent” values.…”

Section: Resultsmentioning

confidence: 61%

Oxidation of ethidium-based probes by biological radicals: mechanism, kinetics and implications for the detection of superoxide

Michalski

Thiebaut

Ayhan

et al. 2020

Sci Rep

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Hydroethidine (HE) and hydropropidine ($$\hbox {HPr}^{+}$$ HPr + ) are fluorogenic probes used for the detection of the intra- and extracellular superoxide radical anion ($$\hbox {O}_{ {2}}^{\bullet -}$$ O 2 ∙ - ). In this study, we provide evidence that HE and $$\hbox {HPr}^{+}$$ HPr + react rapidly with the biologically relevant radicals, including the hydroxyl radical, peroxyl radicals, the trioxidocarbonate radical anion, nitrogen dioxide, and the glutathionyl radical, via one-electron oxidation, forming the corresponding radical cations. At physiological pH, the radical cations of the probes react rapidly with $$\hbox {O}_{ {2}}^{\bullet -}$$ O 2 ∙ - , leading to the specific 2-hydroxylated cationic products. We determined the rate constants of the reaction between $$\hbox {O}_{ {2}}^{\bullet -}$$ O 2 ∙ - and the radical cations of the probes. We also synthesized N-methylated analogs of $$\hbox {HPr}^{+}$$ HPr + and HE which were used in mechanistic studies. Methylation of the amine groups was not found to prevent the reaction between the radical cation of the probe and the superoxide, but it significantly increased the lifetime of the radical cation and had a substantial effect on the profiles of the oxidation products by inhibiting the formation of dimeric products. We conclude that the N-methylated analogs of HE and $$\hbox {HPr}^{+}$$ HPr + may be used as a scaffold for the design of a new generation of probes for intra- and extracellular superoxide.

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Intramolecular Hydrogen Transfer Reactions of Thiyl Radicals from Glutathione: Formation of Carbon-Centered Radical at Glu, Cys, and Gly

Cited by 30 publications

References 50 publications

Thiyl Radical Reactions in the Chemical Degradation of Pharmaceutical Proteins

Thiyl Radical Reactions in the Chemical Degradation of Pharmaceutical Proteins

Sequence-Specific Formation of d-Amino Acids in a Monoclonal Antibody during Light Exposure

Oxidation of ethidium-based probes by biological radicals: mechanism, kinetics and implications for the detection of superoxide

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