Indian Journal of Chemi stry Vo l. 4 1 A, October 2002, pp. 2096-2 100

Photooxidation of substituted purines in presence of peroxydisulphate in aqueous

solution

Midudhula Sudha Swaraga & Mundra Adinarayana*

Department of Chemi stry, Osmani a Uni versity,

Hyderabad 500 007. India

Rece i ved 4 JIII/1I0 rV 2002 ; r evised 6 M il." 2002

Photooxi c!a ti on of purine bases. viz .. caffeine. theobromine, thcophylline. xanthine. hypoxanthine. adenine anc! guanine in aq ueous solution has been carried ou t in presence of peroxydi sulph:l1e (PDS). Peroxydisul phate is acti vated to SO/- at 254 nill . The reacti ons are foll owed by measuring the absorbance of pu rine bases at their respecti ve Am", ' The ra tes of reactions are calcu lated under different ex perimental conditi ons. The li ght intensity is Illeasured using peroxydi sul phate solution as st:lIldard chemica l actinometer. Using reacti on ra te and li ght intensity at 254 nill . the quantu lll yields are ca lcul ated . The rates o f pholOox idati on of pur ine bases are fou nd to increase with increase in IPDS I and independent of rpurine]. The increase of li ght intensity has been found 10 increase the rate of oxidati on. The quan tum yields are found to depend on Ipuri ne] but independent of IPDSj and li ght intensity. On the basis of ex perimental results a proba ble mechani sm is suggested in whi ch peroxycli sul phate on photolysis gives sul phate radical ani on whic h initiates the reac tion by capturing an electron from C8 positi on of purine to form purine radical cation. Thi s radica l cati on deprotonates and undergoes further oxidation to give C(8) hydroxy purine.

Caffeine is a potent scavenger of hydroxy l radicals. It has been reported I that the reac tion rate constant of

hydroxy l radi cal w ith caffe ine is 6 .9x l O'> mor l dm' S·I

obta ined in pulse radio ly sis . C affe ine reac tio n with O H radicals generated by Fento n reacti o n or by the reac tion of C r(V) with H20 :" and in vestigated by e lec tro n spin resonance (ES R) spin trapping technique shows that caffeine effecti ve ly scavenges OH radical

with a rate constant of approx imate ly 5.9x 10lJ mor l

dm1 S· I which is comparable with those of othe r

effici ent OH radi cal scavengers. Caffe ine and theobromine are naturally occurring

purines present in coffee and tea. Because of high consumption of caffe ine co ntaining beverages, we thought it is worthwhile to understand the antioxidant properties of caffe ine and theobromine. In thi s directio n we have undertaken a systematic kinetic study of the oxidation of caffe ine and theobromine by SO/- . To get an insight into the site of attack of SO/-

on the purine ring syste m, oxidati on of o ther substituted purine syste ms viz., theophy lline, xanthine and hypoxanthine have also been undertaken. Furth er

the oxidati on of adenine and g uanine by SO-l°- has been carried out to explore the possibility of using caffeine , theobromine and other substituted purines as antioxidants on DNA model systems.

Experimental Caffe ine, theo bro mine, theophy lline, xanthine,

hy poxanthine, adenine and guanine were fro m sig ma and used as recei ved. The soluti o ns of purines were al ways prepared afresh using di still ed water. T he solutio ns o f potass ium salt of pe roxydi sul phate was prepared using doubly d istilled water and standard ised using ce rimetry using re rro in ind icator. Pe roxydi sulphate solutio n was added to a measured excess of ferro us a mmo nium sulphate , and back­ti trated with a standard ceri c ammonium su lphate sol uti on. At roo m temperature thi s react io n is rap id enoug h for analytica l purposes and equi va lency of ferrous ion to peroxydisulph ate is 2 to I. T he co ncentration of subst ituted purines are determined by

meas uring the absorbance at the ir respecti ve A""" from the know n mol ar absorptio n coe ffi ci ent va lues . Irradiations were carri ed out in a quantum y ie ld reactor model QYR-20 using hi gh-pressure mercury lamp. [n general, intensity measureme nts were carried out using fe rrioxal ate acti no metry. I n a typi ca l reac tion , purine and PDS soluti o ns were mi xed in a speci a ll y des ig ned I cm path length cuve tte , whi ch is suitable, both fo r irradi at io n in the reactor as we ll as absorbance measu re ments . The absorbance measurements were carri ed out on Hitachi UV -visible spec trophotomete r model 34 10. The progress of the reactions were foll owed by measuring the absorbance

at the respective A max of substitu ted purines by interrupting irradiati on at reg ular inte rval s of time . The reacti on rates with respect to substituted purines were calculated from the plots o f absorban ce versus time using a compute r program . The quantum yie lds were calculated from the rates and calculated light intensity absorbed by PDS at 254 nm . Thi s is the wave length at which peroxydi sul phate is activated to radical reactions. The light intensity absorbed by PDS at 254 nm was calculated fro m the equation given below:

NOTES 2097

Table I- Effect of [PDS] and [substrate] on the rate of ox idation of purines by sulphate radical anion (S04-) in aqueous soluti on Light intensity = 2.16 x JO I5 quanta S- I; pH = 7.5 : Temp = 300 K

104x [PDSI 10' x 108 x rate (mol dm -\ -1)

(Illol dlll·J ) [substrate] Aden ine Guan ine Caffeine T.brollline T.phylline Xanthine H. .wnthine Guanosine (Illo l dlll -J )

2.000 5.000 0.3000 0.6523 0.7500 0.4890 0.2050 0.4 153 0.3200 I. MOO 4.000 5.000 0.5223 0.8560 1.4800 0.9800 0.4640 0.7500 0.5530 3.0000 6.000 5.000 0.7200 1. 1180 2.2800 1.5320 0.7860 0.9814 0.9088 4.9500 8.000 5.000 1.0000 1.5000 2.7000 2.2420 1.0600 1.3700 1.1 200 6.0200 1.000 5.000 0.1200 0.2 125 0.3680 0.2250 0.1660 0.2090 0.1687 0.8000 1.000 2.000 0.1500 0.2315 0.3060 0.2304 0. 1800 0.2220 0.2200 0.9 120 1.000 1.000 0.1700 0.2400 0.3060 0.2660 0. 1700 0.24 10 0.2080 0.9000

Table 2- Effect of light intensity on the rate of oxidation of purines by sulphate rad ical anion (S04'- ) in aqueous solution [PDS] = 5 x J()"4M: I Purine] = 5 x 10-5M ; telllp =300 K: pH = 7.5

l,x IO I) lOS x rate/Illo l dnf.1 S-I Quanta S· I Adenine Guan ine Caffe ine T.brollline T.pilvlline Xanthine /-I .. wntiline Guanosi ne

2.160 0.349 1. 148 1. 186 1.054 2.553 0.480 1.5 I I 2.483 1.560 3. 140 0.580 1.948 3.633 2.101

E PDS II'DSI X I I I'DS = - ---.:....:::.::.:.....-----

. ] I E pDS IPUSI + Epli riIlC[PUnn e

IpDs = intensity of light absorbed by PDS; It = total intensity of light at 254 nm obtained from PDS act inometry ; EpDS = molar absorption coefficient of PDS at 254 nm (24.1 dm}mor1cm-I

); and Epll rine = the molar absorption coefficient of caffeine, theobromine, theophy lline, xanthine, hypoxanthine, adenine and guanine, the values are 4590, 4642, 4740, 7142.8, 10186, 12557 and 1100 dm}mor1cm-1 at 254 nm respective ly.

The HPLC system used for analysis of products includes Shimadzu LC-10AT equi pment with a dual piston-pump system, a so lvent programmer and a Reodhyne injector model 7725 fitted with 20 )..l l loop. A prepacked octadecylsi lyl silica gel ODS hypersil column 25 cm x 0.46 cm, mean particle size 5 )..lm was used . The column effl uents were monitored at 280 nm, us ing variable wavelength SPD-I0A UV­visible detector equipped with 8 )..ll flow cell and attached to a C-R7 Ae plus chromatopac integrator. Samples were eluted with aqueous solutions containing 10% (v/v) methanol and buffered with 10

1.062 1. 120 0.800 3.441 1.765 1.760 1.000 4.533 1.990 2.367 1.200 5.700

mM KH2P04 solution adjusted to pH 7.0 . Before use the phosphate buffer was filtered through a millipore type HA 0.45 )..lm membrane fi lter. All mobile phases were degassed using a vacuum pump. The so lvent flow rate was kept constant at 0.5 mllmin and all the HPLC runs were carried out at ambient temperature.

Results and discussion

The rate of oxidation of substituted pu rines have been calculated from the absorbance versus ti me data. The plot of absorbance versus time is found to be linear up to 80% completion of the reaction indicating zero order dependence on [purine]. The rate of oxidation of purines have been found to increase with increase in [PDS] (Table I) and the plot of log (rate) versus log [PDS] was found to be linear with unit slope indicating first order dependence 0 11 [PDS I. The effect of light intensity on the rate of oxidation of purine has been stud ied and found to increase with increase of light intensity (Tab le 2). The quantulll yields are calcu lated from the rate of oxidation of purine and the light intensity absorbed by PDS at 254 nm. The quantum yields are found to be independent of [PDS] at constant [purine] and li ght intensity while they are fo und to increase with increase in [purine] at constant [PDS] and light intensity (Table 3). The quantum yields are fou nd to depend on light intensi ty at constant [purine] and constant [PDS] (Table 4).

2098 INDIAN J CHEM, SEC A. OCTOBER 2002

Table 3--Effect of [PDS] and [substrate] on quantum yields of ox idation of purines by sulphate radi cal anion (S 0 4' in aqueous so lution

Light intensity = 2. I 6 x 10'" quanta S·I ; pH = 7.5; temp = 300 K

104 X 10' x ~

[PDS] [substrate I Aden ine Guanine Caffeine Tbrolllille Tphy/lille Xanthine H.xallihille Guanosine (11101 dill") (11101 dm" )

2.000 5.000 0.3200 0.3450 0.3052 0.20[ I 0.0863 0.2176 0.2853 1.9460 4.000 5.000 0.2890 0.4152 0.3073 0.1422 0.[000 0.2300 0.2500 1. 8000 6.000 5.000 0.2675 0.3625 0.2804 0.21 86 0.1143 0.2110 0.2920 1.9800 R.OOO 5.000 0.2807 0.4046 0.2920 0.2450 0.1 [86 0.2400 0.2565 1.8280 1.000 5.000 0.26[2 0.4247 0.2965 0.1830 0.[380 0.2620 0.2997 [.8970 1.000 2.000 0.1 320 0.1787 0. [000 0.0762 0.0607 0.1097 0.[554 0.8700 1.000 [.000 0.0753 0.0936 0.05[3 0.0430 0.0300 0.0620 0.0842 0.4320

Table 4--Effect of li ght intensity on the quantum yields of oxidation of purines by sulphate radical ani on (S04'- ) in aq ueous soluti on [PDS] = 5 x 1O.4M; [Pu rine] = 5 x 1O" M; Temp = 300 K; pH = 7.5

<I>

l,x lO l 5 Adenine Guanine Caffeine Tbrolllin e Tphyllille Xanthine H. xanthille Guanosine Quanta S· I

2. [60 0.248 0.445 0.305 O. [79 0.[84 0.286 0.288 1.6500 2.553 0.289 0.500 0.352 0.222 0.228 0.382 0.295 1.8500 3.[40 0.292 0.523 0.420 0.245 0.237 0.418 0.301 [.9000 Tbromine = theobromine; Tphylline = theophylline; and Hxanthine = hypoxanthine

The itTadiated aqueous solution of caffeine and PDS has been analysed in HPLC using UV-vis ible detector at 280 nm. Two peaks were observed with retention times 11.05 min and 13.28 min in which 11.05 min cotTesponds to 1,3,7-trimethy luric acid

(product of caffeine) which was further confirmed by authentic sample while 13.28 min corresponds to caffeine. The itTadiated aqueous solution of xanthine and PDS has been analysed in HPLC using UV- visible detector at 280 nm and the retention times 8.63 min and 11.29 min in which 8.63 min corresponds to uric acid (product of xanthine) which was further confirmed by authentic sample while 11.29 min corresponds to xanthine. It has been reported that OH radical s attack purines, viz., xanthine, isocaffeine, guanine and adenine at C8-position6

.t). The product of oxidation of caffeine

by OH radicals has been reported to be 1,3,7-trimethy luric acid formed via C8-0H adduct radicals. Sulphate radical anion is known to react with purines by electron transfer, producing one electron oxidised species as primary product i.e. radical cation2

. The sulphate radical anion which is produced on photolysis of peroxydisulphate in the initiation step in the present investigation might react with purine by adding at C8 position. According to Pullman" the

calculated charge density and localisation energy values of electrophi li c attack at various carbon atoms in purine ring suggest that C8 is more favourable for electrophilic attack The effect of light intensity on the quantum yields indicate that it might be involved mainly in the activation of PDS to sulphate radi cal

anion in the initiation step. The rates are found to be independent of [purines] (Table I) suggesting that the sulphate radical anion might react with purine in a fast

step to give C8 adduct. The aqueous solution of substrates (purines) in the absence of PDS has been irradiated for 2 to 3 h and could not find any change in the absorbance at their absorption maxima (AmaJ which indicates the absence of any reaction of the substrates only on shining the light. The increase in quantum yields with increase in [purine] (Table 3)

suggests that the excited state of puri ne mi ght be acting as a sensitizer to transfer energy to PDS to produce sulphate radical anions. The life time of excited singlet state of purine is of the order of a few picoseconds4 while that of excited triplet stateS is around 1-20 J.ls . Further in purine system the yields of intersystem crossing has been reported to be quite high, and therefore, the excited triplet concentration

of purine would be higher during photolysis process in our system. It is, therefore, proposed that the

NOTES 2099

X h· hv X h' ant me _ ant me*

2-

Xanthine ' + S20S - 2S04 + Xanthine

+ S04

Xanthine

Oxidali on j - H'

S04-

fiN N :):

H

AN I )-OH H

U ric ac id

Scheme 1

sensitization of PDS by purine occurs in the second step. The increase in rates of oxidation of purines with

increase in [PDS] (Table 1) suggests that PDS is involved in the rate controlling step. The magnitude of the values of the rate of oxidation of caffeine, theobromine, theophylline and xanthine are found to be not very much different in spite of methyl group substitution at N I , No, and N7 in caffeine, theobromine and theophylline respectively indicating that the reaction site in all the cases might be the same which is not influenced by the methyl group substitution. This further indicates that N 1• No and N7 cannot be the possible sites of attack by S04°- . The rate of oxidation

of guanosine by S04°- was found to be very close to the purine systems in spite of D-ribose substitution at Ny indicating that Ny is not the site of attack of S04°- . From Table 1 it is clear that there is not much steric

influence of methyl groups present on adjacent carbon atoms of C4 and Cs excluding the possibility of attack of SO/- on C4 and Cs double bond. In view of the above discussions and experimental results , it is clear that C8 is probable site of attack of SOq"- . The mechanism of photooxidation taking xanthine as an example could be written as given in Scheme 1.

Acknowledgement The authors thank Prof P Jayaprakash Rao,

Osmania University for helpful discussions, MSS. is thankful to the CSIR, New Delhi , for awarding a fellowship .

References I KesaV3n PC & Powers E L, Illf j Radiar Bioi, 48 ( 1985) 223 . 2 Telo P J & Vi era A J S C, J chI'lli SoC, Perkill TrailS 2 ( 1997)

1755.

2100 INDIAN J CHEM, SEC A. OCTOBER 2002

3 Pullman B, J chelll Soc , ( 1959) 162 1.

4 Murg ida D H, Bilmes G M & Erra-Ba lse ll s R, Ph%chelll Pli%hio/ . 64 (1996) 777.

5 Kasama K. Takematsu A & Aral S, J pliys,Chelll , 86 (1982) 2420.

6 Santamaria J, Pasquier C, Ferradini & Pucheault J. Adv £.rp Med Bio/, 167A (1984) 185.

7 Vieira A J C S & Steenke n S, J chilli Ph."." 93 ( 1996) 235. 8 Vieira A J C S & Steenken S, J Alii chelll Soc, 11 2 ( 1990) 6986. 9 Vie ira A J C S, Calldeias L P & Steen ken S, J chilli Ph.".\'. 90

( 1993) 88 1.

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