The biological chemistry of thiols reactions with biologically-relevant

The biological chemistry of thiols: reactions with biologically-relevant oxidants reactions of radicals formed on oxidation Peter Wardman University of Oxford, Gray Cancer Institute Supported by

Overview l Thiol oxidation products & reactivity of oxidants n thiol ionization a key property: p. H-sensitive chemistry n non-enzyme-based oxidants are mainly radicals n enzyme-based oxidants utilize H 2 O 2 cofactor l Thiyl radical is a key precursor of products n a moderately strong oxidant in its own right l Addition/transformation routes of thiyl radicals n conjugation with thiolate constitutes a ‘redox switch’ n isomerization of cis fatty acids to trans n intramolecular transformation of GSH thiyl radicals

Biological thiols and their oxidation products

Biologically-important thiols Adult human has ~ 30 g glutathione (typical cytosolic concentration is 5– 10 m. M) l Cysteine is the most abundant thiol moiety l Glutathione is a cysteinyl peptide and the most abundant non-protein thiol l Lipoate is an example of a dithiol, can reduce GSH

Oxidation of thiols and radical intermediates

A key property in thiol chemistry: dissociation to thiolate

Thiol ionization (dissociation of S–H): the most important single property? l The S–H bond of thiols dissociates with p. Ka (p. H where 50% dissociated) in the range ~ 7– 10: RSH Á RS– + H+ thiol p. Ka H 2 S ~7· 1 (to form HS–) cysteine ~8· 5, 8· 9 (-NH 3+, -NH 2) glutathione ~8· 9, 9· 1 (-NH 3+, -NH 2) l About 3% of glutathione (GSH) is dissociated to the thiolate form (GS–) at p. H 7· 4 l Many reactions of thiols with oxidants will be p. H-dependent around physiological p. H because thiolate form is usually oxidized much faster than the undissociated thiol

Effects of thiol dissociation on rates of reactions in the physiological p. H range

Example of thiolate(p. H)-dependent reactivity Reactivity of GSH increased exactly 100–fold between p. H 6– 8 l Acetaminophen (paracetamol, Tylenol) oxidized in the liver to a quinoneimine l GSH adds rapidly to double bond, protects against adduct forming with protein thiols l Only thiolate anion reactive, reaction p. H-dependent at p. H < thiol p. Ka l Reaction accelerated by glutathione. S-transferases Coles et al. 1988

p. H-Dependent reaction of NO 2 • with thiols NO 2 • + RS– ® NO 2– + RS • l Half-life of NO 2 • in presence of 5 m. M GSH is only ~ 7 µs at p. H 7· 4 Estimates of rate constants from simplified analysis that under-estimated reactivity at higher p. H, hence slope < 1 cysteine glutathione Ford et al. 2002

Oxidation of thiols (thiolate) to form thiyl radicals

There are several potential oxidants of thiols non-radical oxidants or Cu(I) / Cu(II) or Asc. H– O 2 NO • • OH NO 2 • Fe(III) O 2 • – Fe(II) HOCl Cl–, MPO free-radical oxidants N 2 O 3 NO 2– MPO NO • ~30% • OH ONOOH H+ H+ ~70% NO 3– ONOO– H 2 O 2 ~65% NO 2 • × 2 O 2 • – ± SOD O 2 ONOOCO 2– ~35% • – • OH ~65% CO 2 CO 3 • –

Reactivity of oxidants forming thiyl radicals Oxidant + RSH/RS– ® product + RS • (+ H+) Oxidant Rate constant / M– 1 s– 1 (glutathione) at p. H 7. 4, room temperature • OH 1· 3 ´ 1010 (Quinitiliani et al. 1977) NO 2 • 1· 9 ´ 107 (Ford et al. 2002) CO 3 • – 5· 3 ´ 106 (Chen & Hoffman 1973) O 2 • – 2· 2 ´ 102 (Jones et al. 2002) Half-life of radical is about 0. 7 / (k [GSH]) seconds

Nitric oxide (without O 2) oxidizes GSH with concentration-dependent kinetics l GSSG and nitrous oxide are products l Higher reactivity is reported at high [NO • ] (Aravindakumar et al. 2002) compared to low [NO • ] (Hogg et al. 1996) l This can be explained by an equilibrium step (Folkes & Wardman 2004): GSH Á GS– + H+ GS– + NO • (+ H+) Á GSN • OH 2 GSN • OH ® GSSG + HONNOH ® N 2 O + H 2 O l The rate is then proportional to [NO • ]2[GSH]

Reaction of ~ 9 µM NO • with GSH, ~ 27°C Folkes & Wardman 2004 Anaerobic solutions!

Thiyl radicals as a route to nitrosothiols l Formation of S-nitrosothiols can be envisaged to occur by a two-step process: RSH + oxidant ® RS • + product RS • + NO • ® RSNO l Radical-coupling reaction is poorly characterized n rate constant 2· 8 ´ 107 M– 1 s– 1 reported (Hofstetter et al. 2006) n if correct, reaction would be too slow to compete with reaction of RS • with ascorbate in tissue and/or urate in plasma (at least in cytoplasmic/aqueous compartments)

Reactions of non-radical oxidants Oxidant Rate constant / M– 1 s– 1 (glutathione) at p. H 7. 4, 25 °C (*37°C) HOCl >1· 0 ´ 107 (Folkes et al. 1995) N 2 O 3 ~6· 6 ´ 107 (Keshive et al. 1996) ONOO– ~6· 0 ´ 102 (Koppenol et al. 1992) H 2 O 2 * ~0· 9 ´ 100 (Winterbourn & Metodiewa 1999) l Oxygen can be incorporated into products: GS– + H 2 O 2 ® GSOH + OH– GSH + ONOO– ® GSOH + NO 2– GSOH + GSH ® GSSG + H 2 O l HOCl can give a sulfonamide and ‘dehydro’ GSH (Harwood et al. 2006) and GSCl and GS • (Davies & Hawkins 2000)

Enzyme-based oxidants use H 2 O 2 cofactor and often generate thiyl radicals l e. g. Horseradish peroxidase, prostaglandin H synthase l Cpd I and II intermediates are one-electron oxidants l Thiyl radical spintrapped (Harman et al. 1986) l No thiyl radicals from GSH peroxidase

Thiyl radicals a product of radical ‘repair’ (including drug radicals) Carboncentred Oxygencentred Sulfurcentred

Thiyl radicals are oxidizing agents and react with ascorbate and urate

Reaction of GS • with ascorbate l Absorption of ascorbate radical at 360 nm after generating GS • by pulse radiolysis: GS • + Asc. H– ® GSH + Asc • – l Rate constant 6· 0 ´ 108 M– 1 s– 1 at p. H 7 (Forni at al. 1983) implies half-life of GS • is ~ 3 µs with 0· 4 m. M ascorbate l Thiyl radicals products of general radical ‘repair’, so radical from the ascorbate ‘sink’ is an indicator of radical stress ESR signal from Asc • – in human skin illuminated with UVA light (Haywood et al. 2003)

Reaction of GS • with urate and stepwise radical transformation l Thiyl radicals from GSH oxidize urate (UH 2–): GS • + UH 2– ® GSH + UH • – k ~ 3 ´ 107 M– 1 s– 1 at p. H 7· 4 (Ford et al. 2002) l In turn the urate radical oxidizes ascorbate: UH • – + Asc. H– ® UH 2– + Asc • – k ~ 1· 4 ´ 107 M– 1 s– 1 (Willson et al. 1985) Urate and ascorbate are the dominant radical scavengers in blood plasma because the GSH concentration is only ~ 1 µM

Thiyl radicals react with thiolate and oxygen to ‘switch’ or modulate redox properties

Conjugation (addition) reactions of thiyl radicals with thiolate and oxygen l Addition reactions can act as a redox ‘switch’, or to weaken the oxidizing power of thiyl radicals n thiolate addition to form disulfide radical-anion (a reductant and source of superoxide) GS • + GS– Á (GSSG) • – + O 2 ® GSSG + O 2 • – n oxygen addition to form less reactive peroxyl radical GS • + O 2 Á GSOO • l These reactions can be important in cells in vitro but might be less important in vivo because GS • reacts with ascorbate preferentially n but protein thiol groups in proximity may enhance S–S bond formation (cf. oxy. R + H 2 O 2, Demple 1999)

GSOO • is a weaker oxidant than GS • + CPZ ® GS– + CPZ • + (CPZ = chlorpromazine) GS • + O 2 Á GSOO • K ~ 3200 M– 1 (Tamba et al. 1986) GSOO • + CPZ ® GSOO– + CPZ • + l Oxygen slows down rate of oxidation of chlorpromazine by GS • (Wardman 1990) l GSOO • is at least 10 fold less reactive than GS • kobs = k. GS/(1+K[O 2])

Thiyl radicals add to double bonds – can catalyse isomerization

Cis/trans isomerization of fatty acids l Thiyl radical is a catalyst

Some thiyl radicals can also switch from oxidizing to reducing by intramolecular rearrangement

Base-catalysed intramolecular transformation of GS • l Deprotonation of –NH 3+ moiety renders the adjacent C–H group susceptible to H abstraction by –S • l Half-life ~ 0. 5 ms at p. H 7. 4 (Grierson et al. 1992) l Resulting carbon-centred radical reducing, will add O 2 to form superoxide via a peroxyl radical-adduct

Conclusions l Thiols are important antioxidants by: n scavenging oxidizing radicals directly in some circumstances n ‘repairing’ free radical damage l However, the thiyl radical products of radical scavenging or ‘repair’ by thiols are not inert: n thiyl radicals are oxidizing agents n they act as damage transfer agents to O 2, urate and (especially) ascorbate radical ‘sinks’ n they catalyse cis/trans isomerization of fatty acid double bonds

Some books, reviews & illustrative references While some of these are now dated, they still provide a good overview of the key chemistry of thiyl radical generation and fate Chatgilialoglu, C; Ferreri. Trans lipids: the free radical path. , C. , Acc. Chem. Res. , 2005, 38, 441 -8. Folkes, L. K. ; Wardman, P. Kinetics of the reaction between nitric oxide and glutathione: implications for thiol depletion in cells. Free Radic. Biol. Med. 37: 549556; 2004. Ford, E. et al. Kinetics of the reactions of nitrogen dioxide with glutathione, cysteine, and uric acid at physiological p. H. Free Radic. Biol. Med. 32: 1314 -1323; 2002. S-Centered Radicals (Alfassi, Z. B. , ed. ), Wiley: Chichester, 1999. ISBN: 0 -471 -98687 -9 Biothiols in Health and Disease (Packer, L. ; Cadenas, E. , eds. ), Marcel Dekker: New York, 1995. ISBN: 0 -8247 -9654 -3 Wardman, P. ; von Sonntag, C. Kinetic factors that control the fate of thiyl radicals in cells. Methods Enzymol. , 251: 31 -45; 1995. Schöneich, C. et al. Oxidation of polyunsaturated fatty acids and lipids through thiyl and sulfonyl radicals: reaction kinetics, and influence of oxygen and structure of thiyl radicals. Arch. Biochem. Biophys. 292: 456 -467; 1992. Sulfur-Centred Reactive Intermediates in Chemistry and Biology (Chatgilialoglu, C. ; Asmus, K. -D. , eds. ), Plenum Press: New York, 1990. ISBN: 0 -306 -43723 -6