Reaction of Hypochlorous Acid with Hydrogen Peroxide and tert- Butyl Hydroperoxide. *H NMR Spectroscopy and Chemiluminescence Analyses Jürgen Arnhold11, Oleg M. Panasenkob, Jürgen Schiller1, Klaus Arnold3, Yurij A. Vladimirovb, Valeri) I. Sergienkob a Institut für Medizinische Physik und Biophysik. Medizinische Fakultät, Universität Leipzig, Liebigstr. 27. D-04103 Leipzig. Bundesrepublik Deutschland b Research Institute of Physico-Chemical Medicine. M. Pirogovskaya la. RUS-119828, Moscow. Russia Z. Naturforsch. 51c, 386-394 (1996); received January 26/February 27. 1996 Hypochlorous Acid. Hydrogen Peroxide, tert-Butyl Hydroperoxide, Singlet Oxygen. Free Radicals, 'H NMR Spectroscopy, Chemiluminescence In contrast to the well-known reaction of hypochlorous acid with hydrogen peroxide, no singlet oxygen is formed as the result of reaction between hypochlorous acid and fm-butyl hydroperoxide. The reaction with hydrogen peroxide yielded a quadratic dependence of light intensity on reactant concentration, a drastic enhancement of luminescence yield using D20 as solvent and only an emission of red light, that are typical characteristics of emission result­ ing from two molecules of delta singlet oxygen. Other chemiluminescence properties were observed using tert-butyl hydroperoxide. There was a linear dependence of light intensity on reactant concentration using rm-butyl hydroperoxide in excess with a decline of emission at higher concentrations. 'H-NMR spectroscopic analysis revealed di-rm-butyl peroxide, fm-butanol and also rm- butyl hypochlorite, acetone and acetate as products of the reaction between hypochlorous acid and fm-butyl hydroperoxide. The formation of di-rm-butyl peroxide is only possible assuming a re/Y-butyloxy radical as primary intermediate product of this reaction. Our results demonstrate that alkoxy radicals derived from organic hydroperoxides can participate in lipid peroxidation induced by hypochlorous acid. On the other hand, singlet oxygen did not influence the yield of peroxidation products. Changing H20 for D20 in suspension of egg yolk phosphaditylcholine no differences in accumulation of thiobarbituric acid reactive products were observed.

Introduction and thioether groups (Winterbourn, 1985; Arn­ Stimulated neutrophils generate the powerful hold et al, 1991) the formation of chloramines oxidant hypochlorous acid in a myeloperoxidase- (Weiss et al, 1982), enzyme inactivation (Wasil et catalysed reaction between hydrogen peroxide al, 1987; Klebanoff, 1988) and also the initiation and chloride anions (Thomas, 1979; Albrich et al, of lipid peroxidation. An increase of primary 1981). Hypochlorous acid contributes to tissue in­ (diene conjugates, hydroperoxides) and secondary jury in a number of diseases including rheumatoid (thiobarbituric acid reactive compounds) products arthritis (Schiller er c//., 1995; 1996), coronary heart of lipid peroxidation in liposomes and lipoproteins disease and others (Fliss, 1988; Klebanoff, 1988; upon the action of hypochlorous acid has been de­ Smith et al, 1989). An involvement of low density tected by several groups (Stelmaszynska et a l, lipoproteins modified by hypochlorous acid into 1992; Evgina et al, 1992; Panasenko et a l, foam cell formation during arteriosclerosis is also 1994a,b). under discussion now (Hazell et al, 1993; 1994). Although hypochlorous acid reacts in lipid sys­ Hypochlorous acid has manifold effects on cell tems with different targets only its reaction with constituents. Among these are the oxidation of organic hydroperoxides previously accumulated functional sites of proteins, especially of sulfhydryl by autoxidation or other processes leads to a pro­ motion of new lipid peroxidation (Panasenko et al, 1995). Other possible reactions of hypochlo­ rous acid such as the chlorohydrin formation with Reprint requests to Dr. Jürgen Arnhold. olefinic double bonds of phospholipid molecules Fax: 0341-9715709. (Winterbourn et al, 1992: Arnhold et al, 1995) or

0939-5075/96/0500-0386 $ 06.00 © 1996 Verlag der Zeitschrift für Naturforschung. All rights reserved. D J. Arnhold et al. ■ HOC1 and Hydroperoxides 387 the oxidation of molecules with an aldehydic func­ copy-grade) were obtained from Fluka, Switzer­ tion did not contribute to additional peroxidation land. H 20 2 was a product from Merck, Germany. reactions (Panasenko et al., 1995). On the other Hexamethylethane was purchased from Aldrich, hand, the chlorohydrin formation diminishes the Germany. Sodium hypochlorite was from Sigma, number of double bonds in lipid systems and thus Germany. Luminol was obtained from Boehringer, also the amount of available substratum for lipid Mannheim, Germany. 2-Thiobarbituric acid was a peroxidation. Consequently, sufficient products of product from Serva, Germany. 7erf-butyl hypo­ lipid peroxidation are only detectable at relatively chlorite was prepared bubbling chlorous gas low concentrations of hypochlorous acid (Pana­ through an alkaline solution of re/Y-butanol senko et al., 1994a). (Metzger, 1989). In the present paper we focused our interest on the reaction mechanism between hypochlorous Solutions acid and organic hydroperoxides and the role of A stock solution of NaOCl was kept in the dark reaction products to promote lipid peroxidation. at 4 °C. Its concentration was determined at pH 7erf-butyl-hydroperoxide and cumene hydroper­ 12 using £290 = 350 M _ 1cm _1 (Morris, 1966). It was oxide have been found to promote additional pro­ diluted with 0.14 mol/1 NaCl, 10 mmol/1 phosphate duction of thiobarbituric acid (TBA) reactive pro­ immediately prior to use and adjusted to pH 7.4. ducts if they are incorporated into egg yolk In some cases the buffer used for dilution was pre­ phosphatidylcholine liposomes (Panasenko et al., pared with D 20. 1995). This increase in TBA-reactive compounds A stock solution of H 20 2 was prepared in phos­ can be inhibited by the free radical scavenger bu- phate buffer using H20 or D 20. Its concentration tylated hydroxytoluene. On the other hand, hy­ was determined spectrophotometrically using drogen peroxide did not influence the yield of e 230 - 74 M - 1cm _1 (Beers et al., 1952). TBA-reactive products (Panasenko et al., 1994a), although hydrogen peroxide is known to yield sin­ Liposome preparation and incubation with glet oxygen in its reaction with hypochlorous acid hypochlorous acid (Khan et al., 1970; Held et al., 1978). Singlet oxy­ gen itself is assumed to be a potent agent to induce 1 ml of egg yolk phosphatidylcholine dissolved peroxidation reactions (Thomas et al., 1980; Fran­ in chloroform ( 6 mg/ml) were given in a round- kel et al., 1982). bottom flask and evaporated to dryness using a The objection of the present paper is to compare rotary evaporator. Multilamellar liposomes were the reaction of hypochlorous acid with hydrogen prepared by dissolving the lipid film in 3 ml 0.14 peroxide and with organic hydroperoxides to es­ mol/1 NaCl, 10 mmol/1 phosphate (pH 7.4) and tablish differences between these two reactions vortexing rigorously for 30 seconds. and to characterize mechanisms responsible for In order to induce peroxidation aliquots of lipo­ initiation of lipid peroxidation. Using chemilumi- some samples (final concentration 2 mg/ml) were nescence detection and 'H-nuclear magnetic reso­ incubated with NaOCl at 37 °C for 40 min. Final nance spectroscopy results are obtained favouring concentrations of NaOCl varied from 0.05 to 3 the appearance of free radicals immediately dur­ mmol/1. Control measurements revealed that the ing the reaction of hypochlorous acid with or­ pH value remains at 7.4 after the addition of ganic hydroperoxides. NaOCl. A number of experiments was made using buffer and NaOCl solutions prepared on D 20. Material and Methods Malondialdehyde was determined by its reac­ Chemicals tion with thiobarbituric acid (Uchiyama et al., 7e/t-butyl hydroperoxide (70% solution in 1978). H 20), cumene hydroperoxide, rm-butanol, di -tert- Chemiluminescence butyl peroxide, rm-butyl methyl ether, 2 -methyl- propene, egg yolk phosphatidylcholine, D 20, 200 [.il of NaOCl solution (1-50 mmol/1, final CDCI3 and all organic solvents (in UV-spectros- concentration) in phosphate buffer was placed 388 J. Arnhold et al. ■ HOC1 and Hydroperoxides into plastic vials of the luminometer AutoLumat LB 953 (Laboratorium Prof. Dr. Berthold. Wild- bad, Germany). Then 50 |il of H 20 2 (final concen­ tration 2 - 1 0 ”* mol/ 1) or tert-butyl hydroperoxide (final concentration 2 - 1 0 “ 1 mol/ 1) was added by means of an injector device. Total light intensities were determined using an integration time of 10 s. In some cases a cut-off filter (A>600 nm) was placed between the photomultiplier and reaction tube. Only red light photons were counted by this approach.

NMR measurements NaOCl, mmol/l Solutions of tert-butyl hydroperoxide either pre­ Fig. 1. Chemiluminescence intensities in the result of re­ pared in phosphate buffered saline or D20 were action of different amounts of NaOCl and hydrogen per­ incubated with NaOCl at 25 °C for 15 min. The oxide (2 -1 0 '1 mol/1, final concentration). 50 |ol H20 2 was concentration ratios between rm-butyl hydroper­ injected into 200 ul NaOCl solution made in 0.14 mol/1 NaCl, 10 mmol/l phosphate, pH 7.4. Photons were oxide and hypochlorous acid are indicated in the counted over 10 s. A cut-off filter (X>600 nm) was placed Results section. Then 1 ml of the reaction mixture between reaction tube and photomultiplier in some ex­ was thoroughly mixed with 1 ml CDCI 3. After a periments (full symbols). H20 2 and NaOCl solution were prepared with H20 (circles) or D20 (squares). short centrifugation to allow phase separation, the Means of four measurements are given. S.D. was lower water and chloroform phases were analysed by than 6%. 'H-NMR spectroscopy. Proton spectra were obtained on a Bruker AMX-NMR spectrometer at 300.13 MHz. The spectra were accumulated 64 times. No line-broad- to a solution of hypochlorous acid as a function of ening or Gauß-broadening was used. reactant concentration. No significant differences Chemical shifts were referenced to sodium 3- were found measuring the total luminescence or (trimethylsilyl)propane-l-sulphonate in aqueous those at wavelengths higher than 600 nm using a solution or to the CHC1 3 resonance in deuterated cut-off filter. In both cases a square dependence chloroform. of light emission intensity on the reactant concen­ tration is found. Changing H20 for D20 the light emission is Results drastically enhanced in reaction between hy­ drogen peroxide and hypochlorous acid. D20 is Chemiluminescence resulting from reaction of known to prolong the lifetime of singlet oxygen hypochlorous acid with hydrogen peroxide from 2-4 [is to 20 jas (Merkel et al., 1972; Rodgers Hypochlorous acid and hydrogen peroxide react et al., 1980). The same range of intensities of light in a strong 1:1 (molar ratio) stoichiometry to yield emission in D20 is shifted to lower reactant con­ singlet oxygen (Held et al., 1978). Although some centrations by about an order of magnitude in details of this reaction are under discussion, it is comparison to H20 (Fig. 1). The emission in D20 well known that free radicals are not involved in consists also completely of red light and has a this process (Held et al., 1978). The formation of square dependence on reactant concentration. singlet oxygen can be followed by appearance of The red nature of luminescence, its quadratic a red chemiluminescence due to dimol emission of dependence on reactant concentrations, and the excited oxygen molecules (Khan et al., 1970: Ka- enhancement of light emission by D20 are charac­ nofsky. 1989). Fig. 1 shows the luminescence yield teristic features of the dimol emission of singlet after the addition of excess of hydrogen peroxide oxygen. J. Arnhold et al. ■ HOC1 and Hydroperoxides 389

Chemiluminescence resulting from reaction of butyl hydroperoxide would explain the deviation hypochlorous acid with organic hydroperoxides of the linear dependence between light intensity and NaOCl concentration using higher amounts of Hydrogen peroxide was then replaced by tert- hypochlorous acid in Fig. 2. butyl hydroperoxide or cumene hydroperoxide. A A likely candidate for this primary intermediate chemiluminescence was also measured in the reac­ product is the terf-butyloxy radical (CH 3)3C O . It tion between tert-butyl hydroperoxide and hy­ is known to abstract a H from tert-butyl hydroper­ pochlorous acid. Using an excess of te/7-butyl-hy- oxide (Metzger, 1989): droperoxide and variable concentrations of

NaOCl a linear dependence between light inten­ (CH 3)3C -0 + (CH3)3COOH sity and NaOCl concentration has been found up (CH3)3COH + (CH 3)3COO. to approximately 10 - 3 mol/1 (Fig. 2). Then the lu­ minescence raises more slowly and decreases at These data show that the light emission in the higher concentrations of hypochlorous acid. The result of reaction between ter/-butyl-hydroperox- application of a cut-off filter (X > 600 nm) into the ide and hypochlorous acid is not caused by singlet equipment drastically diminishes the light emis­ oxygen. Differences in chemiluminescence mecha­ sion. Most of this emission is of non-red nature. nisms using hydrogen peroxide or tert-butyl hydro­ Only 1% of total light intensity can come through peroxide in their reactions with hypochlorous acid the cut-off filter. D20 did not enhance the lumi­ confirm previous results (Panasenko et al., 1995) nescence yield. A decrease of light emission was of influence of these hydroperoxides on the pro­ found in this case. Similar results were obtained duct yield in lipid peroxidation induced by hy­ using cumene hydroperoxide (data not shown). pochlorous acid. Whereas hydrogen peroxide did The linear dependence between light intensity not cause additional peroxidation of unsaturated and the concentration of NaOCl in its reaction phospholipids, tert-butyl and also cumene hydro­ with rm-butyl hydroperoxide supports the view peroxides favour this deterioration process. that a primary intermediate product of these reac­ tants or one of its subsequent products is formed in an excited state. A direct reaction of this pri­ Accumulation of TBA-reactive products in D 2O mary intermediate product with the original tert- Singlet oxygen is formed in the result of reaction between hydrogen peroxide and hypochlorous acid. It is known to promote the lipid peroxidation reaction (Thomas et al., 1980; Frankel et al, 1982). These results are in contrast to our previous obser­ vation (Panasenko et a l, 1994a). A possible reason could be the very short lifetime of singlet oxygen. To increase the lifetime of singlet oxygen egg yolk phosphatidylcholine liposomes were prepared in D 20. Fig. 3 shows the accumulation of TBA-re- active products in these liposomes in the presence of H20 or D 20. Lipid peroxidation was initiated by addition of sodium hypochlorite. However, there were no differences in the yield of peroxida­ Fig. 2. Chemiluminescence intensities in the result of re­ tion products using H20 or D 20. These experi­ action of different amounts of NaOCl and tert-butyl hy­ ments confirm our previous data (Panasenko et al, droperoxide (2 • 10_ 1 mol/1. final concentration). 50 |il 1994a) obtained by incubation of liposomes with /m-butyl hydroperoxide was injected into 200 |.il NaOCl solution made in 0.14 mol/1 NaCl, 10 mmol/1 phosphate, hydrogen peroxide that singlet oxygen does not PH 7.4. Photons were counted over 10 s. A cut-off filter play any role in initiation of a lipid peroxidation (Ä>600 nm) was placed between reaction tube and pho­ induced by hypochlorous acid. tomultiplier in some experiments (full symbols). Means of four measurements are given. S.D. was lower than Therefore, an other mechanism besides the for­ 6%. mation of singlet oxygen is responsible for en­ 390 J. Arnhold et al. ■ HOC1 and Hvdroperoxides 0.8 + CI______.1

0.6 o> + 0 -0 + , . 1 , E ö £ 0.4 c + OH J < Q ^ 0.2 + OOH I

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.0 0 1 2 3 Chemical shift (ppm) NaOCI, mmol/l Fig. 4. ’H NMR spectra of /m-butyl hydroperoxide, tert- butanol, di-fe/Y-butyl peroxide and rm-butyl chloride in Fig. 3. Concentration of TBA-reactive products (in deuterated chloroform. nmol/mg phospholipids) in phosphatidylcholine lipo­ somes (2 mg/ml) as a function of NaOCI concentration. Liposomes were prepared in 0.14 mol/1 NaCl. 10 mmol/ Tert-buly\ hydroperoxide yielded in CDC1 3 an 1 phosphate, pH 7.4 and incubated for 40 min with intense singlet at 1.245 ppm for all methyl protons NaOCI. Puffer and NaOCI were prepared on H20 or D20 . All data are given as means and S.D. (n -4). and a much less intense singlet at 7.329 ppm for the proton of the hydroperoxide group. Methyl protons of terf-butanol and tert-butyl chloride gave singlets at 1.252 and 1.60 ppm, respectively. Addi­ hanced lipid peroxidation after the reaction of hy- tionally, a very small resonance appeared at 1.382 pochlorous acid with organic hydroperoxides. for the OH group of terf-butanol. Di-/m-butyl per­ oxide was characterized by an intense resonance 'H-NMR analysis of reaction products between at 1.193 ppm. A mixture of both tert-butyl hydro­ tert-butyl hydroperoxide and hypochlorous acid peroxide and di-terf-butyl peroxide yielded two well separated resonances at chemical shifts indi­ In order to reveal further details of the reaction cated above (data not shown). mechanism between rm-butyl hydroperoxide and Tert-butyl hydroperoxide dissolved in D20 or hypochlorous acid the products of this reaction phosphate buffer on the basis of D20 was treated were analysed by 'H-NMR spectroscopy. If tert- with different amounts of hypochlorous acid for 15 butyl hydroperoxide would react like hydrogen min. After that deuterated chloroform was added. peroxide with hypochlorous acid products with a Both phases were separated and analysed by ‘H- single tert-butyl group should be formed. On the NMR spectroscopy. A typical 'H-NMR spectrum other hand, if free radicals would be formed in the of the water phase is given in Fig. 5. There is an result of reaction between tert-butyl hydroperox­ intense signal at 1.24 ppm for protons of tert-butyl ide and hypochlorous acid products of recombina­ groups. Three small signals appeared at 1.90, 2.20, tion of free radicals should appear. and 3.47 ppm. By comparing with resonances of 'H-NMR spectra of rm-butyl hydroperoxide. known substances and signal enhancement after te/7-butanol, tert-butyl chloride and di-terf-butyl the addition of these substances the signals were peroxide are shown in Fig. 4. All spectra were re­ attributed to acetate, acetone and methanol, corded using deuterated chloroform as solvent. In respectively. this solvent different chemical shifts for the tert- Much more complex are the 'H-NMR spectra butyl group of these substances can be obtained. of chloroform phase of reaction products between On the other hand, using D20 as solvent, all these hypochlorous acid and rm-butyl hydroperoxide protons have the same chemical shift at 1.24 ppm (Fig. 6 ). The resonance for methyl protons of tert- (data not shown). butyl hydroperoxide at 1.245 ppm decreases con- J. Arnhold et al. • HOC1 and Hydroperoxides 391

Table I. Chemical shifts and fine structures of 'H NMR signals in the CDC13 phase of reaction products between tert-butyl hydroperoxide and HOC1/OC1- . Tert-butyl compounds Chemical Formula Fine Inten­ shift structure sity

1.193 (CH3)3C -0-0-C (C H 3)3 singlet +++ 1.22 (CH3)3COCl singlet ++ O 1.245 (CH3)3COOH singlet H,C-C-CH, 1.252 (CH3)3COH singlet +++ 1.32 non identified singlet traces 1.54 H.O singlet 1.60 (CH3)3CC1 singlet traces CH3COO CH,OH 2.15 c h 3c o c h 3 singlet +++ ~ J ______3.47 c h 3o h singlet traces 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.78 non identified singlet + 3.81 non identified singlet traces Chemical shift (ppm) 7.24 CHC13 singlet Fig. 5. 'H NMR spectrum in D20 of reaction products of tert-butyl hydroperoxide and NaOCl. 7erf-butyl hy­ droperoxide (10 mmol/1) was incubated with NaOCl (10 mmol/1) for 15 minutes. All solutions were diluted with 0,14 mol/1 NaCl, 10 mmol/1 phosphate, pH 7.4 made on from relatively simple molecule groups because D20. After incubation 1 ml of reactants was thoroughly mixed with 1 ml CDC13. they are singlets. Signal identification was per­ formed by the comparison of chemical shifts with resonances of known substances and signal en­ tinuously with increasing concentrations of hy- hancement after the addition of these compounds. pochlorous acid (data not shown). Surprisingly, New signals at 1.193, 1.22 and 1.253 ppm were at­ marked amounts of rm-butyl hydroperoxide were tributed to di-ter/-butyl peroxide, terr-butyl hypo­ still present using a twofold excess of hypochlo- chlorite and ter/-butanol, respectively. Whereas rous acid. There are only traces of tert -butyl hydro­ only traces of di-rm-butyl peroxide were detected peroxide at a fivefold excess of hypochlorous acid. using equimolar or lower amounts of hypochlo­ Seven new resonances appeared in chloroform rous acid, the yield of this product raises markedly phase of samples treated with hypochlorous acid. using an excess of hypochlorous acid. An intense They are listed in Table I. All signals should arise resonance was observed for acetone at 2.15 ppm in CDCI3. The acetone resonance appears already at the lowest concentration of hypochlorous acid and raises continuously at higher HOC1/OC1" con­ centrations. Methanol was found in traces at 3.47 ppm. Resonances at 1.32, 3.78, and 3.81 ppm could not be exactly assigned. Signal intensities for tert- butyl hydroperoxide, water and chloroform were not indicated in Table I. Tert-b\xty\ hydroperoxide is the original compound. Water is contained in traces after phase separation in the CDC1 3 layer. Chloroform is also found in low amounts in 4.0 3.5 3.0 2.5 2.0 1.5 1.0 CDCI3. Chemical shift (ppm) Furthermore, the ability of terf-butanol, di -tert- Fig. 6. 'H NMR spectrum in CDC13 of reaction products butyl peroxide and acetone to react with hy­ of rm-butyl hydroperoxide and NaOCl. 7m-butyl hy­ pochlorous acid has been studied. Whereas tert- droperoxide (10 mmol/1) was incubated with NaOCl (10 butanol and di-terr-butyl peroxide did not show mmol/1) for 15 minutes. All solutions were diluted with any reaction with hypochlorous acid under our ex­ 0,14 mol/1 NaCl, 10 mmol/1 phosphate, pH 7.4 made on D20. After incubation 1 ml of reactants was thoroughly perimental conditions, acetone yielded acetate and mixed with 1 ml CDC1> chloroform in a slow reaction with hypochlorous 392 J. Arnhold et al. ■ HOC1 and Hydroperoxides acid. The latter reaction is known as the so-called (CH 3)3C - 0 + (CH 3)3COOH haloform reaction: (CH 3)3COH + (OT^COO. CH3-CO-CH3 + 3 HOC1 — The re/7-butylperoxy radical is more stable than CH3COOH + CHCI3 + 2 H20 . ferf-butyloxy radical. The recombination of two rerr-butylperoxy radicals will also yield di-/m-bu- 'H-NMR spectra of CDCI3 solutions of 2,2',3,3'- tyl peroxide according to a Russell mechanism tetramethylbutane (CH 3)3CC(CH 3)3, tert-butyl (Russell, 1959). methyl ether (CH 3)3COCH3, and 2-methylpro- According to the scheme in Fig. 7 rm-butyl hy­ pene (CH 3)2C=CH 2 were also recorded. They droperoxide is involved into two reactions. First of yielded singlets at 0.85 ppm (2,2',3,3'-tetramethyl- all it yields the rerf-butyloxy radical as the result butane), 1.17 and 3.19 ppm (terf-butyl methyl of reaction with hypochlorous acid. Secondly, tert- ether) or 1.71 and 4.19 ppm (2-methylpropene). butyl hydroperoxide reacts also with the tert-buty­ Therefore, these compounds could not be iden­ loxy radical. tified as reaction products between tert-butyl hy­ A formation of di-ferr-butyl peroxide from droperoxide and hypochlorous acid. (CH 3)3C and (CHs^COO seems to be unlikely In some cases the reaction between fm-butyl because the appearance of other recombination hydroperoxide and hypochlorous acid was studied products as (CH 3)3C-C(CH 3)3 should be also ex­ using higher concentrations of chloride or after pected for this reaction sequence. Furthermore, a bubbling argon through all solutions to remove bubbling of argon through all solutions to inhibit oxygen. Neither of these approaches leads to sig­ the formation of (C ^^C O O from (CH3)3C and nificant changes in the product composition (data 0 2 did not result in a lower yield of di-terf-butyl not shown). peroxide. These results demonstrate that (CH3)3CO radi­ Discussion cals are formed in the result of reaction between NMR and chemiluminescence investigations of tert-butyl hydroperoxide and hypochlorous acid. the reaction between tert-butyl hydroperoxide and Such radicals are assumed to be a promoter of free hypochlorous acid indicate the formation of a free radical lipid peroxidation (Small et al., 1979; radical intermediate. One the of main reaction Schöneich et al., 1990). Therefore, our previous products is di-rm-butyl peroxide. It arises from the finding (Panasenko et al., 1995) of an enhance­ recombination of two rm-butyloxy radicals. ment of the production of TBA-reactive products in liposomes composed of egg yolk phosphati­ 2 (CH 3)3C -O ' — (CH 3)3C-O - O- C(CH3)3. dylcholine treated with hypochlorous acid in the A scheme of this reaction sequence and some presence of ferr-butyl or cumene hydroperoxide is other proposed reactions of tert-butyloxy radical is caused by the appearance of RO radicals. Such given in Fig. 7. 7erf-butanol can also arise from radicals are also formed in the metal-catalysed de­ (CH3X3C-O by abstraction of a proton from the composition of hydroperoxides (Vladimirov et original tert-butyl hydroperoxide al., 1972).

(CH 3)3C -0 -0 -C (C H 3) 3

(CH3)3COOH + (CH3)3CO* HOCI/OCI

(CH3)3COH

Fig. 7. Formation of di-rm-butyl peroxide ((C H ^ C -O -O -C X C H ^ ) and revf-butanol ((CHu^COH) in the result of reaction of tert-butyl hydroperox­ ide ((CH^COOH) with hypochlorous acid. (CH^CO and (CH^COO in­ dicate rm-butyloxy radical and /m-butylperoxy radical, respectively. J. Arnhold et al. ■ HOC1 and Hydroperoxides 393

The exact mechanism of formation of oxides differs considerably in comparison to its re­ (CH3)3CO from rm-butyl hydroperoxide under action with hydrogen peroxide. The reaction with the influence of hypochlorous acid remains un­ hydrogen peroxide yields singlet oxygen as the known. One possible pathway is the homolytic dis­ main product. Free radicals are not involved in ruption of (CH3)3COOH into (CH3)3CO and this process (Held et al., 1978). On the other hand, OH. On the other hand, the attack of hypochlo­ free radicals are formed during reaction of HOC1/ rous acid on tert-butyl hydroperoxide can be ac­ OC1- with tert-butyl or cumene hydroperoxides. companied by the formation of (CH 3)3COOCl, Singlet oxygen is not generated in this reaction. whose homolytic disruption will be the source for These properties are also responsible for the dif­ rerr-butyloxy radical. ferent behaviour of hydrogen peroxide (Pana­ Electron spin resonance experiments to detect senko et al., 1994a) or tert-butyl hydroperoxide free radicals are under progress. They are not in­ (Panasenko et al, 1995) in peroxidation of unsatu­ cluded in this paper. rated phosphatidylcholine induced by HOC1/OC1". Two other main products of the reaction be­ Only tert-butyl hydroperoxide has been found to tween tert-butyl hydroperoxide and hypochlorous promote an additional accumulation of thiobarbi­ acid are tert-butyl hypochlorite and acetone. There turic acid-reactive substances. are several pathways for their formation. Tert-bu­ Organic hydroperoxides are always present in tyl hypochlorite can be formed from a direct reac­ biological membranes at low concentrations. Free tion between hypochlorous acid and either tert-bu­ radicals generated as the result of reaction be­ tyl hydroperoxide or rerf-butanol. A third tween HOC1/OC1- and organic hydroperoxides possibility is the interaction between the tert-buty- can initiate new oxidation processes. loxy radical and a chlorous atom. Acetone can also be formed in the result of sev­ Acknowledgements eral reactions. A ß-scission of terf-butyloxy radi­ This work was supported by the German Minis­ cals yields acetone. The decomposition of tert-bu­ try of Research and Technology (Grant 01 ZZ tyl hypochlorite is also accompanied by the 9103/9-R-6) and by the Deutsche Forschungsge­ formation of acetone. Finally, acetone can result meinschaft (Grant INK 23/A1-1). One of the au­ from a direct reaction between tert-butyl hydro­ thors (O.M. Panasenko) obtained a grant from the peroxide and hypochlorous acid. Alexander von Humboldt Foundation, Bonn, Ger­ Acetone itself is known to react with hypochlo­ many. A grant of the Graduiertenkolleg (Molecu­ rous acid to yield acetic acid and chloroform. lar and cell biology of connective tissue) was These results demonstrate that the reaction provided for J. Schiller by the Deutsche For­ mechanism of HOC1/OC1" with organic hydroper- schungsgemeinschaft.

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