Kinetic and Spectral Investigation of the Electron and Hydrogen Adducts of

Dihydroxy and Dimethyl Substituted Pyrimidines: A Pulse Rsdiolysis

and Product Analysis Study

Kinelic and Spectral Investigation ... .70

Abstract

The reactions of hydrated electron (eAa,) and hydrogen atom (H') with 4,6-

dihydroxy-2-methyl pyrimidine (DHMP), 2,4-dirnethyl-6-hydroxy pyrimidine (DMHP),

5,6dimethyl uracil (DMU) and 6-methyl uracil (MU) have been studied at different pH

values using pulse radiolysis technique. The second order rate constants obtained for

the reaction of eL,, with these systems are in the range 5-10 x 10' dm3 rnolL1 s" at near

neutral pH. At basic pHs, the rate constant values were considerably reduced due to

the electrostatic effect between e,, and pyrimidine anion. The transient absorption

spectra of the electron adducts of DHMP, DMHP, DMU and MU have distinct

absorption maxima at around 300-320 nm. The decay pattern of the electron adduct

spectrum of DHMP was different from the other three pyrimidines. The initial spectrum

at pH 4.5 was found to undergo a first-order transformation and to form a dfferent

spectrum after about 40 ps with a maximum around 380 nm and a broad absorption

between 490 and 520 nm. Based on the spectral characteristics and the yields of

methyl viologen radical cation (MV') resulted from the electmn transfer reaction

between the electron adducts and MV~', it is proposed that a protonated (at oxygen)

electron adduct of DHMP is initially formed at pH 4.5. This undergoes a proton

catalysed transformation at lower pH and forms a reducing C(6)-ylC(5)l-I radical. A

phosphate catalysed protonation at C(5) also yields similar radical. Such preferential

protonation at C(5) is more predominant only with dihydroxy pyrimidine systems. At pH

9 and 13, formation of a radical monoanion of DHMP (pK, 213) is proposed. The

possible attack of e-, is proposed to be at the N(1) or N(3) of DMHP. The resulting

electron adduct has a pK, value around 6.0. Similar properties for the electron adducts

of DMU and MU (electron attack at O(4)) are proposed. The phosphate induced

transformation of the initial electron adduct were not observed in presence of 0.3 mol

dm" phosphate with DMHP, DMU and MU (i.e, k < lo4 s-'1. The second order rate

constants for H' with DHMP, DMHP, DMU and MU were in the range 1-20 x 1 o8 dm3

rnol" i'. The rate constant values were affected by the pK, values of the parent

pyrimidines as H' attack is electrophilic in nature. The hydrogen adduct spectra were

generally identified as their absorption maxima at 310-380 nm and at 460-510 nrn.

Formation of C(6)-ylC(5)H adduct radical, the same radical that formed after the H+ and

phosphate catalysed transformation of the electron adduct, is proposed for DHMP. The

possibility of the formation of C(5)-yl and C(6)-yl H adducts of DMHP, DMU and MU are

discussed.

High pressure liquid chromatography connected with electrospray mass

spectrometer (HPLC-ES-MS) has been used to analyse the products obtained from the

Kinetic and Sweciral Investination ... .7 1

reaction of e-,, with DHMP and DMHP and the results revealed that the products are

mainly derived from the C(6)-ylC(5)H radicals. These products are proposed to be

formed from the disproportionation and dirnerisation of the C(6)-yl radicals. A possible

reaction mechanism is proposed for the product formation.

G(-Substrate) and G(U) for the electron reaction were calculated from the

sample of aqueous solution of selected pyrimidines irradiated using 6D~o-y-source by

the analysis using high pressure liquid chromatography (HPLC).

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

M.R. Hoffmann and C.T. Aravindakumar, "Kinetic and Spectral Investigation of the Electron and Hydrogen Adducts of Dihydroxy and Dimethyl Substituted Pyrimidines: A Pube Radiolysis and Product Analysis Study." J. Phys. Org. Chem., 2001 (submitted).

2. T.L. Luke, H. Mohan and C.T. Aravindakumar, "Kinetic and Spectral Investigation of the Electron Adducts of Dimethyl and Dihydroxy Substituted Pyrimidines." Proceedings of the National $mposium on Radiation and Photochemistry, Vishakhapatanam, June 4 - 8, f 997.

Kinetic and Spectral Investigution ... .72

As outlined in chapter I , radiation chemical studies of DNA model

systems such as nucleobases and nucleosides are important in understanding the

chemical basis of radiation induced lesions in DNA. Several reports'-7 on the

radiation chemical studies of purines and pyrimidines are available and most of

these studies are directed to the understanding of the reactions of water derived

free radicals with DNA model systems. Such studies are carried out in dilute

aqueous solutions where the radiolysis of water provides the primary free

radicals (reaction 1 .19).

The reactions of hydroxyl radical ('OH) and hydrated electron (c,) with

purines, pyrimidines and their nucleosides are often very fast and are diffusion

control~ed.~ The fornation of electron adducts and their fast protonation (k t 1 0' $ I )

at heteroatoms in solution state are reported with purine bases (e.g. adenine, guanine

and hypoxanthine).9-1 The canversion of the initially formed heteroatorn

protonated electron adducts into carbon protonated electron adducts is predominant

(at least in the pulse radiolysis scale) only in purine nucleo~ides.~~-'~ These

transformation reactions are catalysed by both OH- and phosphate buffer.

However, the rate of protonation at the carbon site of various nucleosides were

different, owing to the substituent effect. Both kinetic and thermodynamic

factors were known to control this transformation phenomenon as can be seen in

severd recent report^.'^-'^

On the other hand, the protonation of the initially formed electron

adducts of pyrimidines occurs at different rates in solution state. For example,

protonation of the electron adduct of cytosine by water is reported to be

Kinetic and Spectral Investigation .. . .73

6 - 1 14 considerably fast (k-3.5 x 10 s ) but that of uracil derivatives is relatively

S ~ O W (k < 5 lo5 s - ~ ).l5-I7 In the case of uracil and its methyl substituted

derivatives, the conversion of the oxygen protonated electron adduct to carbon

protonated electron adduct (C(6) protonation) can be catalysed by phosphate

buffer (scheme 3.1). But the protonation at C(6) was observed to depend on the

site of methyl substitution in the pyrimidine ring and the rate constants varied

from 1.6 x 1 o7 dm3 mol-I i' for 1,3-dimethyl thymine to < 10' dm3 mol" i' for

6-methyl. uracil. The oxygen protonated and the carbon protonated radicals

can be differentiated from their redox behaviour using the oxidants methyl

viologen (MV~'), p-nitroacetophenone (pNAP) or tetranitromethane (TNM).I7

Scheme 3.1: Protonation of pyrimidine electron adduct at oxygen and carbon sites by water and phosphate.

Although both C(5) and C(6) are potential sites of protonation, it is generally

observed that C(6) protonation is more favoured in the case of thymine, uracil and

cytosine." On the other hand, C(5) protonation catalysed by H+, phosphate and OK,

is reported to be more favoured in the case of 4,6dihydroxy pyrimidine, using an

esr study in liquid state." It is important to note that the preferential protonation

Kinetic and Spectral Investigation ... .74

at C(5) catalysed by H', phosphate or OH- is not yet clearly established by

pulse radiolysis studies of any pyrimidine derivatives. Therefore, we have

selected methyl substituted dihydroxy pyrimidine, an isomer of cytosine, along

with dimethyl mono hydroxy pyrimidine, dimethyl uracil and methyl uracil, and

studied the possibility of transformation reactions at different concentrations of

H', OH- and phosphate. The effect of substituents as well as their position in

the pyrimidine ring on the transformation reaction was investigated.

Furthermore, it will be shown that 4,6-dihydroxy-2-methyl pyrimidine is an

ideal system to demonstrate the preferential protonation at C(5) position using

the oxidant, methyl viologen (MV~'), which is not a major process with the

electron adducts of other pyrimidine derivatives reported so far. The selected

systems are 2,4-dimethyl-6-hydroxy pyrimidine (DMHP), 4,6-dihydroxy-2-

methyl pyrimidine (DHMP), 6-met hyl uracil (MU) and 5,6-dimethyl uracil

(DMU). The kinetic and spectral parameters at different pH values are also

determind. The oxidant, methyl viologen (MV~') is used to investigate the

redox nature of the electron adducts.

The end product analysis after the reaction of water derived free radicals

with biomolecules is often a difficult task due to the Iow concentrations of the

products ( 1 -10 x lo4 mol dm-3 under low dose experiments). However, a

considerable progress is made in this field with the help of GC-MS in the case

of 'OH reactions with purines and On the other hand,

practically little information is available about the end products of e-,, reaction

with purines and pyrimidines. Therefore, an attempt is made to investigate the

Kinetic and Spectral Investigation .... 75

end products of the reaction of e-,, with DHMP and DMHP with the help of

HPLC connected with electrospray mass spectrometer (HPLC-ES-MS). To our

knowledge, this is the first attempt to analyse the products resulting from the

reaction of electron with a DNA model system using HPLC-ES-MS which has

several advantages over other conventional techniques.

3,l Acid Base Properties

DHMP is reported to have three different pK, values (0.21, 6.35 and 12.9)

and exists predominantly in the monoketoImonoenol The pK, values of

DMWP were experimentally determined as 3.15 and 9.9. Two pK, values (2.7

and 9.8) were determined in the case of DMU. The reported pK, value of MU

is 9 . 5 ~ ~ ~ and the experimentally determined value was 9.5.

3.2. Reactions of Hydrated Electron (e-,,)

3.2.1 Kinetics

The second order rate constants of the reaction of hydrated electron with

DHMP, DMHP, DMU and MU in N2 saturated aqueous solutions containing

either 0.2 mol dm-3 2-methyl propan-2-01 or propan-2-01 to scavenge 'OH, were

determined by following the decay of the hydrated electron at 720 nrn as a

function of substrate concentration. The pseudo first order decay constant (bbs)

versus concentration plots gave straight line graphs with very good correlation

coefficients (10.99). A typical &I,, versus concentration plot obtained in the case

of DMHP is shown in Figure 3.1. The rate constants of the neutral forms are

diffusion controlled (Table 3.1) and these values are in good agreement with the

Kinelk und Spectr(ll Investigation . .. .76

rate constants reported for other pyrimidines.8 However, at higher pH values

where the pyrimidines are in their mono anionic form, the rate constant values

were significantly reduced except in the case of DHMP where the difference is

only marginal. The lowering of rate constants at higher pH values, as shown in

Table 3.1, represents the electrostatic effects resulted from the pyrimidine anion

and hydrated electron. Similar trends in the rate constant values were reported

with mono and dianionic forms of both purines and pynmidines.6

0 0.5 1 1.5 2 2.5 3 3.5

[DMHP] 1 lo4mol dm3

Figure 3.1: Plot of the rate of build-up (bb,) versus concentration observed at 3 10 nrn after delivering electron pulses to N2 saturated solution of 1 o - ~ mol dmu3 2,4-dimethyl-6-hydroxy pyrimidine (DMHP) containing 0.2 mol dm" 2-methyl propan-2-01 at pH 6. Inset: Decay of eLaq in presence of 10" mol dm-) DMHP.

Kinetic and Spectral Investigation .... 77

Table 3.1: Second order rate constants (bb) obtained for the reaction of el,, with the selected pyrimidines at different pH values.

Pyrimidine PH k / dm3 mol-' S-I

4,6-dihydroxy-2-methyl

Pyrimidine (DHMP)

2,4-dimethyl-dhydroxy

Pyrimidine (DMHP)

5,6-dimethyl uracil (DMU)

6-methyl uracil (MU)

'From ref. 15

3.2.2 Spectra

a) 4, &dihydrtq-2-methy&yrim idine @HMP)

The time resolved absorption spectra obtained at pH 4.5 from the

reaction of e-, with DHMP in the presence of propan-2-01 is shown in Figure 3.2.

Propan-2-ol was used in all of the spectral measurements to avoid any contribution

h m the reaction of H' with the selected pyrimidines. The spectrum recorded at 3 p

afler the pulse has an absorption maximum at 320 nm with a shoulder at around 380

nm. While this spectrum showed a decay at 320 nm (k = 5.5 x 1 o4 s'l), a further

growth in absorbance was observed above 350 nm &- = 4 x 1 o4 s") indicative of

a transformation of the initial species. As can be seen h m Figure 3.2, the

transformed spectrum has a A- around 380 nm and a broad absorption between 460

and 550 m. The time resolved spectra were also recorded in presence of phosphate

buffer (0.3 rnoldm") at pH 7.5 (Figure3.3). It was observed that the decay at

Kinetic and Spectral Investigation .... 78

Figure 3.2: Transient absorption spectra recorded in N2 saturated aqueous solutions of DHMP (1 n 10" rnol dm") containing propan-2-01 (0.2 rnol dmA3) at 3 ps (m) and 40 ps (0) after the pulse at pH 4.5. The dose / pulse was 16 Gy.

Figure 3.3 Transient absorption spectra recorded in N2 saturated aqueous solutions of DHMP (1 x 10" rnol dm-') containing propan-2-01 (0.2 mol dm") at 2.5 ps (m), 15 ps (A) and 40 ps (a) after the pulse in presence of phosphate buffer (0.3 rnol dmL3) at pH 7.5, dosdpulse w 16.5 Gy.

Kinetic and Spectral Investigation .... 79

320 nrn and the build-up above 350 nm are greatly enhanced (kJsonm = 3.6 x lo5 s-',

k490nm = 2.0 x 1 6 S-'1. The hansient spectrum recorded at 3 ps and 40 p after the

pulse at pH 4.5, the transformed spectrum recorded in presence of phosphate

buffer measured at 1 5 ps after the pulse at pH 7.5 and the typical traces at 320

and 490 nm in the absence of phosphate and at 485 nm in the presence of

phosphate are shown in Figure 3.4.

The spectral characteristics at pH 9 was different from that at pH 4.5.

The observed first order decay at 320 nrn and the slow build-up of absorbance

above 350 nm at pH 4.5 was not visible at pH 9, but the spectrum showed two

distinct maxima at 3 10 nm and at 360 nm. Similarly, the spectrum recorded at

3 ps after the pulse at pH 13 (Figure 3.5) was found to decay without showing

any delayed build-up unlike at lower pH. The absorption trace of the

intermediate obtained at 3 10 nm at pH 9 is shown in the inset of Figure 3.5.

In order to understand the nature of the electron adducts, the electron

transfer reaction of the electron adducts with MV*' at different pH values have

been investigated. The formation of methyl viologen radical cation (MV") was

monitored at 605 nm. 2-methyl propan-2-01 was used as the 'OH scavenger

instead of propan-2-01 in all these experiments to avoid the reaction of

formed from reaction of the 'OH with propan-2-01, with M V ~ + . The yields,

G(Mv"), and the second order rate constants for the electron transfer reaction

are compiled in Table 3.2. The G values were calculated by taking a typical E

value of MV*' as 12,800 dm3 mol-' cm-I at 605 nm.26 The G value of MV"

obtained at pH 4.5 was 2.8 x mol J-'. Expecting about 20% of the direct

Kinetic and Spectral Investigarion ... ,813

reaction of e-, with MV~' at this experimental condition ([Mv"] = 5 x lom5

mol dni3, [DHMF] = 2 x rnol dmA3, k (ePq + MV~C) = 5.4 x 10'*drn3 rnol-' df,

the observed G(Mv'+) corresponds to the total yield of the electron adduct.

Similarly at pH 9, the observed G value 2.8 x lom7 rnol J - I indicate the

quantitative reaction of the electron adduct with MV". At pH-1 3, a G(Mv*')

value of 3.2 x rnol J' is obtained. At this pH, a considerable part of H' will

be converted into 6, @(H. +ow) = 2.3 x 10' dm3 mol-' s ) . Calculating an

yield of e-,, at this pH as 3.2 x lo-' rnol J - I , the observed G(Mv'+) indicates a

quantitative reaction of the electron adduct.

Figure 3.4: Transient absorption spectra recorded in N2 saturated aqueous solutions of DHMP ( 1 x loF3 rnol dmm3) containing propan-2-01 (0.2 rnol dm'3) at 3 ys (*) and 40 ps (0) aRer the pulse at pH 4.5 and the spectrum recorded in presence of phosphate buffer (0.3 rnol dm") at 15 ps (A) after the pulse at pH 7.5, doseipulse = 16.5 Gy. Inset: Traces of the intermediates obtained at 320 nm (a) and 490 nm (b) in the absence of phosphate and at 485 nrn (c) in the presence of phosphate.

Kinetic and S~eclral Invesii~aliun ... .8 1

Figure 3.5: Transient absorption spectra recorded in N2 saturated aqueous solutions of DHMP ( 1 x rnol dm") containing 2-methyl propan 2-01 (0.2 mol dm-3) at 3 ps after the pulse at pH 9 (A) and pH I3 (a), dosdpulse - 16 Gy.

The attack of e-,, is likely to occur at the electron affinic oxygen, O(4)

or O(6) of the keto form of the pyrimidine. The resulting radical anion (ketyl

radical) can be protonated by water andlor H'. The nature of the initial spectrum

recorded at 3 ps after the pulse at pH 4.5 with a h,, at 320 nm is in agreement

with the electron adduct spectra of other pynmidines reported earlier.16~1726 We

propose that the species absorbing at 320 nm corresponds to the protonated

electron adduct of DHMP as the protonation of the electron adducts of

pyrimidines at oxygen is normally very fast with p ~ , values around 7.2.26

Moreover, the feasibility of protonation at two identical C(4) and C(6) oxygen

(as shown in scheme 3.2) makes the protonation more efficient compared to uracil

Kinetic and Spectral Investigation .... 82

electron adduct. Therefore in the present case it is likely that the radical anion must

be getting protonated with a half life < 1 ps (this is the minimum time required

to measure the initial spectrum using our experimental set-up) and the proposed

structure is I (scheme 3.2) which is in resonance with 11. Such fast protonation is

justified based on the earlier reports on the protonation at oxygen or nitrogen in the

case of uracil (k 5 x 10' 5') and cytosine14 (k N 3.5 x lo6 c'). The

h t order nature of the decay of the initial spectrum at its and the oomponding

delayed build-up at tt h 350 nm indicate a clear first order transformation of the

initially formed protonated electron adduct to a different

Table 3.2: The yields and rate of formation of MV" obtained from the reaction of the electron adduct of DHMP, DMHP, DMU and MU at different pH values

Pyrimidine

4.5

DHMP 9.0

13.0

DMHP

6.0 DMU

12.0

--

*These are averages of at least three values.

# k' = kEdectron adduct -I- MV~'] (These were determined from the pseudo first-order build-up of MV'+ kom a single concentration of M V ~ ' and hence are approximate).

. . Kinelic and Spectral Investigarion .... 83

species with a h,,,, around 380 nm and a broad absorption band between 490

and 550 nm. It is interesting to note that the nature of this transformed spectrum

and the spectrum recorded at 15 ps after the pulse in presence of phosphate

buffer (0.3 rnol dmm3) is very similar (Figure 3.4) which implies that the

transformation can be catalysed by both H' and phosphate. However, the rate of

transformation was almost ten times higher in presence of phosphate buffer. A

phosphate catalysed conversion of an oxygen protonated electron adduct to

carbon protonated (C(5) and C(6)) electron adduct is a well established reaction

in the case of uracil, thymine and some of their substituted deri~atives.'~,'~ On

the other hand, it appears that such transformations catalysed by H+ is not very

common, but restricted to only dihydroxy pyrimidines as reported in an esr

study by Novais and n teen ken.'^ Therefore we can clearly understand that the

transformed species is a carbon protonated electron adduct of DHMP. Since

such carbon protonation can be at C(5) andor C(6), as observed with uracil and

thymine,I6 one has to clearly estimate the percentage distribution of these

protonated radicals. The results obtained with MV" gave a detailed account of

the contribution of the reducing radicals. The observed G value of MV'+

(i .e. 2.8 x lo-' mol J-') at pH 4.5 clearly indicates that the protonated electron

adduct is reducing in nature. This is understandable from the proposed structure

of the protonated electron adduct (I) in which the unpaired spin density is at

carbon. This is in agreement with the earlier reports with uracil and its

The GIMV'') value, further, leads to the conclusion that even

the transformed species at pH 4.5 is reducing in nature. If we assume that the

4 - 1 transformed protonated electron adduct (kt = 4 x 10 s ) is a non-reducing

Kinetic and Spectral lnvesrigation .... 84

radical (as reported with uracil and thyminei6), one can calculate the G value of

(eqn. 3.1) from the competition between the reaction of electron adduct (EA)

with MV2+ (&on 3.1 ) and the transformation of the electron adduct (reaction 3 -2).

where k2 is the second order rate constant, kt is the rate of transformation and

EA' represents the transformed electron adducts.

2.8 x mol J-I is the total yield of the electron adduct. According to this

calculation the expected G value of MV" is 2.4 x rnol J-I. But the

observed G(MV+) is 2.8 x 1 o - ~ mol I-' (Table 3.2). This indicates that the

transformed species is also reducing in nature with respect to M V ~ + . In order to

further confirm the nature of the transformed electron adduct, the G(MV") is

detemined in presence of 0.3 rnol dm-3 phosphate at pH 7.5. The observed G value

was 2.8 x lom7 mol s'. In presence of phosphate the rate of transformation is ten

times higher. Based on these observations it is proposed that the transformed

species is a C(6)-ylC(5) protonated form of DHMP (111). The structure 111 is in

resonance with IV. Both 111 and IV can be reducing in nature with respect to

MV~'. The ketonic form of structure IV reported in the case of dihydroxy

Kinefic and Spectral lnvesiiga lion .... 85

pyrimidine,'8 is also probable. However, the speed of this enol-keto

transformation could not be determined. Jt is interesting to note that the phosphate

IV

Scheme 3.2: Proposed mechanism of the reaction of e-, with DHMP at pH 4.5.

catalysed carbon protonation in the case of uracil and its methyl derivatives

leads to a non reducing C(5)-yl radical whereas in the present case it is a C(6)-yl

radical. An additional. support for the formation of III is its spectral similarity

with H adducts. (see section 3.3 for more explanation). However, a

simultaneous formation of a possible C(5)-yl radical (non-reducing) as was

reported with other can be fully ruled out at pH 4.5 based on the

G (MV") value. Furthermore, it is important to note the reported esr study with

Kinelic and Spectral Investigation .... 86

dihydroxy pyrimidines that at pH 4-5 the observed esr spectrum corresponds to

H adducts. l8 This report is clearly in line with our observation on DHMP that in the

initial stage, an oxygen protonatd electron adduct is formed which is later

transformed in presence of H' to a C(5) protonated electron adduct which is similar

to the H adduct. The difficulties in the detection of oxygen protonated electron adduct

in the previous reportI8 may be due to the longer time scales that normally usad in

experiments, the C(5) protonation must have been completed by then.

At pH 9, DHMP is predominantly existing in its dqmtonated form

(p& = 6.35). Therefore, the reaction of e, at thls pH must be considered as the

addition of e-, to the monoanion of DHMP, the reduction in the second order

rate constant at pH 9 is a clear indication of this reaction (Table 3.1). Such

addition reaction would definitely lead to the formation of a radical dianion as

shown in scheme 3.3. The radical dianion can be protonated by water so that a

radical monoanion can be formed. We propose that the species with absorption

maxima at 3 10 and 360 nrn at pH 9, is the monoanionic form of the electron

adduct of DHMP (V) as represented in scheme 3.3. The supporting evidence for this

argument came from the esr and conductance studies on dihydroxy pyrimidine

systems between pH 6 and 12.18 The cunductance measurements at pH 8.5-9.5

yielded a quantitative production of O K which clearly supported the protonation

reaction.''

Kinetic and Spectral Investi~ution .... 87

Scheme 33: Proposed mechanism of the reaction of e-, with DHMF in basic pH

Furthermore, the esr study indicated the formation of a ketyl type radical

monoanion whose unpaired spin is strongly delo~alised.'~ Our results with

MV" gives a clear indication of the reducing property of the radical anion (V)

at pH 9. At this pH the observed G(Mv'+) value (i.e, 2.8 x mol J-')

represents the full yield of electron adduct (Table 3.2). Therefore, it is expected

that the unpaired spin in DHMP radical anion (V) is more localised towards the

ketyl carbon as reported in other similar pyrimidine electron adduct ~tudies.'~"'

As can be seen from Figure 3.5, that the spectral nature of the intermediate at

pH 13 is similar to that at pH 9, indicating the similarity of the species existing

at these two pHs. Since DHMP has its second p~~ at 12.9, the reaction of e'.,, at

Kinelic and Spectral Investigation .... 88

pH 13 is with a mixture of about 56% dianionic form and 44% monoanionic

form of DHMP. The attack at the dianionic form would give rise to a radical

trianion in the initial stage which could be immediately protonated by water.

Since the spectral features at pH 9 and I3 are very similar, it will be logical to

assume that the trianion radical is getting protonated twice to form a monoanion

radical of DHMP with a structure similar to that obtained at pH 9 (scheme 3.3).

The similar spectra obtained both at pH 9 and at 13 may be an indication of a

much higher pKB value (> 13) for the monoanion radical of DHMP. The esr study

on dihydroxy pyrimidine systems reported the presence of a radical dianion

protonated at C(5) (catalysed by OH-) and its subsequent protonation leads to a

neutral radical similar to the H-adduct.I8 The C(5) protonated dianion could be

detected only at [OH] 1 0.2 mol h m 3 . 1 8 However, the percentage distribution of

the radical monoanion and the neutral H adduct of DHMP (existing at pH>9, if

formed) could not be obtained using M Y 2 + in the present case as both the structures

can be reducing in nature with respect to The quantitative yield of MV+ at

pH 13 when the G(e-q) is 3.4 x lo-' mol J' due to the conversion of H' to 6, by

OH, reveals once again the fact that of the electron adducts are reducing in nature

and the unpaired spin is located at C(2), C(4) or C(6) positions. Further, the

protonation at C(6) leading to the formation of a C(5)-yl radical of DHMP, as

observed with uracil and thymine,I6 is negligible in the case of DHMP. The

quantitative yield of is in line with such a conclusion.

-- Kinetic and Spectral Investigalion . .. .89

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

Figure 3.6: Transient absorption spectra obtained in N2 saturated aqueous solutions of DMHP (1 x low3 mol dmm3) containing propan 2-01 (0.2 mol at pH 6 at 2 ps (A), 20 ps (*) and 40 ps (+) after the pulse, dosdpulse 15 Gy.

250 3 s 450 550

h l n m Figure 3.7: Transient absorption spectra obtained in N2 saturated aqueous

solutions of DMHP (1 x 1v3 mol dmm3) containing propan 2-01 (0.2 mol dm-3) at pH 12.5 (A) and 4.5 (a) at 2 ps after the pulse, dosdpulse = 15 Gy. Inset: Dependence of absorbance of the intermediate on pH at 430 nm. The inflection point is at pH 6.

- Kinetic and Spectral .hvestigation .. . -90

The time resolved spectrum obtained at pH 6 at 2,20 and 40 ps affer the

pulse is shown in Figure 3.6. The noticeable change in the spectral feature is

only a relatively faster decay of absorbance at the h,, compared to other parts

of the spectrum. The electron adduct spectrum showed a single distinct

absorption maximum at 310 nrn at pH 4.5 (Figure 3.7) as well as at pH 6 and

was very similar with slight difference in the absorbance values. The time

resolved spectra at both pH values did not show any indication of a

transformation reaction as was observed with DHMP, but underwent a second

order decay, The nature of the spectrum at pH 12 was also similar but the

absorbance at the A,, was significantly higher (Figure 3.7). A plot of pH

versus absorbance of the electron adduct (Figure 3.7, inset) at 430 nm showed

an inflection point around 6.0. The spectrum obtained in presence of 0.3 mol

dm-3 phosphate did not show much difference with that obtained without

phosphate indicating the absence or a relatively slow transformation reaction in

the pulse radiolysis scale (Figure 3.8). A typical trace obtained at 320 nm is

shown in the inset of Figure 3.8.

The G (MV'+) for DMHP at pH 6 was 2.6 x mol J-' (Table 3.2). It

is calculated that 9% of the G value is from the direct reaction of e,, with MV"

under our experimental conditions. Therefore the actual yield of MV"

corresponds to 92% of the total electron adduct. A nearly similar value is

obtained at pH 12.5 as well. The second order rate constants obtained for the

reaction of the electron adduct with MV" are also summarized in Table 3.2.

Kinelic and Spectral hwstigczrion . . . -9 1

0 J 1 250 300 350 400 450 500

Xlnm

Figure 3.8: Transient absorption spectra recorded in N2 saturatd aqueous solutions of DMHP (1 x 10" mol dm") containing propan-2-01 (0.2 mol dm-3) in presence of phosphate buffer (0.3 mol dm-3) at 2.5 ps (A) a d 40 ps (m) after the pulse at pH 7.5, dosdpulse = 16.5 Gy. Inset: Absorption trace of the intermediate obtained at 320 nm.

From the spectral behaviour of the electron adduct of DMHP at pH 4.0,

6.0 and 12.5, it can be assumed that at low pH the electron adduct is protonated.

The protonationldeprotonation equilibrium, determined at 430 nm (Figure 3.7),

gave an inflection point around 6.0. Therefore, the species existing at higher pH

such as at pH 12.5 (the spectrum is identified with its A,, at 3 10 nm) is the

deprotonated form of the electron adduct. The initial. fast decay of absorbance

observed in the spectra at pH 6 (see Figure 3.6) at the h,, may be an indication

of this protonation. A similar behaviour of the electron adduct of uracil and

1,3-dimethyl thymine is already In aqueous medium DMHP is like1 y

to be in its enolic form. This assumption is based on a recent study where we

Kineric and Spectral Investiga~ion .... 92 -

have shown that both DMU and MU undergo a rapid deprotonation at near

neutral pH whereas DHMP and DMHP did not show such a phenomenon. This

is observed only with pyrimidines containing H at N(l) position and is

explained on the basis of the deprotonation of the N(1)H. This is an additional

support for the conclusion that DMHP exists in its enolic form at C(6).

Therefore, the expected sites of attack are at N(1) or N(3) and the resulting

radical anion can be easily protonated by Hz0 / H* (reaction 3.3).

Although DMHP is in its deprotonated form at pH 12.5, the radical

dianion resulting from the e-, attack is likely to get protonated by water, similar

to the dianion of DHMP, leading to the radical monoanion which undergoes

protonation only at lower pH. Both protonated and deprotonated forms can be

reducing in nature with respect to MV'+ since the unpaired spin density is at

carbon. The high G(Mv'*) values at lower and higher pHs are the result of this

reducing property. A point of concern, at this stage, is the slightly less value of

G(MV+) both at pH 6 and at pH 12.5. It follows that a minor part of the

electron adduct does not posses reducing property. The most probable

explanation to this is the simultaneous formation of a C(5)-ylC(6)H radical

which is not reducing in nature as reported in the case of uracil and its

derivatives. 16*17

Kinetic and Spectral Investigation ... .93 -

c) 5,6-dimethyl uracil (DMU) and 6-methyl uracil (MU)

0.009 1

Figure 3.9: Transient absorption spectra recorded in N2 saturated aqueous solutions of DMU ( 1 x 10" mol dm") containing propan-2-01 (0.2 rnol dm") at pH 12(0) and pH 6 (A) at 30 ps after the pulse, dosdpulse = 16.7 Gy. Inset: Absorption trace of the intermediate obtained at 300 nm at pH 6 .

The electron adduct spectra of DMU and MU were recorded in presence

of 0.01 rnol dm" phosphate buffer in order to minimise the absorption from the

possible formation of DMU anion resulting from the reaction of radiolytically

produced OH- and DMU. The DMU and MU anions are reported to have A,,, at

295 and 285 nm respectively.28 The transient spectra at pH 6 and 12 have shown

a single h,, at 300 nm but they showed a clear difference in their absorbance

values (Figure 3.9). The absorption trace of the intermediate obtained at 300 nm

at pH 6 is shown in the inset of Figure 3.9. The experiments with M V ~ * gave a

G (MV'') at pH 6.0 of 2.8 x rnol J-' which corresponds to the total yield of

- Kinetic and Spectral Investigation . .. - .94

the electron adducts and a similar yield 2.9 x lou7 mol J - ' was calculated at pH

12.0 as well. Thus the electron adduct of DMU also appears to have similar

properties as in the case of DHMP.

The protonated electron adduct and its deprotonated forms can be

distinguished from the difference in their absorption coefficient at the h,,

(Figure 3.9). The preferred site of attack in this is proposed to be at O(4) and

O(2). It is reported in the case of uracil that the O(2) protonated electron adduct

has an absoption maximum less than 280 nm at pH 5 . 1 . ~ ~ Therefore, it can be

concluded that the species with A,, at 300 nm is the 0(4) protonated electron

adduct of DMU which is in its deprotonated form at higher pH as shown in

reaction 3.4.

The high yield of MV+ at pH 6 and 12.5 (see Table 3.2) is in agreement

with the above explanation. Both protonated and the deprotonated foms can be

reducing with respect to MV~'. In this case too the phosphate induced

protonation at carbon [C(5) and C(B)] is relatively slow and this was confirmed

fiom the absence of any spectral changes -between 380-520 nm in presence of

0.3 rnoI dm? phosphate. The transient spectrum obtained with MU is also

similar to the spectrum of DMU and hence similar electron adduct protonated at

Kinetic and Spectral lnvestigulion ... .95

0(4) is proposed at pH 6 (Figure 3.10). The absorption trace obtained at 290 nrn

is shown in the inset of Figure 3.10 which shows only a second order decay.

The speckurn showed a single k,, at 295 nm. In an earlier study by ~ a ~ o n ~ ~

reported that the transient spectra at neutral and basic pHs and the present

spectrum at pH 6 is in agreement with this earlier report. In addition to this, we

have carried out the reaction of the intermediate with MV~+ and the G(Mv'+)

value obtained at pH 6 (i. e. 2.7 x 1 o - ~ mol 1') and at pH 1 2 (i. e. 3.4 x 1 o - ~ rnol J ' ) clearly indicate the quantitative formation of the reducing radical, the electron

adduct protonated at O(4) (at pH 6) and its deprotonated fom (at pH 12) which

is in line with the earlier report.26

250 350 450 550 hlnm

Figure 3.10: Transient absorption spectra recorded in N2 saturatsd aqueous solutions of MU ( 1 x mol dm-3) containing propan-2-01 (0.2 rnol dm") at pH 6 (2 ps after the pulse), dosdpulse = 14.5 Gy. Inset: Absorption trace of the intermediate obtained at 290 nm.

Kinelic and Spectral Investigation .. . .96 -

3.3 Reactions of H Atom: Kinetics and Spectral Studies

The reaction of H' with DHMP, DMHP, DMU and MU were studied at

pH 1 as well as at near neutral pH. The reaction at neutral pH was studied in

N20 saturated solutions containing 2-methyl propan-2-01 (0.2 mol dm'3) to

scavenge 'OH. The second order rate constants, determined from the rate of

build-up at the respective absorption maxima of H adduct are given in Table 3.3.

As can be seen from the table that the pK, values of the substrates have a

profound effect on the rate of reaction. The birnalecular rate constants at pH f

for all the pyrimidines were considerably lower than that at higher pH except in

the case of DHMP, where the values at pH 1 and 4.5 were comparable. The

comparatively low rate constants at lower pH values are understandable due to

the fact that H attack is electrophilic. Since DHMP has its pK, value only at

0.2 1 the rate constant value remained unaffected.

The absorption spectra obtained for the reaction of H* with DHMP,

DMHP, DMU and MU are given in Figure 3.1 1 (a-d). The H adduct spectrum

of DHMP showed a single absorption maximum at 380 nm and a broad

absorption band between 460 and 580 nm at pH 1 and at 4.5. The absorption

maxima obtained with DMHP at pH 1 are 3 15 and 5 10 nm while that at pH 6

were 3 10 and 505 nm. The H adduct of DMU at pH 1 has maxima at 3 1 0 and

460 nm, while that at pH 6 are 305 and 460 nm. The spectral parameters are

presented in Table 3.3. The H adduct spectrum of MU has also similar

properties. The H adduct spectra of all these compounds were found to undergo

a second order decay at their absorption maxima.

Kinetic and Spectral ~nvestigation ... .97

Table 3.3: Second order rate constants (k), absorption maxima (A,,) and molar absorptivities (E) obtained for the reaction of H' with DHMP, DMHP, DMU and MU at different pH values.

DHMP 1 .O 2.8 x lo9 380 5 10

4.5 9.2 x los 380 5 10

DMHP 1 .O 8.7 x lo8 315

5 10

6.0 2.0 x 1 o9 310 505

DMU 1.0 1.7 x lo8 3 10 460

6.0 2.6 x lo9 305

460

MU 1 .O 6.0 x lo8 310

460

The preferred site of attack of H' in uracil derivatives are the C(5) and

C(6) position?-" Therefore, similar sites of attack are likely in the case of

DHMP. It is thus assumed that C(6)-yl radical is formed from the attack of H' at

the C(5) position of DHMP as H* is an electrophile and therefore, it can attack

at the more electron rich C(5) position (scheme 3.2). The difference in the

absorbance at lower and higher pHs as can be seen in Figure 3.1 1, must be due

to the protonation of the parent pyrimidine. This difference is more prominent

with DMHP at pH 1.0 where it is fully in its protonated form. At this point, it is

interesting to compare the transformed electron adduct spectrum at pH 4.5 and

the spectrum at pH 7.5 in presence of phosphate (Figure 3.4) with the H adduct

spectrum at pH 4.5 in the case of DHMP (Figure 3.1 1(a)). It can be seen that

Kinetic and Specrral Investigatioion ... -98

these three spectra are similar and hence i t is logical to assume that the

intermediate species are also similar.

"I- .-.-. --.---

I

Figure 3.11: H adduct spectra recorded at 2 ps after the pulse of a) DHMP at pH 4.5 (0) and at pH 1 (A); b) DMHP at pH 6 (0) and pH 1 (A); c) DMU at pH 6 (0) and 1 (A) and d) M U at pH l(A). (All experiments at near neutral pH were carried out in N20 saturated aqueous soIutions and that at pH 1 was in N2 saturated aqueous solutions containing typically 1 x mol substrate at dosdpulse = 16 Gy. The absorbance values are calculated for a typical G(H) value of 3.3.

Similar to DHMP, formation of C(6)-ylC(5)H radicaI is proposed in the

case of DMHP which exists in its deprotonated form at pH 6. However, in the

case of DMU and MU, both C(5)-ylC(6)H and C(6)-ylC(5)H radicals are

Kinetic and Spectral Investigation ... .99

proposed to be formed. Based on the preferential formation of C(5)-ylC(6)H

radical in the case of thymine," i t can be expected that a similar trend is likely

in DMU and MU. It can be further seen from the H adduct spectrum of DMU

(Figure 3.1 1 (c)) that at h<300 nm there i s a disproportionate difierence

between the spectrum at pH 6 md pH 1 . This difference is mainly due to the

interference of the DMU anion spectrum which has a A,, at 295 nm as

discussed in section 3.2.2 (c).

The G(-substrate) values for the reaction of e-,, with selected

pyrimidines were determined by monitoring the degradation at the respective

absorption maxima of the parent pyrimidines (lo4 mo1 dm-') at different

absorbed doses using UV-VIS spectrophotometer. The absorption maxima for

DHMP, DMHP, DMU and MU are 255, 230, 267 and 260 run, respectively.

The overlay spectra obtained from UV-VIS spectrophotometer for the

degradation of DMHP by ePq ( 1 0" rnol dm') DMHP and 0.2 mol dm" 2-methyl

propan-2-01 saturated with h) using Coy-source at pH 6 is shown in Figure 3.12.

The doses used are 0 to 5 kGy. The concentration of the substrate decreases

with increasing dose. At 5 kGy the absorbance value becomes almost steady.

The G values for the substrate decay obtained are given in Table 3.4. The G(-S)

values are around 1 and represent only about 36 % of the expected value which

is around 2.8 x 10" mol J - I . A possible explanation to this reduced value is the

fact that the products formed after the e"a, attack may also contribute to the

absorbance at the wavelengths used for G(-S) calculation (200-300 nm).

Kinetic and S~ectvul lnvestinaiion .... 100

The electron transfer fiom the pyrimidine electron adducts to 5-brornouracil

was also investigated by monitoring the release of free uracil (fiom

5-bromouracil) using HPLC (Figure 3.13) after y-radiolysis of a solution

typically contained 10" mol dm-' pyrimidine and 1 o4 mol dm" 5-brornouracil at

neutral pH. The yield of uracil (G(U)) values obtained from this study compiled

in Table 3.4. These values are much lower than those obtained fiom the electron

transfer reaction of the eIectron adducts with MV" which are almost

quantitative ( G ( M ~ + ) 1 2.8 x 10" rnol J").

Figure 3.12: The overlay spectra obtained from UV-VIS spectrophotometer for the degradation of 10') rnol dm" DMHP induced by Cq using 60~o-y- source at pH 6; A-F corresponds to 0, 1, 2, 3, 4 and 5 kGy respectively.

Kinetic and Spectral investigation . . . . I0 1

Figure 3.13: HPLC chromatogram obtained for the DHMP (1 x 10'~ mol dm3) containing 2-methyl propan-24 (0.2 mol dm-3) and 5-bromouracil (lo4 mol dm+') saturated with Ar ai pH 4.5 irradiated using 60~o- y-source. 1 ml/min 10% methanol in water is used as the eluent. The retention time of DHMP, U and BrU are 2.32, 3.19 and 8.14 min. respectively.

The possible reactions in presence of 5-BrU are given in reaction

3.5- 3.9. The el, reacts with 5-BrU and pyrimidine to form the electron adduct

of 5-BrU and pyrimidine. As both e-, and Py*- can react with 5-BrU, the

concentration of 5-BrU was kept at lo4 mol dm-3. The uracil radical is formed

from 5BrU'- and it subsequently reacts with 2-methyl propan-2-01 to give free

unaltered uracil.

e-,, + BrU -------r, BrUm-

e-aq f PY - Pya-

Py'- + BrU --------) BrU '- + Py

BrU*- - Br-+U*

U* + (CH3)3COH U + 'CH2C(CH3)20H

Kinetic and Sneclral Investination ... ,102

Table 3.4: The yields of substrate and the yield of uracil obtained from the reaction of the electron adduct of DHMP, DMHP, DMU and MU at near neutral pH.

Substrate G(-S) x lom7 mol J-' G(U) x mol P'

DHMP

DMHP

DMU

MU

Both e-, and the electron adducts can, therefore, react with BrU and

give rise to uracil. Although, the G(U) E G(e-,)) - 2.8 x lo-' rnol J- I , we get

only 46% for DHMP, DMHP and MU and 71% for DMU. From the

competition kinetics, between the reaction of e-,, with DHMP and the e-,, with

5-BrU it can be understood that about 32% direct reaction of e-, with 5-BrU at

this experimental condition ([5-BrU] = lo4 rnol dm", [DHMP] = loL3 mol dm"

I -1 8,27 and k(Caq + 5-BrU) = 1.6 x 10" dm3 mol- s ). This means that the observed

G(U) constitute about 0.4 x rnol I' from the direct ePq reaction and the

remaining 0.9 x lo-' mol I-' from the reaction of the electron adduct. A more or

less similar values are obtained with other pyrimidines as well (see Table 3.4)

except with DMU where a slightly higher G(U) was observed. This value is

unexpectedly low considering the fact that the electron adducts (or their

protonated forms) are reducing in nature and this ability is very clear from the

G(Mv*') value (see section 3.2.2). No convincing reason is gven to this

reduction in the GQJ) values at this stage. It can only be assumed that the 5-BrU

is not an efficient electron acceptor from the electron adducts of the selected

Kinetic and Spectral Invcstigaiion .... 103

pyrimidines. It is also not clear that the electron adducts of these pyrimidines

can undergo any other reactions other than electron transfer with 5-BrU. Further

studies are required to understand such reactions. Almost same value is obtained

for DMHP and MU. For DMU about 13% is due to the reaction of e-, with

5-BrU. The calculated expected value is 2.45 x mol J - I . We got the value of

G(U) is 2.0 x lo-' mol J-' and therefore the calculated observed value is

1.75 x lo-' mol PI .

Qualitative product analysis was carried out aRer prolonged y-radiolysis

(dose - 2000 Gy) of argon saturated solutions containing either DHMP or

DMHP (1 x lom3 rnol dmJ) in presence of 2-methyl propan-2-01 (0.2 mol dmm3)

using HPLC-ES-MS. Even with the prolonged irradiation it was observed that

only a minor portion of the substrate has bsen consumed and the products peaks

were too feable. However, a series of product peaks were obtained with varying

d z values. From the mass peaks, the most probable mlz values were selected

for most probable products. The identified products, their molecular weight and

the mlz values obtained from the MS data are summarized in Table 3.5 and 3.6,

respectively, for DHMP and DMHP. HPLC chromatogram obtained for DHMP

at pH 4.5 is shown in Figure 3.14.

Kinetic and Specrral Invmttgalion . . . ,104

Figu .re 3.14: HPLC chromatogram obtained for DHMP (1 x mol dmm3) containing 2-methyl propan-2-01 (0.2 mol dm-3) saturated with Ar at pH 4.5 irradiated using 60~o-y-source. 0.5 ml/rnin Millipore-Milli-Q water is used as the eluent.

From the identified products, it is clear that these products are the results

of a bimolecular reaction of the protonated electron adducts III (from DHMP)

and VI1 (from DMHP). The structure VII is the mesomeric form of the

protonated electron adduct of DMHP (see reaction 3.3). The structures of these

electron adducts were demonstrated with the help of MV~' using the pulse

radiolysis experiments (see section 3.2.2). Furthermore, these products are

proposed to be resulted from mainly disproportionation and dimerisation

reactions as shown in scheme 3.4 and 3.5- In the case of DHMP at pH 4.5, the

disproportionation reaction leads to the products a and b (scheme 3.4, reaction

3.10) where b is the starting compound. Indication is obtained for the formation

of a product like c, which could be the result of an unstabIe intermediate C(6)-

dihydroxy product as shown in reaction 3.1 1. This can be resulted fiom the

oxidation of I11 followed by addition of OH-. Such an oxidation is possible

Kinetic and Spectral Investigalion ... ,105

either by the disprotonation reaction of III or by the reaction of radiolytically

formed HZ&. The formation of dimers is another important reaction pathway

which can lead to products with the structure d, e and f. e and fare formed via

stepwise elimination of water forming the initial dimer as shown in scheme 3.4.

As there is practically little information is available about the product formation

from the reaction of e-,, with pyrimidine, no fruitful comparison of these

Table3.5: Products identified from the reaction of e-,, with DHMP by HPLC-ES-MS analysis at pH 4.5.

Products Molecular Molecular formula Weight M/z

Kinetic and S~ectral Investi~arion .... 106

Figure 3.15: Typical ES-MS obtained fiorn the HPLC chromatogram after the reaction of e-, with DHMP at pH 4.5 (Eluent = water, Flow rate = 0.5 mllmin) A) in -ve ionization mode (RT = 0.9 min), the d z value -125 is attributed to the compound b and B) in +ve mode (RT =1.19), the mlz value +236 is attributed to the product e. The products included in Table 3.5 were obtained by scanning the entire region of the chromatogram.

Kinetic and Swectrul lnvesti~ation . .. ,107

dispmpotionation

b

Scheme 3.4: Proposed mechanism of the formation of products fiom the protonated electron adduct of DHMP at pH 4.5.

products could be made. However, a stepwise elimination of water from the

dimeric products of pyrimidine is reported using conventional chromatographic

t ~ c h n i ~ u e . ~ ~

DMHP also gave similar product pattern like DHMP at pH 6 (scheme 3 -5).

The products g and h (where h is the starting compound, DMHP) are the result of a

disproportionation reaction. The mlz vdue corresponds to the dirner i is however

was not seen in the MS data. On the other hand the products j and k resulting from

the successive water elimination of the dimeric product i were seen (Table 3.5).

The only possible reason for its absence is likely its instability leading to the fast

Kinetic and Spectral Investigurion . . . .I08

water elimination (reactions 3.18 and 3.19). The formation of 1 (reaction 3.20

and 3.2 1) can also be understood as explained in the case of DHMP.

Table 3.6: Products identified fiom the reaction of eVq with DMHP (pH 6 ) by HPLC-ES-MS analysis

Products Molecular Molecular formula Weight Mlz

g. 5,5-dihydro-6-hydroxy-2,4- C~NZOHIO 126 -125, +127 dimethyl pyrimidine

h. 5-hydro-6-hydroxy-2,4-dimethyl C6N20Hs 124 -123, +I25 pyrimidine

i . Bis(5,5-dihydro-6-hydroxy-2,4- c1zN402Hig 250 Not detected dimethyl pyrimidined-yl)

j . 2,4-dimethyl-6-(5,5dihydro-6- el 16 232 -232 hydroxy-2,4-dimethyl pyrimidine- ~ - Y U

Kinetic and Spectral Inws figation ... ,109

Figure 3.16: Typical ES-MS obtained from the HPLC chromatogram after the reaction of e,, with DMHP at pH 6 ( Eluent = water, Flow rate = 0.5 ml/min) A) in -ve ionization mode (RT = 5.3 min), the m/z value -123 is attributed to the compound h and B) in +ve mode (RT = 8.5), the d z value +I27 is attributed to the product g. The products included in Table 3.6 were obtained by scanning the entire region of the chromatogram.

Kinetic and S~ectral Invesiiaation .... 1 10

di:

VII

Scheme 3.5: Proposed mechanism of the formation of products from the protonatsd electron adduct of DMHP at pH 6 .

3.5 Conclusion

The preferentid protonation at C(5) position of the electron adduct of

DHMP by H' and phosphate is demonstrated from its spectral features and with

the help of its reaction with the oxidant, methyl viologen using pulse radiolysis

technique. Similar species is formed on H' attack at the C(5) position of

DHMP. The formation of such C(5) protonated C(6)-yl radical is only a minor

process in other pyrimidine derivatives. The electron transfer from a carbon

protonated electron adduct of thymine to molecular oxygen or nitro aromatic

radiosensitizers is a restricted process." The present study demonstrate that the

Kinetic and Spectral Invesligarion ... . I 1 1

nature of the substituents at the pyrimidine ring has a profound effect on the

redox properties of the electron adducts as the C protonated electron adduct of

DHMP transfers an electron to radiosensitizers such as MV~'. The C(5) and

C(6) protonation of the electron adducts of DMHP, DMU and MU by phosphate

is found to be a very slow prww at least in the pulse ladiolysis s a l e ( k < 1 o4 s-I).

Product analysis from the reaction of e-, with pyrimidine has been a relatively

unattended area and this study identified a number of important products

resulted from mainly disproportionation and dimerisation of the protonated

electron adducts of DHMP and DMHP. These data, reported the first time with

HPLC-ES-MS, is expected to provide some important guide lines for the

identification of the end products resulting from the electron attachment of

DNA model systems in future.

Kinetic and Spectral Invesrigcltion . . . . 1 1 2

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