Reactions of and e: Reactions of and

The second-order rate constants of the reaction of 'OH with these systems are of the order of 2-9 x lo9 dm3 mol-' s" at near neutral pH. The differenc...

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Chapter 4

Reactions of Hydroxyl Radical (OH) with Hydroxy and Methyl Substituted Pyrjmidiin e : Dehydration Reactions of Their OH-Adducts and End-Product Analysis

Abstract Reactions of hydroxyl radicals ('OH) with 4,6-dihydroxy-2-methyl pyrimidine

(DHMP), 2,4dimethyl-6-hydroxy pyrimidine (DMHP), 6-methyl uracil (MU) and 5,6dimethyl uracil (DMU) have been studied by pulse radiolysis and steady-state radiolysis

techniques at different pH values. The second-order rate constants of the reaction of 'OH with these systems are of the order of 2-9 x lo9 dm3 mol-' s" at near neutral pH.

The difference in the spectral features of the intermediates at near neutral pH and at higher pH (10.4) obtained with these pyrimidines are attributed to the deprotonation of

the OH-adducts. The G(TMPD") obtained at pH+, from the electron transfer reactions

of

the

oxidizing

intermediates with

the

reductant,

phenylenediamine (TMPD), are in the range 0.2-0.45

x

N,N,N',N1-tetramethyl-p-

lom7mol J-Iwhich constituted

about 3-7.5% oxidizing radicals. These yields were highly enhanced at pH 10.4 in the

case of DHMP, DMU and MU (G(TMPD") = 3.8-5.2 x

mol

J-I

= 66-89 % oxidizing

radical). Based on these results, it is proposed that a non-oxidizing C(6)-ylC(5)OH radical adduct is initially formed at pH 6 which is responsible for the observed transient spectra. The high yield of TMPD" at higher pH is explained in terms of a base

catalysed conversion (via a dehydration reaction) of the initially formed C(6)-ylC(5)OH adduct (non-oxidizing) to C(5)-ylC(6)OH adduct which is oxidizing in nature. G(-pyrimidines) and G(-F~(CN~%) were determined by steady-state radiolysis. Glycolic product peaks from MU and DMU showed a significant enhancement in presence of K3(Fe(CN)6)when anatysed by HPLC. Qualitative analyses of the products resulting from the OH-adducts of DHMP (at pH 4.5) and DMHP (at pH 6) were carried out using HPLC-ES-MS and a variety of products have been identified. Glycolic and dimeric products were observed as the major end-products. The product profiles of

both DHMP and DMHP have shown that the precursors of the products are mainly the C(6)-ylC(5)OH and the H-adduct radicals. The identified products are formed mainly by disproportionation and dimerisation reactions of these radicals. The mechanistic aspects are discussed.

--

Publications from this chapter: 1. T. L. Luke, T. A. Jacob, H.Mohan, H. Destaillats, V. M. Manoj, P. Manoj,

J. P. Mittal, M.R. Hoffmann and C.T. Aravindakumar, "Properties of the OH-Adducts of Hydroxy, Methyl, Methoxy and Amino Substituted Pyrimidines: Their Dehydration Reactions and End-Product Analysis", J. Phys. Chem. A, 200 1. (submitted)

Hecrctions ?f Hydroxyl Radical .... 1 I6

2. T.L. Luke, H. Mohan and C.T. Aravindakumar, "Reactions of Hydroxyl Radical with Methyl and Hydroxy Substituted Pyrimidines: A Pulse Radiolysis Study." Proceedings of the Forth Biennial Symposium on Radiation and Photochemistry (TSRP-981,Bombay, Jan 14- 19, 169, 1998.

3. T.L. Luke, T.A. Jacob, V.M. Manoj, C.T. Aravindakumar, H. Mohan, J.P. Mittal, H.Destaillats and M.R. Hoffmann, "Reaction of Hydroxyl Radical with 2,4-dimethyl-6-hydroxypynmidine: A Pulse and Product Analysis Study." Proceedings of the National Symposium on Radiation and Photochemistly, Roorky, Feb. 2 1-23,2,2001.

Reaciions of Hydroxvl Radicnl .... 1 I 7

Being the major DNA damaging agent, the reaction of hydroxyl radicals

('OH) with DNA model systems such as the nucteobases is the most widely pursued research area in the past many decades. 'OH reacts with both purines

and pyrimidines at almost diffusion controlled rate (>lo9 dm3 mol-I s-I).' It generally undergoes addition at C(4), C(5) and C(8) positions of the purine

ring. The resulting OH adducts in the case of 2'-deoxyguanosine were reported to have both oxidizing [G(4)OH) and reducing CG(5)OH and G{8)OH]

properties with respect to some known oxidants and redu~tants.~'~ A more or less similar distribution of both oxidizing and reducing OH adducts was reported in the case of adenine.' In purine nucleosides and nucleotides, 25% of

the products were reported to be formed by the primary attack of 'OH at the sugar moiety.4

In the case of pyrimidines, the main reaction is also the addition at C(5)-C(6) double bond while the double bond between N(3) and C(4) of

cytosine is a potential additional sitesm9The major OH radical adducts are, therefore, C(5)-ylC(6)-hydroxy and C(6)-ylC(5)-hydroxy radicals, The former

has

oxidizing

properties

with

respect

to

N,N,W,N1-tetramethyl-p-

phenylenediarnine (TMPD) and the latter has reducing properties with respect to tetranitromethane (TNM). The ratio of the oxidizing to reducing radicaIs depends on the nature of the substitumts present in the C(5) and C ( 6 ) position

of the pyrimidine ring whereas such substituent effect is not predominant with pyrimidines substituted at N(1)

The deprotonation of the OH adduct

is normally occurred at N(1) when there is a hydrogen at N(i) position. Based

Reuctions ofHydroxyl Radicnl ....1 18

on the redox properties of the radicals, it has been identified that the distribution of C(6)-yl radical in uracil is 80% while that of thymine, cytosine and

I-methyluracil are 56,87 and 65% respectively.8A third type of radical derived by a H-abstraction reaction from the methyl group of pyrimidine has also been reported

In the case of methyl substituted pyrimidines eg. thymine and

1-methyl uracil, in addition to C(5) and C(6)OH adducts, two types of methyl radicals may be formed by the abstraction of H from C-methyl and N-methyl

groups.9 An interesting property of the reducing radicals (C(6)-ylC(5)OH) of pyrimidines is their base catalysed dehydration reaction to yield oxidizing radicals (C(5)-ylC(6)OH) which can oxidize TMPD to TMPD*+.6 '7 In order to look at this important transformation reaction in basic pHs more closely, methyl

and hydroxy substituted pyrimidines were selected and investigated the effect of various substituents as well a s their positions in the pyrimidine ring on the

dehydration reaction. TMPD has been utilized to determine the ratio of oxidizing to non-oxidizing radicals and to demonsirate the transformation reaction. The selected pyrimidines include 4,6dihydroxy-2-methyl pyrimidine

(DHMP), 2 , 4 d i m e t h y l - y d x y pyrimidine (DMHP), 5,6-dimethyl uracil (DMU) and 6-methyl uracil (MU). Both kinetics of the reaction and the spectral nature of the intermediates were also investigated at different pH values.

One of the difficult areas in the field of free radical chemistry of

biomoIecules is the end product analyses. This is mainly due to low concentrations of the products (= 1

x

1 0 ' ~rnol

or less) and hence can not

be easily analysed using the normal analytical techniques. GC-MS analysis after rotary evaporation followed by sylilation of the dry products has made some

--

progress in this area.'%owever,

Reuctions af Hydroxyi Radical ... . 1 t 9

such studies are restricted only to few DNA

model systems. In the present study, we have used HPLC connected to Electrospray Mass Spectrometer (HPLC-ES-MS), and analysed the end products directly in aqueous medium resulting from the reaction of 'OH with

DHMP and DMHP. Some glycolic products are separately analysed by HPLC.

4.1

Pulse Radiolysis The bimolecular rate constants of the reaction of *OH with the selected

pyrimidines were found to be diffusion controlled (2-9 x 1 0 dm3 ~ mol'l s-I) at

near neutral pH and the values are tabulated in Table 4.1. These were

determined from the plot of the rate of formation of the intermediates (kbs) at their absorption maximum versus the concentration of the substrate. A typical build-up trace of the intermediate is shown in the inset of Figure 4.1. The hb, versus concentration plots gave straight line graphs with good correlation

coeficient (20.99) with all the compounds. A typical kobs versus concentration

plot obtained with DHMP at pH 10.4 is shown in Figure 4.1. The high rate constants obtained for these compounds are in good agreement with other pynmidines reported earlier.

Reactions of Hvdroxvl Hudiccrl .... I20

ldnd&

-1

Figure 4.1:

kOb,versus concentration plot at 290 nrn for DHMP saturated with N20at pH 10.4. Inset: A typical build-up trace obtained at 290 nm.

Table 4.1:

Second order rate constants and the A,,

of the transient

intermediates obtained for the reaction of *OHwith the selected pyrimidines.

Pyrimidine 4,6-dihydroxy-2-methyl pyrimidine (DHMP)

kz /

lo9

(dm3mo12'sA1) 5.6#, (3.7)"

PH 6

pH 10.4

kmX(-1

xmx(rim)

420

290

390

2,4-dimethyl-6-hydroxy pyrimidine (DMHP)

6-methyl uracil' (MU)

* from ref 15. The pseudo first order rate constants were measured at 435 nrn and 410 mn, respectively, for DMU and MU. # Measured at pH 4.5. $

", ,

Reactions of Hydrox-vi Kndicd ... . I 2 I

The transient spectra obtained with DHMP at pH 4.5 is characterised by its

single h;,at around 420 nrn and this undergoes a bimolecular decay. The spectrum obtained at 2 p after the pulse at higher pH ( 10.4) showed three absorption maxima at 290, 390 and 480 nm (Figure 4.2). The initial spectrum undergoes a very fast decay, with a slow build-up of absorption around 290-300 nm. These spectral

features are in agreement with an earlier report on the OH adduct of DHMP.

The spectrum with DMHP showed two absorption maxima at 325 and

450 rnn at pH 6 (Figure 4.3). The nature of the spectrum was similar at higher pH as well. In both cases the spectra were found to undergo a second order decay. The transient spectra obtained with MU showed two absorption maxima at 285 and 410 MI at pH 6. The b e resolved transient spectra obtained with hlLT at pH 10.4

showed two absorption maxima at 3 10 and 440 nm (Figure 4.4). These two peaks were slightly red shifted at higher pH compared to those at pH 6 . The initial spectrum was found to decay by first order kinetics (k3l0,

=

1.9 x lo6 5") and the spectrum

recorded at higher time scales (>40 p)has only a single A-

at 440 nrn.Similarly,

the transient spectrum obtained with DMU has two absorption maxima at 295 and

410 nrn at pH 6. At pH 10.4, it showed two absorption maxima at 3 10 and 425 nm (Figure 4.5). The spectra recorded at higher time scales (340 p) have only a single

& at 425 nm. An observable difference in the decay kinetics of the transient spectra h m both MU and DMU at pH 6 was their first order type decay at the lower

(285 and 295 nm respectively for MU and DMU). It is now known that the peaks

obtained at 285 and 295 nm are not from the OH adducts but from the ionised

form of MU and D M U . ' ~More explanations are given in discussion part.

Reactions of Hvdroxvi Radical ...,122

hl nm Figure 4.2: Transient absorption spectra obtained in NzO saturated solutions of DHMP (1 x mo1 dm-3) at 1 ps after the pulse at pH 4.5 (A), at 2 (a)and 90 ps (m) after the pulse at pH 10.5 (dose per pulse = 15 Gy).Inset: (a) intermediate trace obtained at 290 nm at pH 10.4 (b) dependence of absorbance of the intamdate on pH at 420 nm.

Figure 4.3:

Transient absorption spectra obtain& in NzO saturated solutions of 2,4-dimethyl-6-hydroxy pyrimidine (DMHP) ( 1 x 1oP3mol at 2 ps (0) after the pulse at pH 6 and 3 ps (A)after the pulse at pH 10.4 (dose per pulse = 15 Gy). Inset: 'The intermediate trace obtained at 330 nm at pH 10.4.

Figure 4.4: Transient absorption spectra obtained in N20saturated solutions of 6-methyl uracil (MU) ( I x lop3rnol dm") at 1.5 ps after the pulse at pH 6 (O), at 2 ps and 40 ps (A) at pH 10.4 (dose per pulse = 15 Gy) Inset: intermediate trace obtained at (a) 3 10 and (b) 440 nm at pH 10.4, (c) the TMPD" build-up at 565 nrn obtained with MU (2 x mol dm-3)in the presence of TMPD (5 x 1o-' mol dm-3) at pH 10.4; dose per pulse = 5 Gy.

(a)

Figure 4.5: Transient absorption spectra obtained in N 2 0 saturated solutions of 5,6-dimethyl uracil (DMU) (1 x rnol dm-3 at 1 ps (A) after the pulse at pH 6 , 3 (0)and 85 ps (+) after the pulse at pH 10.4 (dose per pulse = I 5 Gy). Inset: the TMPD" build-up at 565 nm obtained with DMU (2 x lo-' mol dm") in the presence of TMPD rnol dm-3);dose per pulse = 5 Gy. (5 x

Reuutions of Hydroxyl Rudicul

124

The reaction of 'OH was carried out at low doses in presence of TMPD

which is an effective reductant and can be oxidized to TMPD" by transferring one electron to an oxidizing intermediate6" The build-up of TMPD'' has been monitored with solutions contained 2 x 1 0-3rnol dm-3 pyrimidine and 5 x 10."

mol dm" TMPD at 565 nm. It is observed in the case of DHMP, MU and DMU that at near neutral pH, there is a feable absorption build-up of TMPD" whereas at higher pH, there is a strong and well defined build-up of TMPD'*. A typical trace at pH 4.5 and 10.4 in the case of DHMP is shown in Figure 4.6. The yields

of the r d c a l cation, G(TMPD"), were calculated for all these pyrimidines at low

w

i

m

50 ps I div

Tim

Figure 4.6: The TMPD" build-up at 565 nm obtained with DHMP (2 x in the presence of TMPD (5 x mol dm3)at i) pH 6 rnol and ii) pH 10.4; dose per pulse = 5 Gy.

pHs (4.5-6) and at higher pHs (-10.4), and are summarized in Table 4.2. The percentages of oxidizing radicals were thus calculated for all the pyrimidines

based on the maximum G(TMPD'+) as 5.7 x

mol J-' and are also tabulated

in Table 4.2. The G(TMPD**) values are almost the same at pH 6 and pH 1 0.4

for DMHP (0.3-0.9~10-'rnol P')(Table 4.2). However, in the case of DHMP,

Reactions of Hj{droxy[ Radical....125

DMU and MU, a much higher yield of TMPD'* (3.8-5.2

x

1o-? mol

J-I)

is

obtained at higher pH (Table 4.2). The TMPD" build-up at 565 nm obtained in

the case of MU and DMU are shown in the inset of Figures 4.4 and 4.5 respectively. 'OH generally undergoes addition reaction at C(5)-C(6)double bond of

the pyrimidines according to earlier reports.'-' The resulting transient spectra are characterised by their absorption maxima around 320 and 430 nm. Based on the

reactions of the OH adducts with TMPD a comparatively high yield of the reducing radicals (C(5)-C(6)yl radicals) were reported to be formed with a number of uracil deri~atives.~In agreement with these reports we propose a

similar 'OH addition at the C(5) and C(6) positions of the selected pyrimidines at pH 6 as well as at pH 10.4 (reaction 4.1). An important point which must be

noted at this stage is about the A,,,

in the case of DMU and MU at pH 6 . The

transient at the lower absorption maxima at 295 and 285 nm, respectively, from

DMU and MU were found to undergo a very fast decay. We have recently identified these peaks as due to the deprotonated DMU and MU (at N(1)) which result from the reaction of radiolytically formed OH- in N 2 0 saturated solution

even at this short time scale and the fast decay is only a (re)protonation.'2"3In the present case too, a similar deprotonated DMU and M U (at N(1)) are

reported to be formedi2and therefore, the peaks at 295 and 285 nm cannot be from the OH adducts. It must be noted that the deprotonation by radioIytically produced OH- would take place only with those pyrimidines having a hydrogen at

the N(1) position. This means that among the selected pyrimidines, only

Reactions of~vdroxylAudicul .... 126

DMU and MU show this phenomenon. However, the absorbance due to the deprotonated DMU and MU at pH 10.4 will not appear as these compounds are

already in the deprotonated form (pKs are 9.8 and 9.5 for DMU and MU, respectively) and therefore any ground state absorption will be taken care by the optical detection set-up. The phenomenon of deprotonation and their fast

(re)protonation of pyrimidines with hydrogen substituted at N(1) is well documented. l2

Table 4.2: The G(TMPD)) ( x 10-'rno11-') values and the percentage of oxidizing radical obtained for the selected pyrimidines at pH 6 and 10.4.

Pyrimidine

Percentage of oxidizing radicals at pH*

G(TMPD*+)at pH

..

-

DHMP

0.44'

5.18

7.5

89.3

DMHP

0.37

0.32

6.3

5.5

DMU

0.34

3.83

5.9

66.0

MU

0.19

4.76

3.2

82.0

* Percent~gesare calcuiated based on a maximum yield of the intermediates, G(intermediates) -- 5.7x 10.' mo1 J-I . ' at pH 4.5. The G(TMPD'*) values at near neutral pH, as given in Table 4.2, show a

clear account of the yield of oxidizing radicals. At pH 6 the G(TMPD'+) values obtained for DMHP, DMU and MU are 0.37

and 0.19

x 1

x

o - rnol ~ J-' respectively and 0.44

loL7mol x

PI,

0.34

x

moI J-'

lo-' mol I-' for DHMP at pH

4.5.Such low yield of oxidizing radicals is in agreement with the earlier reports on the OH adducts of thymine and uracil

The oxidizing property

of the C(5)-ylC(6)OH radical can be understood as its mesomeric structure is

either an oxygen centered radical (at C(4)-0) or a nitrogen centered (at N(3)).

Reactions rfHvdruxy1 Radicul ...,127 -

Therefore, this gives an additional support for the assignment of the intermediate

spectra to the formation of C(6)-ylC(5)OH adduct (see reaction 4. I) in the case of all the selected pyrimidines which could act as a reducing radical as was

reported in the case of uracil and thymine.8 Furthennore, there can be a possibility for H-abstraction reaction from the methyl group of these compounds leading to ally1 type radicals as reported in the case of thymine.* However, these radicals cannot be reducing unlike thymine. In the case of thymine, the abstraction of H from the methyl group leads to the formation of

ally1 type radicals. This is transformed into its mesomeric form, C(6)-yI radical, and it is reducing in nature with respect to tetranitromethane (TNM). The transient absorption spectra at higher pHs (- 1 0.4) were different

from that at lower pHs either by their higher absorption coefficient or by a shift in their absorption maxima (including additional peaks like in the case of

DHMP). Such differences in the case of uracil, thymine and cytosine were explained in terms of the formation of deprotonated OH adducts at higher p ~ s . 5 7 In ' i the present cases too, such an interpretation is logical. The absorbance versus pH plot in the case of DHMP (Figure 4.2 (b)) gave a clear pK

Reactions oftiydrvqd Radicul .... 128

type curve with inflection point around pH 6.2. DHMP has pK, values as 0.21,

6.35 and 12.9. Therefore, it can be concluded that the species existing at pH 10.4 is the deprotonated form of the OH adduct of DHMP. The decrease in the

absorbance value at pH>11 (Figure 4.2) is attributed to the lose of 'OH as 'OH

0'- + H+(see reaction 1.30). Based on these observations and

on previous reports,'*" the spectral differences at lower and higher pHs, are

attributed to the protonation-deprotonation reaction of the OH adducts. However, in the case of DMHP, there was no observable difference in the absorption spectra at pH 6 as well as at 10.5 (Figure 4.3) and hence it must be assumed that the pK, value of the OH adduct is higher than pH 10.4 and that the spectrum existing at both the pHs are the neutral OH adduct of DMHP.

As shown in Table 4.2, the percentage of oxidizing radicals calculated

from the G(TMPD'+) values were almost constant for DMHP at lower and higher pH values, but for DHMP, DMU and MU the calculated percentages of oxidizing radicals were 89, 66 and 82% respectively at higher pH. The high

difference in the G(TMPD") value at lower and higher pHs gives an indication of the change in the oxidizing property of the intermediate radicals. A two component build-up of TMPD''

absorption has been reported with uracil at

pH>9 where the initial component depended on the concentration of TMPD.'

The second component, on the other hand, was dependent only on the pH (and not on the concentration) indicating a slow transformation (in the pulse radiolysis scale) of the initial non-oxidizing species into an oxidizing species.

Therefore, in the present case, we have monitored the build-up of TMPD*' at a

higher time scale with the pyrimidines at pH

- 10.5 and the G(TMPD")

values

are presented in Table 4.2. me mechanism of the change in the oxidizing properties which are observed in the case of DHMP, DMU and MU are

proposed on the basis of the conversion of the initially formed (deprotonated) OH adduct (C(6)-ylC(5)OH) to C(5)-ylC(6)OH at higher pH values. In the case

of cytosine, this occurs by the dehydration of C(5)OH adducts (reducing in nature) which ultimately leads to the formation of a radical site at C(5) or

oxygen at C(2) p~sition."~Therefore, a similar conversion of the initially formed C(6)-ylC(5)OH to an oxidizing radical at basic pHs is proposed as

shown in the case of DHMP (scheme 4.1).

Scheme4.1: Proposed mechanism of the conversion of (deprotonated) C(6)-ylC(5)OH to C(5)-ylC(6)OH at higher pH. A similar type of dehydration reaction of the C(6)-ylC(5)OH radical in

basic medium is proposed for DMU and MU. In the case of DHMP, EPR evidence is available for the formation of an oxyradical at basic p ~ s 'and l this assignment is in good agreement with our observation of a high G(TMPD*+)

-

--&

Kecrc!ions of Hydroxyl Rudiuul.. .. 1 30

value at pH 10.4 (Table 4.2). The relatively low value of G(TMPD") obtained

with DMU could be understood based on the possible formation of two kinds of ally1 type radicals (A and B) from a H-abstraction from the methyl groups. It

could be probable that the C(5)-yl radical (A) is formed in high yield compared to C(6)-yl radical (B). The radical A could be reducing, but cannot be

transformed into an oxidizing radical. The distribution of these radicals, on the other hand, cannot be determined using the common oxidants or reductants as

both the C(6)-ylC(5)OH and the ally1 type radical A (due to its rnesomeric form A') can be reducing in nature while C(5)-ylC(6)OH and the ally1 radical B (due to its mesomeric form B') can be oxidizing in nature. The redox properties of

similar radicals such as from thymine and I-methyl uracil are already well d~cumented.~ N-methyl centered and C-methyl centered radicals are proposed for 1 -methyl uracil and thymine respectively and these are reducing in nature.

Reactions ofHydroxyl Radical .... 13 I

The fast first-order decay of the transients at higher pHs is, therefore,

proposed as the dehydration of the initially formed reducing radicals in the case of DHMP, DMU and MU. It is also important to note that such transformation

reactions have been observed only with DHMP, DMU and MU and not with

DMHP.The major structural difference of these compounds with DMHP is that these contain either a keto or hydroxy group at the C(4) of the pyrimidine ring. It is therefore obvious that the chemical environment in the case of DMHP is not conduciQe for an O H elimination reaction. Some additiona1 spectral features obtained with DHMP, DMU and MU at high pHs further gave some

indication of the optical absorption spectra of the oxidizing radicals. In the case

of DHMP the initial spectra had undergone a fast decay at the absorption maxima 290,390 and 480 nm, the later spectrum (90 ps after the pulse) appears to have a single prominent peak at 300 nm. The absorption trace at 290 nm (in the inset of Figure 4.2) has shown a slow build-up. This trace was found to

undergo a second-order decay after about 500 ps (data not shown). Therefore, it is likely that the radical which is formed after the dehydration reaction of

C(6)-ylC(5)OH of DHMP, contribute to the absorption build-up around 300 nm. Such a difference in the time resolved absorption spectra in the case of both MU

and DMU was also prominent as shown in Figure 4.4 and 4.5. The initial

spectra were found to have two absorption maxima where the later spectra (85 and 40 ps, respectively, for MU and DMU after the pulse) have only a single A,,

which undergo a bimolecular decay. This spectral features also give

some indication of the contribution of absorption from the oxidizing radicals of DMU and MU.

---

.-

Reactions ofHydroxy1 Rudiucll ... . I 32 -

4.2

Steady-State Radiolysis

4,2.1

Degradation ofthe Pyrimidines Induced by 'OH The degradation of the parent pyrimidines induced by 'OH at near

neutral pH is monitord using UV-VTS spectrometry after irradiating N 2 0 saturated pyrimidines (1 x I o - rnol ~ dm-') at different doses. Absorbance of the

irradiated sample is noted at 255,230,260 and 267 nrn for DHMP, DMHP, MU and DMU respectively. A typical degradation profile in the case of DMHP is

shown in Figure 4.7. From the plot of absorbance versus dose the G(-S) was calculated and these values are given in Table 4.3. However these values are

much lower than the expected values as the G('0H) is 5.7 x

rnmol

s'. This

could be due to the fact that even the products formed from the reaction of 'OH may also absorb at the UV region and hence the calculated G(-S) may widely be

different from the actual G(-S).

Table 4.3:

G(-S) (x 10.' mol J-') and G ( - F ~ ( c N ) ~ )( ~ xmol J - I ) obtained from the reaction of 'OH with DHMP, DMHP, DMU and MU (1 x 10" mol dmm3). The G(-F~(cN)~)~is determined f h m the pyrimidine solutions containing K3(Fe(CN)6)( I x 10" rnol dm'3).

Pyrimidine

PH

G(-S)

G(-Fe(CN)6)

DHMP

4.5

2.0

6.2

DMHP

6.0

2.0

5.1

DMU

6.0

3.1

5.0

MU

6.0

2.8

4.6

Reuclions of Hydroxyl Radical .... 133

-

Figure 4.7: The overlay spectra obtained from UV-VIS spectrophotometer for the degradation of 1 0 ' ~ rnol dm" DMHP induced by 'OH using 60~o-ysource at pH 6; A-F corresponds to O,O.5, 1, 1.5,2 and 2.5 kGy respectively. 4.2.2

Reaction of K3(Fe(CN)6) with the OH-Adducts The above reaction is carried out in the presence of a strong oxidant,

potassium ferricyanide ( 1

x 1 o4

mol dm-'). The decrease in absorbance of

F ~ ( c N } ~at-420 m is monitored by W-VIS specropphotometry. G(-F~(CN)6'1is calculated from the pIot of dose versus absorbance (Figure 4.8) and the values are given in Table 4.3. K3(Fe(CN)6) is a strong oxidant which can abstract electron from any reducing carbon centered radicals from purines and

The observed high G(-F~(cN)?-) is, therefore, attributed to the

reaction of F ~ ( c N ) ~with ~ - the carbon centered reducing radicals. It must be noted that under the present experimental conditions not only OH adducts, but

H adducts can also be formed. In the case of DHMP, the reaction of H atom gives rise to a C(S)HC(S)-yl radical which is a reducing radical as described in

Reactions of ff vdr-axy( Radica[.... I34

-.

chapter 3 (see section 3.3). This means that in addition to the formation of reducing

radicals from the 'OH attack which makes up to about 90% (an assumed yeld equal to the non-oxidizing radical, see Table 4.2), the reducing radicals fiom H-attack also contribute to the reaction with F ~ ( c N ) ~This - . meam that the total G-value of the

reducing radicals would be about 5.8 x lom7 mol

the OH adduct and 0.6

x

J-I

( i.e.. 5.2 x 1o-?rnol 5-' from

10'~ mol J'' from H adducts). However, a slightly

higher value of 6.2 x lom7 m01 J" (see Table 4.3) has been obtained for DHMP. This means that a part of the carbon centered oxidizing radicals (with respect of the reductant TMPD) may also undergoes electron transfer reaction with

F ~ ( c N ) ~Being -. a very powerful oxidant such a reaction with F ~ ( c N ) ~is- not

unlikely. A report by steenken3also points to this possibility.

50

100

ISO

Dose I Gy

Figure 4.8:

Dose dependent decay (F~(cN)~)'-at 420nm obtained in N 2 0 saturated solution of DMHP (1 x mol dm'3) in presence of K3(Fe(CN)6)( I x 1o ' ~m01 dm-3)

Reactions of Hydrox.yl Radical.. .. I 3 5

On the other hand, the G(-F~(cN)~~') obtained with DMHP, DMU and

MU

are relatively lower compared to DHMP. It is known in the case of uracil.

that the H-adducts are not reducing in nature as the radical site is at C(5) position.14Based on this report as well as h-om the interpretation of the H adducts in chapter 3 (see section 3.31, the H adducts of DMU and MU appears

not to contribute to the decay of F ~ ( c N ) ~Based -. on the results with TMPD (see Table 4.2) a G ( - F ~ ( c N )=~5.2 ~ ~x lo-' mol P' is expected in these cases.

The observed values are consistent with this explanation though these are slightly less than the expected. However the value obtained with DMHP ( G ( - F ~ ( c N ) ~ ~=-5.1 ) x 100' mol

J-I)

is relatively lower as in this case a part of

the H-adduct may also contribute to the decay of F ~ ( c N ) ~This ~ - . discrepancy

could not be understood at this stage. 4.2.3

Formation of Glycolic products from the OH adducts of M U and

DMU In the reactions of 'OH with pyrimidines, glycols and pyrimidine dimers are

reported as two important products.'0 Glycols are f o d with G value 0.9 x l~.?rnol

J' in the case of 1,3dimethyl uracil.l o HPLC analysis has been carried out with

M U and Dh4U in search of glycol in presence of potassium ferricyanide. Triply distilled water was used as an eluent and the glycol was monitored at 210 nm. Those peaks which showed a significant enhancement in presence of F ~ ( c N ) ~ ~ -

is assumed as the glycolic peak and its approximate concentration is determined using dihydrouracil as the standard8 Dihydrouracil was used as a standard for

MU and DMU glycol assuming that the peak area would not be very different from the glycol. Such an assumption has been made by Jovanovic and sirnic8 in

500

0

1000

I500

2000

Dose I Gy

Figure 4.9: Linear dependence of the formation of glycol h m MU (1 0" mol dmm3, N20saturated) on different doses in the presence (A) and in the absence (e) of potassium ferricyanide (lo4 mol dm").

which glycolic products from thymine, uracil and 1-methyl uracil were identified. The area of the glycol peak should be enhanced in presence of K3(-Fe(CN)6)consistent with the fact that the OH adduct (reducing in nature) reacts with F ~ ( c N ) ~followed ~by water addition leading to the formation of glycol as shown in scheme 4.2. Based on these measurements the G(g1ycol)

obtained for M U is 0.36

x

10.' mol J-' and 0.68 x lo-' rnol J" in the absence

and in the presence of ferricyanide and that for DMU is 0.15 x 1o - mol ~ J-' and 0.24 x

mol P' . Concentration of glycol from MU versus dose is plotted in

the presence and in the absence of ferricyanide (Figure 4.9) and the above G-values were determined from these plots.

Reactions oflfvdroxyl Radical.... I 37

Scheme 4.2:

4,2.4

Formation of glycol from &methyl uracil in the presence of potassium ferricyanide.

Analysis of the end products from the OH adducts of DHMP and DMHP using HPLC-ES-MS

Qualitative HPLC-ES-MS analysis of N 2 0 saturated solution of DHMP (pH-4.5) DMHP IpH-6) after y-irradiation gave a large number of mass peaks

with varying mlz values. The electrospray method does not provide a good

estimation of the relative yields for the various products observed, based on the intensity of the mass signals detected. The sensitivity towards ionization in the

spray may be intrinsicaIly very different for each analyte, and is also strongly

affected by the matrix. Therefore, no attempt is made to estimate the relative yield of the products. Among the observed mass peaks a number of m/z values were selected for the most probable products (protonated or deprotonated under

mild ionization conditions). Representative HPLC chromatogram and mass spectra are given in Figures 4.1 0 - 4.1 2. The identified products along with their

Reactions ofHydroxxvl Radical .... I 3 8

m/z values are summarized in Table 4.4 and 4.5. These products are deduced by investigating the MS data in the entire region of the chromatogram. The

possible mechanism of the product formation resulting from the proposed radical intermediates from DHMP and DMHP is shown in schemes 4.3 and 4.4,

respectively. Table 4.4:

Products identified from the reaction of 'OH with DHMP using HPLC-ES-MS anaIysis at pH 4.5. Products

Molecular Formula

Molecular weight

mlz

250

+250

142

Not detected

128

4-129

1 26

-I25,+127

254

-254

2 18

-217

Renetions of H~~droxyl Radical ... . I 39

mol dm-3 DHMP saturated Figure 4.10: HPLC chromatogram obtained for lom3 with N20 irradiated for 2 kGy by mCby-sourceat pH 4.5. C-I 8 column is used and the eluent is water (flow rate = 0.5 mllmin).

As discussed in section 4.2, C(5)OH adduct is the major radical

intermediate in the case of DHMP and DMHP at near neutral pH. Therefore, the products are proposed to result from the disproportionation and dimerisation reactions of these radicals. Although, the presence of C(6)OH adduct is evidenced by the formation of TMPD*' (see section 4.2) no products which has a likely precursor as C(6)OH were obtained from the product profile. This could

be due to the very low yield of C(6)OH adduct as evidenced by the low yield of TMPD" (see section 4.1). On the other hand, the product mass peaks clearly gave evidence for the formation of products resulting from the H-adducts of

both DHMP and DMHP (schemes 4.3 and 4.4).

Reuctions of Hydroxyl Radicul .... 140

m.. -m

-: 3I 3 I I! .b

I7 m

f I i 1 g1 1 2I 31 31 181 . I I

-

3 Gi ~s s, ~ 1

&

~ l l d-

Figure 4.11: Typical ES-MS spectra obtained from a chromatographic peak with retention time (RT = 3.6 min, Eluent: water) from the gamma mdiolysis of DHMP ( 1 mol dm-3) saturated with N20 at pH 4.5 (A): positive ionisation mode; (B) negative ionisation mode, The signals at d z -1 25 and + 127 are attributed to 6-hydroxy-2-methyl-(3H)-pyrimidin-4-one(DHMP), miz = -2 17 to bis(2-methyl-(3H)-pyrimidin-4-one-6-y1)(the product k) and m/z = + I 29 to 5,5-dihydm-6-hydro-6-hydroxy-2-methyl-(3H)pyrimidin-4-one (the product h). The strong signal at m/z = -97 corresponds to HS04-used in the buffer. The products included in Table 4.4 were obtained by scanning the entire region of the HPLC-chromatogram.

Reactions of ffydr-uxj~lRcrdiccd .... l41

d

I f OH

DHMP

i

I1

Scheme 43: Proposed mechanism of the reaction of 'OH with DHMP at pH 4.5.

A part of the identified products, summarised in Table 4.4 in the case of

DHMP, are proposed to be mainly resulted from the disproportionation reaction

of radicals I and 11 (as shown in scheme 4.3). From the quantitative analysis of the products in the case of 1,3-dimethyl uracil (1,3-DMU) using GC-MS it is

known that the major products resulting from C(5)OH adducts are the glycol and the dimmer." A detailed description of the formation of glycolic products

from the reaction of 'OH in the radiolysis as well as in the fenton reaction uf 1,3-DMU using GC-MS has been reported recent~~.~"he HPLC-ES-MS data

Re~ru~ions of HydrmyI Radical .... 1 42

in the present case shows a similar trend. However, the immediate product afier

the disproportionation (reaction 4.9) of I is the 5,6-dihydro-5,6-dihydroxy-2methyl-(3H)-pyrimidin-4-one (a) and 5,6-dihydroxy-2-methyl-(3H)-pyrimidin4-one (b). The pyrimidine glycols are known to be acid-labile and therefore, can

undergo a water elimination as shown in reaction 4.10. The product c, therefore, is a result of a dehydration reaction of a. Another important reaction that normally occurs with the OH-adducts of pyrimidine is the dimerisation

reaction.10,'50 Reaction 4.11 leads to a dirner of the type d and the subsequent water elimination reaction leads to the products e and f as shown in reaction 4.12 and 4.13. The formation of e and f through water elimination reactions is

' ~ the OH adduct of uracil supported by the report by K.M. Idriss ~ l i where

undergoes a similar water elimination reaction. A probable product with the structure g is also proposed, resulting from the dehydration of a trihydroxy derivative, as shown in reaction 4.14 and 4.15. This product must have a m/z

value of 142 and the mass peaks the rn/z values should be -141 and +143. However, the product b (from reaction 4.9) has also similar m/z values and no additional peak corresponding to this product was observed in the

chromatogram. Therefore, the existence of g could not be confirmed. An alternative mechanism for the formation of the product g could be the reaction of radiolytically formed Hz02 which acts as an oxidizing agent a s shown in reaction 4.19 and forms an unstable trihydroxy substituted derivative. The immediate water elimination reaction might lead to a product like g (reaction 4.20).

Reactions of Hyd-o,ryl Radical .... 143

Other identified products include m/z values corresponding to h, i, j and k which can be explained only through an initial attack of H' with DHMP. It is also formed in the reaction mixture with a G value of 0.6 x

mol J-' under

the reaction conditions that we used. The most likely mechanism for the

formation of the product h is the disproportionation of the H-adduct radical, 11. The attack of H ' at the C(5)-C(6) doubIe bond of pyrimidines, a reaction similar to 'OH, is well under~tood.~' In a recent study, it is demonstrated that C(6)-

ylC(5)H radical is the major H-adduct (11) in the case of DHMP.'~It may be further noted that the product i is the starting compound, DHMP, but is

considered as the product

partner of h from the disproportionation

reaction. The dimerisation of the radical I i may lead to the product j. Although a single water elimination is possible from j, m/z value corresponds to only k

was observed in the mass peak. On the other hand, our assignment of the product k can be rationalised based on the report on the water elimination from a dimeric hydroxy methyl radical resulted from the OH attack on methyl uraciI

where such water eliminated products were identified using chromatographic techniques. l9 It must be further noted that though a substantial details of 'OH

reaction products is available in the literature, practically little information is available on the end products from the reaction of H' with pyrimidines.

Renciions of ff vdroxyl Radical .... 144

The product profile obtained with DMHP shows a similar reaction route as in the case of DHMP (Table 4.5). The identified products are mainly resulted

from the disproportionation of radical 111 and IV (as shown in scheme 4.4).

Radical I11 undergoes dispropotionation to form 5,6-dihydro-$6-dihydroxy-

2,4-dimethyl pyrimidine (I) and 5,6-dihydroxy-2,4-dimethylpyrimidine (m). Table 4.5:

Products identified from the reaction of 'OH with DMHP using HPLC-ES-MS analysis at pH 6. Molecular Formula

Products --

Molecular Weight

mlz

--

5,6-dihydm-5,6-dihydroxy-2,4dimethyl pyrimidine

5,6-dihydroxy-2,4-dimethyl pyrimidine

5-hydroxy-2,4-dimethyl pyrimidine Bis(5-hydro-5,6-dihydro-2,4dimethyl pyrimidine-6-yl) 5-hydroxy-2,4-dimethyl-6-(5hydro-$6-dihydroxy-2,4dimethyl pyrimidine) pyrimidine Bis(5-hydroxy 2,4-dimethyl pyrimidine-6-yl)

246

-245

140

Not detected

5,5-dihydro-6-hydro-6-hydroxy2,4-dimethyl pyrimidine

126

-125, +126

6-hydroxy-2,4-dimethyl pyrimidine

124

-123, +I25

Bis(5,5-dihydro-6-hydroxy-2,4dimethyl pyrimidine-6-yl)

250

+250

214

-213, +215

-.

-.

Reactiorzs qf Hvdt+vxy/Rndical.. .. 145

Figure 4.12: Typical ES-MS spectra obtained from a chromatographic peak obtained for the radiolysis of DMHP (10'~ moI dm'3) saturated with N 2 0 at pH 6 (eluent: water). (A) positive ionisation mode, The signal at d z = + 125 is assigned to 6-hydroxy-2,4-dimethyl pyrimidine (DHMP) and m/z = +283 to bis(5-hydro-5,6-dihydro2,4-dimethyl pyrimidine-6yl) (the product o); (B): negative ionisation mode, retention time RT = 1.2 min. The signal at m/z = - 125 is assigned to 5,5-dihydro-6-hydro-6-hydroxy-2, 4-dimethyl pyrimidine (the product s). The strong signal with m/z = -97 corresponds to HS04 used in the buffer.

Reactium qf fvdrox~vlRadical ...,146 -

-- .

OH

t

S

OH

pr!,

CH,

'CH, U

V

Scheme 4.4: Proposed mechanism of the reaction of 'OH with DMHP at pH 6 . The product, n is the result of dehydration of 1 (reaction 4.24). Reaction 4.25 leads to the dimmer o and the subsequent water elimination reaction leads to the product p and q as shown in reaction 4.26 and 4.27. A product with the

structure r is also speculated (not detected) as a result of the dehydration of a trihydroxy derivative as shown in reactions 4.28 and 4.29.

Reactions of Hydrox,vl Radical.... I 47

Radical IV is proposed to be formed by the initial attack of 14' with

DMHP. S,S-dihydr0-6-hydro-dhydroxy-2,Cdimethyl pyrimidine

(s)

and

6-hydroxy-2,4-dimethyl pyrimidine (t) are formed by the disproportionation of

the radical IV (reaction 4-30). t is expected to have m/z of 124 and the mass peaks showed m/z values as -123 and +125. t is the DMHP itself. The

mechanism of their formation of the dimers o, p, q, u and v from the C(5)OH adduct and from the H-adducts are presented in scheme 4.4.

4.3

Conclusion The addition of 'OH to the 5,6-double bond of pyrimidines gives rise to

a reducing radical (C(5)OHC(6)-yl) and an oxidizing radical (C(6)OHC(5)-yl).

The percentage of oxidizing radicals increases considerably at higher pH for DHMP, DMU and MU.The percentage of oxidizing radicals obtained are 89, 66 and 82% for DHMP, DMU and MU respectively at pH 10.4. This is

explained based on a transformation of non-oxidizing radical to oxidizing radical via, a dehydration reaction of the deprotonated C(6)-yl radical. A variety

of new stable products have been identified using HPLC-ES-MS analysis and a detailed degradation pathway is proposed. The product analysis from DHMP and DMHP gave indications that these products mainly arise from the

dispropotionation and dimerisation of the initialIy formed C(5)OH adduct as well as the H-adducts. To our knowledge, the identification of the products resulted from the H-adducts in NzO saturated aqueQus solutions, is the first report of this kind.

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S. Steenken, Chem. Rev., 89,503, 1989.

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E. Hayon and M.G. Simic, J. Am. Chem. Soc., 95, 1029, 1973.

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D.K. Hazara and S. Steenken, J. Am. Chem. Soc., 105,4380, 1983.

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S. Fujita and S. Steenken, J; Am. Chem. Soc., 103,2540, 198 1.

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G.A. Infante, P. Jirathana, E.J. Fendler and J.H. Fendler, J. Chem. Soc. Furaday Tram. 1, 70(7), 1171, 1974.

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T.A. Jacob, C.T. Aravindakumar, R. Flyunt, J. von Sonntag and C, von Sonntag, J. Am. Chem. Soc., 123,9007,2001.

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D.J. Deeble, S. Das and C . von Sonntag, J. Phys. Chem., 89, 5784, 1985.

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