David J. Deeble

Reactions of OH Radicals with Poly(U) in Deoxygenated Solutions: Sites of OH Radical Attack and the Kinetics of Base Release

Pulse radiolysis of N2O-saturated solutions of poly(U) in the presence of tetranitromethane showed that 81 per cent of the radicals formed are reducing in nature. Using data from other sources it has been estimated that 70 per cent of the OH radicals add to the base at C(5) and 23 per cent at C(6) while only 7 per cent abstract an H-atom from the sugar moiety. To a large extent the C(5) OH adduct radicals attack the sugar moiety of poly(U) thereby inducing strand breakage and base release. G (base release) = 2.9 can be subdivided into three components: (a) immediate (20 per cent), (b) fast (50 per cent) and (c) slow (30 per cent). The immediate base release must occur either during the free-radical stage or as a result of the rapid (t1/2 less than 4 min at 0 degree C) decomposition of a diamagnetic product. The fast and the slow processes are only readily observable at elevated temperatures, e.g. at 50 degrees C the half lives are 83 min and 26 h, respectively (Ea (fast) = 68 kJ mol-1, Ea (slow) = 89 kJ mol-1, A (fast) = 1.5 X 10(7) s-1, A (slow) = 1.9 X 10(9) s-1. It is concluded that there are three different types of sugar lesions giving rise to base release, structures for which are tentatively proposed.

The reactivity of various free radicals with hyaluronic acid: steady-state and pulse radiolysis studies

The reactions of the primary water radicals with the biopolymer hyaluronic acid have been studied by pulse radiolysis. Bimolecular rate constants, expressed in terms of the disaccharide repeating sub-unit of hyaluronic acid, for OH., H. and eaq- were found to be 7 X 10(8) M-1 X s-1, 5 X 10(7) M-1 X s-1 and less than 5 X 10(6) M-1 X s-1, respectively. By comparing the viscosities of samples, gamma-irradiated in the steady state under a variety of conditions, with unirradiated controls, the efficiencies with which selected radicals cause chain breakage have been determined. Efficiencies of 30%, 15%, 0%, 0.2% and 5% were estimated for OH., H., eaq-, methanol radicals and tert-butanol radicals, respectively. The presence of oxygen during irradiation increased the extent of chain breakage by a factor of 1.75.

The SO4(.-)-induced chain reaction of 1,3-dimethyluracil with peroxodisulphate.

The sulphate radical SO4(.-) reacts with 1,3-dimethyluracil (1,3-DMU) (k = 5 X 10(9) dm3 mol-1 s-1) thereby forming with greater than or equal to 90 per cent yield the 1,3-DMU C(5)-OH adduct radical 4 as evidenced by its absorption spectrum and its reactivity toward tetranitromethane. Pulse-conductometric experiments have shown that a 1,3-DMU-SO4(.-) aduct 3 as well as the 1,3-DMU radical cation 1, if formed, must be very short-lived (t1/2 less than or equal to 1 microsecond). The 1,3-DMU C(5)-OH adduct 4 reacts slowly with peroxodisulphate (k = 2.1 X 10(5) dm3 mol-1 s-1). It is suggested that the observed new species is the 1,3-DMU-5-OH-6-SO4(.-) radical 7. At low dose rates a chain reaction is observed. The product of this chain reaction is the cis-5,6-dihydro-5,6-dihydroxy-1,3-dimethyluracil 2. At a dose rate of 2.8 X 10(-3) Gys-1 a G value of approximately 200 was observed ([1,3-DMU] = 5 X 10(-3) mol dm-3; [S2O8(2-)] = 10(-2) mol dm-3; [t-butanol] = 10(-2) mol dm-3). The peculiarities of this chain reaction (strong effect of [1,3-DMU], smaller effect of [S2O(2-)8]) is explained by 7 being an important chain carrier. It is proposed that 7 reacts with 1,3-DMU by electron transfer, albeit more slowly (k approximately 1.2 X 10(4) dm3 mol-1 s-1) than does SO4(.-). The resulting sulphate 6 is considered to hydrolyse into 2 and sulphuric acid which is formed in amounts equivalent to those of 2. Computer simulations provide support for the proposed mechanism. The results of some SCF calculations on the electron distribution in the radical cations derived from uracil and 1-methyluracil are also presented.

Superoxide Radical Reactions in Aqueous Solutions of Pyrogallol and N-propyl Gallate: The Involvement of Phenoxyl Radicals. A Pulse Radiolysis Study

The reactions of O2-. in aqueous solutions of pyrogallol 1 and the antioxidant n-propyl gallate 2 have been studied. In both cases the initial reaction gives hydrogen peroxide and the corresponding phenoxyl radical (k(1 + O2-.) = 3.4 x 10(5), k(2 + O2-.) = 2.6 x 10(5) dm3 mol-1S-1). These phenoxyl radicals have been produced independently by reacting 1 and 2 with Br2-. and their spectra and first pKa values measured (pKa(phenoxyl radical from 1) = 5.1, pKa(phenoxyl radical from 2) = 4.1). It is necessary to correct the observed spectra for the contribution of the H-adducts, formed by the reaction of radiolytically produced H atoms with the substrates (k(1 + H) = 2.5 x 10(9), k(2 + H) = 3.8 x 10(9) dm3 mol-1 S-1). The H-adduct spectra are given. In the reactions of O2-. with the substrates the initial transient absorbances are characteristic of the phenoxyl radicals; however at longer times a new transient absorbing around 500 nm (epsilon congruent to 10(4) dm3 mol-1 cm-1) appears. This is believed to be the deprotonated hydroxy-orthoquinone, formed by the reaction of phenoxyl radicals with O2-. (k congruent to 1.5 x 10(8) dm3 mol-1 S-1, from kinetic curve-fitting). The absorbance due to the hydroxy-orthoquinones decays by first-order kinetics (1.6 x 10(2) in the case of 1 and 1.1 x 10(2) s-1 in the case of 2). This is thought to be mainly the result of the conversion of the hydroxy-orthoquinone into its hydrate. Similar experiments were carried out with catechol and ethyl protocatechuate. The chemistry appears to be similar to that of the pyrogallol derivatives. The rate constant for reaction of these compounds with O2-. is, however, only less than or equal to x 10(4) dm3 mol-1 s-1.

Pulse radiolysis in model studies toward radiation processing

Abstract Using the pulse radiolysis technique, the OH-radical-induced reactions of poly(vinyl alcohol) PVAL, poly(acrylic acid) PAA, poly(methacrylic acid) PMA, and hyaluronic acid have been investigated in dilute aqueous solution. The reactions of the free-radical intermediates were followed by UV-spectroscopy and low-angle laser light-scattering; the scission of the charged polymers was also monitored by conductometry. For more detailed product studies, model systems such as 2,4-dihydroxypentane (for PVAL) and 2,4-dimethyl glutaric acid (for PAA) was also investigated. With PVA, OH-radicals react predominantly by abstraction of an H-atom in α-position to the hydroxyl group (70%). The observed bimolecular decay rate constant of the PVAL-radicals decreases with time. This has been interpreted as being due to an initially fast decay of proximate radicals and a decrease of the probability of such encounters with time. Intramolecular crosslinking (loop formation) predominates at high doses per pulse. In the presence of O2, peroxyl radicals are formed which in the case of the α-hydroxyperoxyl radicals can eliminate HO2-radicals in competition with bimolecular decay processes which lead to a fragmentation of the polymer. In PAA, radicals both in α-position (characterized by an absorption near 300 nm) and in β-position to the carboxylate groups are formed in an approximately 1:2 ratio. The lifetime of the radicals increases with increasing electrolytic dissociation of the polymer. The β-radicals undergo a slow (intra- as well as intermolecular) H-abstraction yielding α-radicals, in competition to crosslinking and scission reactions. In PMA only β-radicals are formed. Their fragmentation has been followed by conductometry. In hyaluronic acid, considerable fragmeentation is observed even in the absence of oxygen which, in fact, has some protective effect against this process. Thus free-radical attack on this important biopolymer makes it especially vulnerable with respect to a reduction of its viscosity, and in rheumatic diseases this effect may be the reason for their painfulnes.

The enhanced stability of the cross-linked hylan structure to hydroxyl (OH) radicals compared with the uncross-linked hyaluronan

Abstract A comparison has been made of the relative stabilities of hyaluronan and hylan to degradation by OH radicals produced by γ-irradiation of aqueous solutions in N2O, when G (yield per 100 eV) for OH radicals is 5.6 and H atoms 0.6. Using low angle light scattering and viscometric methods, the change in molecular weight of the polysaccharides was measured with increasing dose. From the yield/dose curves (expressed as breaks per molecule), the initial G value for hyaluronan degradation is ∼ 4. A further slow post-irradiation decrease in molecular weight is observed, which can be brought to completion by incubating the solutions for 1 h at 60°C. Thereafter, the G value for degradation is ∼ 6. A similar post-irradiation degradation was found for hylan. A technique using tetranitromethane (TNM) has been used to distinguish between two types of radicals formed on the hyaluronan backbone. Radicals of the 1-hydroxy-2-alkoxy type (C-2, C-4, C-2 and C3 of the glucuronic acid) would induce strand breakage by alkoxy elimination. For the equivalent alkoxy radical at C6 of the acetamido monosaccharide, ring opening would occur with formation of a hemi-acetal, leading also to strand breakage. The C-2 and C-3 radicals would eliminate water rather than produce breaks by β-alkoxy elimination. Thus three out of the initially formed radicals would produce breaks by β-alkoxy formation. These can be stabilised with TNM and distinguished. It is concluded that these are the radicals involved in the post-irradiation thermal degradation process. Comparison of hylan and hyaluronan is, therefore, most valid when this post-irradiation process has been completed. Therefore, all G values for degradation were measured after incubation for 1 h at 60°. This investigation establishes the greater stability of hylan (Gdegradation = 2) compared to hylan (Gdegradation = 6). Therefore, in an environment such as supplementation of an inflammed joint where OH radicals are released, hylan is able to retain its integrity as a viscoelastic macromolecule three times better than hyaluronan. Its potential as a viscosupplementation material, or as an inflammatory drug release matrix inserted within the joint is, therefore, greater than non-cross-linked hyaluronan.

Pulse Radiolytic Studies on Uracil and Uracil Derivatives. Protonation of Their Electron Adducts at Oxygen and Carbon

The electron adducts of uracil, 1,3-dimethyluracil and 1,3-dimethylthymine, known to protonate rapidly in aqueous solution at oxygen, are now shown to undergo a slower protonation at C(6) producing a radical centred at C(5), a reaction which can be catalysed by buffer.

The kinetics of hydroxyl-radical-induced strand breakage of hyaluronic acid. A pulse radiolysis study using conductometry and laser-light-scattering.

Abstract Hydroxyl radicals were generated radiolytically in N2O -and N2O / O2(4: 1)-saturated aqueous solutions of hyaluronic acid. The hydroxyl radicals react rapidly with hyaluronic acid mainly by abstracting carbon-bound H atom s. As a consequence of subsequent free-radical reactions, chain breakage occurs the kinetics of which has been followed using the pulse radio lysis technique. In the absence of oxygen, strand breakage was followed by the change in conductivity in duced by the release of cationic counterions condensed at the surface of hyaluronic acid which is a polyanion consisting of subunits of glucuronic acid alternating with N-acetyl-glucosamine. It appears that strand breakage is not due to one single first-order process, however, the con tributions of the different com ponents cannot be adequately resolved. At pH7 the overall half-life is 1.4 ms, in both acid and basic solutions the rate of free-radical induced strand breakage is accelerated (at pH 4.8, t1/2 = 0.6 ms; at pH 10, t1/2 = 0.18 ms). In the absence of oxygen there is no effect of dose rate on the kinetics of strand breakage. In the presence of oxygen in addition to conductom etric detection, strand breakage was also followed by changes in low-angle laser light-scattering. These two techniques are complementary in that in this system the conductometry requires high doses per pulse while the light-scat tering technique is best operated in the low -dose range. In the presence of oxygen a pro nounced dose-rate effect is observed, e.g. at pH 9.7 after a dose of 9.4 Gy the overall half-time is approx. 0.5 s, while after a dose of 6.6 Gy the half-time is approx. 0.23 s. Both the yield and the rate of strand breakage increase with increasing pH, e.g. at pH 7 G(strand breaks) = 0.7 × 10-7 mol J-1 and at pH 10.4, 4.8 × 10-7 mol J-7. The radiolytic yields of CO2, H2O2, organic hydroperoxides, O2·- and oxygen consum ption have been determined in y-irradiated N2O/ 0 2(4: 1)-saturated solutions of both hyaluronic acid and β-cyclodextrin.

Radioprotection of Pyrimidines by Oxygen and Sensitization by Phosphate: A Feature of Their Electron Adducts

The reactions of the electron adducts of thymine, uracil and 1,3-dimethylthymine in the presence of phosphate buffer and low oxygen concentrations have been investigated. Oxygen reacts with the pyrimidine electron adducts and their O(4)-protonated forms to restore the pyrimidine and produce O2-./HO2.. Thus oxygen acts as a radiation protector. In the presence of high buffer concentrations the electron adducts are irreversibly protonated at C(6). On reaction of this radical with oxygen no restitution of the original pyrimidine occurs and the pyrimidine is destroyed. Thus phosphate buffer acts as a radiation sensitizer. Some speculations are made as to the possible relevance of these reactions to biological systems.

The radiation-induced degradation of hyaluronic acid

Abstract Free-radical-induced chain scission in hyaluronic acid in aqueous solution has been studied using pulse radiolysis. In the absence of oxygen (nitrous oxide-saturated solutions) the process of chain breakage was monitored by measuring changes in conductivity resulting from the release of condensed counter-ions (K + ), originally located in the vicinity of the break. The rate of formation of breaks was found to be first order and was catalysed by acid and base (overall half-lives at pH values of 4.8, 7 and 10.2 were 0.6, 1 and 0.1 ms). It would seem that more than two independent reaction pathways are involved in the cleavage processes. In the presence of oxygen (N 2 O/O 2 ), chain scission has been measured by pulse radiolysis monitoring changes in scattered light intensity as well as following conductivity changes. In oxygenated solutions, the kinetics of OH-radical-induced chain scission were found to contain a second-order component; the rate of breakage was base catalysed. Yield-dose plots for chain breaks (N 2 O/O 2 , pulse-irradiated), showed a marked dependence on pH, with G -values (molecules/100 eV) of 0.7, 2.5 and 4.7 at pH values of 7, 9.7 and 10.4, respectively. Steady-state radiolysis (N 2 O/O 2 ) was used to determine G -values for oxygen consumption [ G (-O 2 ) ≈ 6], carbon dioxide formation [ G (CO 2 ) = 0.8 in the absence of O 2 and 1.3 in its presence] and peroxide formation [ G (H 2 O 2 ) ≈ 2; G (organic hydroperoxide)