JAERI-Conf 95-003

6. Biological System (1)

6.1 *Excitation Transfer, Charge Transfer and Hydrogen Abstraction Reaction, Kinetics Studies of Modification of DNA Components

N.Y.Lin

Laboratory of Radiation Chemistry, Academia Sinica, Institute of Nuclear Research, P.O.Box 800-204 Shanghai, China

Abstract Laser induced acetone-sensitized excitation of DNA components offered, for the first time, T-T absorption spectra and direct triplet states kinetics of cytosine, cytidine, and dCMP, adenine, adenosine and dAMP and guanosine . Mechansims and kinetics of electron transfer reactions between triplet acetone and purine bases and triplet thymine and electroaffinic modifiers have been elucidated. Rapid electron transfer from hydroxycinnamic acid derivatives to oxidizing OH adducts of pyrimidines have been performed. Fast reduction and repair of oxidizing damage of pyrimidines have been achieved accordingly.

INTRODUCTION

Extensive studies on the triplet states of pyrimidines and •their nucleosides and nucleotides were performed. However cytosine and purine base and their nucleosides, nucleotides have not yet observed. Very recently a breakthrough for detection of absorption spectra and direct triplet state kinetics of cytosine and its two derivatives, adenine and its two derivatives and guanosine have been achieved at our laboratory.OH adduct of pyrimidines are the major cause of oxidizing damage of DNA.The oxidizing OH adduct can be reduced via electron transfer reaction by N.N.N.N tetramethyl-p-phenyldiamine(TMPD) or ascorbate. Recently the oxidizing OH adducts of thymine, cytosine and polycytidylic acid (polyC) have been reduced and repaired via electron transfer from hydroxycinnamic acid derivatives to the adducts.

EXPERIMENTAL

DNA components including cytosine cytidine and dCMP,

-204- JAERI-Conf 95-003 guanosine and adenine adenosine and dAMP were obtained commercially and used without further purification. Purity of acetone(Ac) and Mncl„ were given elsewhere.

RESULTS AND DISCUSSION

1.Novel triplet states of DNA components. A new breakthrough of direct detection of triplet states of DNA components has been achieved under acetone-sensitization in aqueous solution. Since energy level of triplet acetone is higher than that of DNA bases, nucleoside and nucleotides. And due to very short life time of acetone,the composite spectra of triplet base and dehydrogenated radicals were observed several micosecond after laser pulse. The neat decay trace and T-T absorption spectra were obtained, for the first time, according to subtraction method r 31 suggested by us; These species decay following pseudo-first order kinetics with the apparent decay rate constant (K . ) as below: obs os q The reaction mechanisms can be summarized as follows:

A 248nm , 3A * Ac * Ac 3Ac* + B _JVr > Ac + 3B* V 5a > B 3B* s » B + Q Q:Mn2+ Several sets of direct kinetic parameters of triplet decay (K . K . K and K , ) were obtained upon detailed kinetic o s q obs * analysis. The life times were derived accordingly. 1.1 Triplet state of guanosine Lf J3 1 The absorption spectra after laser photolysis of guanosine aqueous solution containing acetone as photosensitizer show that besides triplet acetone a new transient species appeared. Based on the subtraction method the neat transient absorption spectrum was obt a ined . Its decay follow first order kinetics with 4 -1 -4 K . =9.4x10 s , T1/0 = 7.4 /is at 1x10 M guanosine in the absence of ODS 1 / £t t\ triplet quencher. In addition, the species can be quenched by Mn with rate constant Kq=l.9xl010M-1S~1.

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r 2i 1.2 Triplet states of cytosine.cytidine and dCMP J The transient absorption spectra from laser flash photolysis of aqueous solution containing cytosine and acetone show that besides triplet acetone a new transient species appeared. Based on the subtraction method for the resolution of composite spectra the neat characteristic absorption was also recorded. Its decay follow the first order kinetics and can be quenched by Mn 2+ . Similar transient absorptions have been recorded from laser photolysis of cytidine and dCMP in the presence of acetone. These novel species should be assigned to triplet cytosine cytidine and dCMP after quenching by Mn 2+ . Their kinetics parameters of triplet decay were given in table 1. Table 1 Kinetic parameters of triplet states of cytosine, cytidine, and dCMP

(s-1) (dm mol s ) (dm mol s ) (dm mol s )

3Cyt* 5.5x10 4.2x10 8.0x10 3.8x10' 3Cyd* 7.6x10' 2.4x10 2.5x10* 4.5xlOJ 3dCMP* 9.4x10* 1.8x10 1.7x10* 5.1xl05

The half life time (TT/O) °f triplet cytosine,cytidine and dCMP are 5.0,5.6, and 5.3ms respectively at the concentration of 2x10 -4M in the absence of triplet quencher. Utilizing energy transfer method and taking the molar absorption coefficient of 3 * -1 ~1 Ac as 600 M cm at 300 nm as well as the kinetic parameters given in table 1, the molar absorption coefficient of triplet cytosine, cytidine and dCMP have been estimated to be 200 M~1cm~1(340nm),200M~1cm"1(350nm) and 400M~1cm"1 (450nm) respectively. 1.3 Triplet states of adenine, adenosine and dAMP The transient absorption spectra from laser flash photolysis of aqueous solution containing adenine and acetone show that besides triplet acetone, a new transient species appeared. According to our subtraction method the neat transient absorption

206 JAERI-Conf 95-003 spectrum was recorded. Its decay follow the first order kinetics and can be quenched by Mn 2+ . Similar transient absorptions have been observed following the laser photolysis of aqueous solution of adenosine and dCMP respectively in the presence of acetone. After quenching by Mn 2+ , these novel species should be assigned to triplet adenine, adenosine and dAMP with half life times of 2.7,2.8and 5.8 /is respectively in the absence of triplet quencher. Their kinetic parameters of triplet decay were deduced as shown in table 2. Table 2. Kinetic parameters of triplet states of adenine, adenosine and dAMP

"obs o sq (s_1) (dm3mol"1s_1) (dm"3mol"1s"1)

3 * Adenine 2.6x10 1.1x10" 6.9x10 4.2x108 3 * Adeonsine 2.5xl05 7.9xl04 8.3x10 3.5xl0( 3 8 dAMP* 1.2xl05 3.7xl04 3.6x10 6.0x10

2.Electron transfer reactions between DNA components and modifiers 2.1 Reactions of triplet acetone with DNA components Following the laser photolysis of guanosine aqueous solution containing acetone besides the triplet guanosine a long-lived absorption, quite similar to the transient absorption from reaction of dG with SO 7 radical, had been assigned to G(-H) radical.F5 1 It was suggested that electron transfer from guanosine to triplet acetone occurred . Subsequent hydrogen abstraction occurred via following reaction: [31 3 . * Ac + dG * Ac + dG -» Ac(+H) + dG(-H) After laser photolysis of adenine in the presence of acetone besides the absorption band from triplet adenine a long-lived absorption which decay following second order kinetics and was assumed to be a dehydrogenated adenine radical arising from electron transfer reaction between adenine and triplet acetone: 3Ac*+ A -» Ac + A + -» Ac(+H) + A(-H) Similar transient absorption spectra have been obtained from

-207 JAERI-Conf 95-003 laser photolysis of adenosine and dAMP aqueous solution in the presence of acetone. Based on kinetic analysis the long-lived absorption should be assigned to the dehydrogenated radical of adenosine and dAMP respectively:T4 1 On another hand the free energy change (AG) of these supposed electron transfer reactions were calculated according to Rehm-Weller equation.Calculated AG values indicated that electron transfer from purine bases or its nuclesides and nucleotides to triplet acetone should proceed spontaneously; 2.2 Electron transfer reaction between triplet pyrimidines and electroaffinic modifiers Laser induced electron transfer from triplet thymine or thymidine to a cyclic disulfide compound,lipoic acid,was studied. Laser induced transient absorption spectra of CHoCN solution of thymine or thymidine and lipoic acid indicated that long-lived transient absorption is very similar to the anion radical of lipoic acid (RSSR* ). Based on the quenching of triplet thymine or thymidine by lipoic acid the electron transfer reaction rate constant was deduced to be 1.3xl010M S and 6.9x10 MS for r g I thymine and thymidine respectively. On the other hand laser induced electron transfer reaction between thymine or uracil and tetracyanoethylene(TCNE) was also studied, laser flash photolysis of CH3CN solution of TONE and pyrimidines led to the formation of a species characterized with an absorption band very similar to anion radical of TONE. The electron transfer reaction rate constant are 7.6x10 9-M 1 S- 1 for thymine and 1.7xl010M~1S~1 for uracil^73 3.Pulse radiolysis studies of the interaction of hydroxycinnamic acid derivatives with oxidizing OH adduct of pyrimidines1[8 1 The hydroxyl radical either generated by radiation decomposition of water in biological system or via redox cycling chemicals can cause DNA damage. There are two ways to inhibit the lesion of cell, either via scavenging OH radical or fast repair of the OH adducts. The reaction of OH radical with pyrimidines take place predominantly at C5-C6 double bond. The radical formed by addition of OH to C5 is reductive, while to C6 is oxidative. The reduction of oxidizing OH adduct of pyrimindes were performed by strong reductant TMPD:f 81 The fast reduction and repair of

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OH adducts of pyrimidines by hydroxycinnamic acid derivatives via electron transfer reaction have been performed at our [Q] laboratory; The transient absorption spectrum at Ifis after pulse * radiolysis of 2 mM cytosine and O.lmM sinapic acid aqueous solution saturated with N20 is stemming from OH adduct of cytosine predominately. At 10 fis after electron pulse accompanying the decay of the characteristic absorption band of OH adduct of cytosine a novel band appeared which was assigned to the phenoxyl radical of sinapic acid arising from electron transfer reaction between OH adduct of cytosine and sinapic acid. After capture of an electron the oxidizing OH adduct should be repaired by dehydroxylation or reduced to stable OH adduct .Similar transient absorption spectrum after pulse radiolysis of solution containing cytosine and caffeic acid or ferulic acid has been recorded. The characteristic absorption peak assigned to phenoxyl radical of caffeic acid or ferulic provided evidences of electron transfer from caffeic acid or ferulic acid to OH adduct of cytosine. The electron transfer reaction of hydoxycinnamic acid derivatives with oxidizing OH adduct of thymine have also been studied in the same way. After pulse radiolysis of 2mM thymine and O.lmM sinapic acid or caffeic acid or ferulic acid in aqueous solution saturated with N20 the transient absorptions revealed similar absorption peaks assigned to the phenoxyl radicals of sinapic acid, cafferic acid and ferulic acid respectively. The electron transfer rate constants for reduction of oxidizing OH adduct of thymine by hydroxycinnamic acid derivatives have also been determined (see table.3) Table 3. The rate constants of electron transfer from hydroxycinnamic acid derivatives to oxidizing OH adducts of pyrimidinine

Substrate k[CytOH] k[ThyOH]

Sinapic acid 1.1x10 1.3xl09 Caffeic acid 1.2x10 1.3xl09 Ferulic acid 1.0x10 l.lxlO9

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ACKNOWLEDGEMENT This project was supported by the National Natural Science Foundation of China.

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