Generation Of Thiyl Radicals Research Articles

Generation of Thiyl Radicals by the Photolysis of 5-Iodo-4-thiouridine

The photochemistry of 2',3',5'-tri-O-acetyl-5-iodo-4-thiouridine (3) in deoxygenated 1:1 CH(3)CN-H(2)O pH 5.8 (phosphate buffer) solution has been studied by means of steady-state and nanosecond laser flash photolysis methods. Under steady-state irradiation (lambda > or = 334 nm), the stable photoproducts were iodide ion, 2',3',5'-tri-O-acetyl-4-thiouridine (4), and two disulfides. The disulfides were the symmetrical bis-(2',3',5'-tri-O-acetyl-5-iodo-4-thiouridine) (5) and unsymmetrical 6, which contains both 4-thiouridine and 5-iodo-4-thiouridine residues. The formation of the dehalogenated photoproduct suggests that C(5)-I bond cleavage is a primary photochemical step. Attempts to scavenge the resulting C(5)-centered radical by suitable addends, bis-(N-alpha-acetyl)cystine-bis-N-ethylamide or benzene, were unsuccessful. Analysis of the photoproducts formed under these conditions showed that the S-atom is the reactive center. The photoproduct 4, obtained by irradiation of 3 in CD(3)CN-H(2)O, followed by reversed-phase HPLC isolation using nonlabeled eluents, did not contain deuterium. An analogous experiment performed in CH(3)CN-D(2)O gave deuterated product 4-d with 88% of the deuterium incorporated at C(5). Transient absorption observed upon laser excitation (lambda= 308 nm) of 3 was assigned to the 4-uridinylthiyl radical on the basis of the similarity of this spectrum with that obtained upon laser photolysis of the disulfide: bis-(2',3',5'-tri-O-acetyl-4-thiouridine) 14. On the basis of the results of steady-state and laser photolysis studies, a mechanism of the photochemical reaction of 3 is proposed. The key mechanistic step is a transformation of the C(5)-centered radical formed initially by C(5)-I bond cleavage into a long-lived S-centered radical via a 1,3-hydrogen shift. Theoretical calculations confirmed that the long-lived S-centered radical is the most stable radical derived from the 4-thiouracil residue.

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze the production of deoxyribonucleotides, which are essential for DNA synthesis and repair in all organisms. The three currently known classes of RNRs are postulated to utilize a similar mechanism for ribonucleotide reduction via a transient thiyl radical, but they differ in the way this radical is generated. Class I RNR, found in all eukaryotic organisms and in some eubacteria and viruses, employs a diferric iron center and a stable tyrosyl radical in a second protein subunit, R2, to drive thiyl radical generation near the substrate binding site in subunit R1. From extensive experimental and theoretical research during the last decades, a general mechanistic model for class I RNR has emerged, showing three major mechanistic steps: generation of the tyrosyl radical by the diiron center in subunit R2, radical transfer to generate the proposed thiyl radical near the substrate bound in subunit R1, and finally catalytic reduction of the bound ribonucleotide. Amino acid- or substrate-derived radicals are involved in all three major reactions. This article summarizes the present mechanistic picture of class I RNR and highlights experimental and theoretical approaches that have contributed to our current understanding of this important class of radical enzymes.

Generation and Electron Paramagnetic Resonance Spin Trapping Detection of Thiyl Radicals in Model Proteins and in the R1 Subunit of Escherichia coli Ribonucleotide Reductase

In the Escherichia coli class Ia ribonucleotide reductase (RNR), the best characterized RNR, there is no spectroscopic evidence for the existence of the postulated catalytically essential thiyl radical (R–S•) in the substrate binding subunit R1. We report first results on artificially generated thiyl radicals in R1 using two different methods: chemical oxidation by CeIV/nitrilotriacetate (NTA) and laser photolysis of nitric oxide from nitrosylated cysteines. In both cases, EPR spin trapping at room temperature using phenyl-N-t-butylnitrone, and controls with chemically blocked cysteines, has shown that the observed spin adduct originates from thiyl radicals. The EPR line shape of the protein-bound spin adduct is typical for slow motion of the nitroxide moiety, which indicates that the majority of trapped thiyl radicals are localized in a folded region of R1. In aerobic R1 samples without spin trap that were frozen after treatment with CeIV/NTA or laser photolysis, we observed sulfinyl radicals (R–S•=O) assigned via their g-tensor components 2.0213, 2.0094, and 2.0018 and the hyperfine tensor components 1.0, 1.1, and 0.9 mT of one β-proton. Sulfinyl radicals are the reaction products of thiyl radicals and oxygen and give additional evidence for generation of thiyl radicals in R1 by the procedures used.

Mechanism-based chemopreventive strategies against etoposide-induced acute myeloid leukemia: free radical/antioxidant approach.

Etoposide (VP-16) is extensively used to treat cancer, yet its efficacy is calamitously associated with an increased risk of secondary acute myelogenous leukemia. The mechanisms for the extremely high susceptibility of myeloid stem cells to the leukemogenic effects of etoposide have not been elucidated. We propose a mechanism to account for the etoposide-induced secondary acute myelogenous leukemia and nutritional strategies to prevent this complication of etoposide therapy. We hypothesize that etoposide phenoxyl radicals (etoposide-O(.)) formed from etoposide by myeloperoxidase are responsible for its genotoxic effects in bone marrow progenitor cells, which contain constitutively high myeloperoxidase activity. Here, we used purified human myeloperoxidase, as well as human leukemia HL60 cells with high myeloperoxidase activity and provide evidence of the following. 1) Etoposide undergoes one-electron oxidation to etoposide-O(.) catalyzed by both purified myeloperoxidase and myeloperoxidase activity in HL60 cells; formation of etoposide-O(.)radicals is completely blocked by myeloperoxidase inhibitors, cyanide and azide. 2) Intracellular reductants, GSH and protein sulfhydryls (but not phospholipids), are involved in myeloperoxidase-catalyzed etoposide redox-cycling that oxidizes endogenous thiols; pretreatment of HL60 cells with a maleimide thiol reagent, ThioGlo1, prevents redox-cycling of etoposide-O(.) radicals and permits their direct electron paramagnetic resonance detection in cell homogenates. VP-16 redox-cycling by purified myeloperoxidase (in the presence of GSH) or by myeloperoxidase activity in HL60 cells is accompanied by generation of thiyl radicals, GS(.), determined by HPLC assay of 5, 5-dimethyl-1-pyrroline glytathionyl N-oxide glytathionyl nitrone adducts. 3) Ascorbate directly reduces etoposide-O(.), thus competitively inhibiting etoposide-O(.)-induced thiol oxidation. Ascorbate also diminishes etoposide-induced topo II-DNA complex formation in myeloperoxidase-rich HL60 cells (but not in HL60 cells with myeloperoxidase activity depleted by pretreatment with succinyl acetone). 4) A vitamin E homolog, 2,2,5,7, 8-pentamethyl-6-hydroxychromane, a hindered phenolic compound whose phenoxyl radicals do not oxidize endogenous thiols, effectively competes with etoposide as a substrate for myeloperoxidase, thus preventing etoposide-O(.)-induced redox-cycling. We conclude that nutritional antioxidant strategies can be targeted at minimizing etoposide conversion to etoposide-O(.), thus minimizing the genotoxic effects of the radicals in bone marrow myelogenous progenitor cells, i.e., chemoprevention of etoposide-induced acute myelogenous leukemia.

Heme from Alzheimer's brain inhibits muscarinic receptor binding via thiyl radical generation

An endogenous inhibitor (<3500 Da) of antagonist binding to the muscarinic acetylcholine receptor (mAChR) has been reported to be elevated 3-fold in Alzheimer's disease (AD) brain. This endogenous inhibitor was found to require the presence of reducing agents such as reduced glutathione (GSH) for optimal activity. In the presence of GSH, the inhibitor was shown to generate thiyl radicals which irreversibly inhibited the mAChR. We now report that the inhibitor contains free heme, a well-established source of oxidative stress capable of generating free radicals and causing neurotoxicity. While FeSO 4, microperoxidase and hemin all inhibited antagonist binding to the mAChR, only hemin shared the inhibitor's requirement for GSH. Both the free radical scavengers Trolox and Mn 2+, and the metal chelator, EDTA, blocked the activity of the endogenous AD inhibitor and of hemin. Heme oxygenase-1 (HO-1) markedly reduced the activity of both the endogenous AD inhibitor and hemin, indicating that the endogenous inhibitor contains heme. Mass spectrometric analysis confirmed the presence of free heme and heme fragments in fractions of the endogenous AD inhibitor. The antioxidants estrogen, vitamin E and vitamin C all protected the mAChR from irreversible inhibition by the endogenous inhibitor or hemin. These antioxidants may function to protect the integrity of the mAChR in vivo and may have therapeutic potential in AD where free heme could be a source of oxidative stress.

Synthesis of 12- and 13-Membered Sulfur-Containing Lactones by Homolytic Macrocyclization of Mercaptoacetic Esters with Alkynes

Starting from mercaptoacetic acid esters of 1,2- or 1,3-diols and substituted acetylenes 12- and 13-membered sulfur-containing lactones as 1:1 adducts were synthesized in yields up to 48%. The mechanism of this homolytic reaction, which is initiated by the tripropylborane/oxygen system, includes generation of thiyl radicals and their addition to the triple bond of alkynes.

The reactivity of thiol compounds with different redox states of leghaemoglobin: evidence for competing reduction and addition pathways

Reaction of the ferric form of leghaemoglobin with hydrogen peroxide has been previously shown to give rise to an iron(IV)-oxo (ferryl) species, and a protein radical. Inclusion of a variety of thiol compounds in this system is shown to lead to rapid loss of the iron(IV)-oxo species and the regeneration of the ferric form and/or the formation of novel sulf species formed by nucleophilic attack of the thiol group on the tetrapyrrole ring. The reduction process also results in the generation of thiyl radicals which have been detected by EPR spin trapping. The relative yields of the products produced by these two competing pathways is shown to be highly dependent on the steric and electronic characteristics of the thiol compound. Evidence has also been obtained, in the absence of hydrogen peroxide, for both the reduction of the ferric form of the protein to the oxy-ferrous form, via a process believed to involve the deoxy-ferrous species, and the formation of sulf-leghaemoglobin species. Both of these pathways are again highly dependent on the structure of the thiol, and the former also results in the generation of thiyl radicals. Inclusion of the sulfide anion in place of the organic thiols results in somewhat different behaviour, in that this species appears to both reduce the iron centre and form a complex with the iron atom. This ligation process is reversible, and the sulfide complex is shown to react readily with both strong oxidizing and reducing agents. The behaviour of this protein, which is structurally related to myoglobin, is dramatically different to that demonstrated by myoglobin; this is rationalized in terms of the much more open heme site of leghaemoglobins, and the presence of an electronic gate which hinders access by negatively charged molecules. The contribution of these processes to the maintenance of the leghaemoglobin proteins in the oxy-ferrous form in vivo and the binding of oxygen is discussed.

Generation of Free Radicals from Neocarzinostatin Mediated by NADPH/Cytochrome P-450 Reductase via Activation of Enediyne Chromophore

Neocarzinostatin (NCS) is one of the most potent antitumor agents. It contains a chromophore of a unique enediyne structure as its prosthetic group. Here, we report the generation of free radicals from NCS by the NADPH/cytochrome (cyt) P-450 reductase system. Free radical species, which were generated in the reaction mixture of NCS/NADPH/cyt P-450 reductase, were identified by using electron spin resonance spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The spin adducts of superoxide and hydroxyl radical (DMPO-OOH and DMPO-OH, respectively) were generated in the reaction of NCS with the NADPH/cyt P-450 reductase system. From kinetic analyses of DMPO-OOH generation as a function of the concentration of NCS, the V max/ K m value of the reaction was 0.4 × 10 6 M −1min −1. In addition, we identified thiyl radical generation in this system in the presence of glutathione. These results indicate that NCS is a potent generator of free radicals in cells where cyt P-450 reductase is available. Moreover, it seems that free radicals, generated by enzymatic activation of NCS, are involved in cytotoxicity of NCS as well as in DNA damage.