- -- and the reactions an d d-shodhganga.inflibnet.ac.in/bitstream/10603/7206/11/11...rule cunstants...
TRANSCRIPT
Chapter 5 - _ ___ ___ - _ _ - - - --
Rate constants and product studies of the reactions did of ozone with nucleobases, nucleotides, an re ate
d d- compoun s in aqueous me ium - --
with nitrite andlor buten-3-01 and, the indigo method (when the rate constant was < IU3 dm3 mol-' s-I). Depending on the degree of protonation, the rate constant (in units of' dm3 mol-' s") varies substantially, e.g. in the case of
k = 18 (protonated), k ;- 1.4 x 10' (neutral) and k = 1.5 x lo6 (deprotonated). A similar variation has been found with the other nucleobases. Upon deprotonation, the mechanism of the ozone reaction may also change; r.g. no singlet dioxygen {O,'%) is formed in its reaction with 5-chlorouracil, but when the 5-chloro-uracilate ion predornitlates, it becomes a major product (- 42%). In the case of adenine and its derivatives, and thus also in the case of DNA, .OH is produced (via 02*" an intermediate). For the determination of their intrinsic ozone rate constants, tert-butylalcohol was hence added as .OH scavenger. The intermediates and end products studies uf the reactions of ozone with thymine and thymidim are carried out. The instant total peroxide yield is 100% in both systems. En thymine, 25% H202 is observed h m catalave and R2S a w y and it is increassd upto 4Vh with half life -12 min. Two organic peraxides w m ident5ed as I-hydropffoxymethylen-3-~2-oxo-prg,anoyl)-u and 5-hydroperoxy-5- methyhydmtoin. At high pH, thymine glycol and singkt oxygen are formed. In the
Publications from this chapter
1 . Jacob A. Theruvathu, R. E'lyunl, C. 1'. Aravindakumar, C. von Sonntag, Rate constants of ozone reactions with DNA, i t s constituents and related compounds, J. Chem. Soc. Perkin Trans.2,200 1,269-274.
2. R. Flyunl, Jacob A. Theruvathu, A. Ixitzke, C:. von Sonntag, Ozonation of thyrninc and thymidine, (manuscript under preparation).
Rule constants undproduct studies ... . . .. --- . ,
118
5.1. General
Ozone is gaining importance in drinking-water processing, because it is not
only a good oxidant but also a powerful disinfectant.'"'~''5~''7~'75~186~257~294-296
Mechanistic details of inactivation of bacteria and viruses are as yet not fully
understood. About 10' ozone molecules are required for the inactivation of a
ba~ te r iu rn . ' ~~ . '~~ It has been suggested that this is required for the destruction of the
bacterial cell wall and subsequent leakage of cellular contents.lr2 However, ozone
also causes mutations. 1u8.297~2v8 This may he taken as evidence for damage of its
DNA (with the cell remaining adequately intact), but it can not be excluded that
ozone by-products (e.g. formed in the reaction with thc cell wall) have caused the
mutagenic effect. For example, hydroperoxidm and H,Q are typical ozonation by-
prducts, and the latter is known to be weakly m u t a g r n i ~ . ~ ' ~ ' ~ In contrast, the
inactivation of viruses is considered to be mainly caused by ozone-induced damage
of their nucleic acids, although damage of the proteins that make up the capsid also
occurs. IF' The considerable spread of inactivation half-lives among various
enterovir~ses '~~ may indicate that, depending on the structure of the virus, there is a
varying competition between ozone damage of the nucleic acid (lethal) and the
proteins (less lethal).
There is already a lot of information available on thc reaction of ozone with
nucleic acids and their constjtuents.~~'Y~2".'87.'87-19F~2X[' Ir)l However, in order to reach a
better understanding o f the inactivation of viruses and bacteria by ozone, a detailed
Rule cunstants and avoduct studies.. . . I19
study covering the rate constants and mechanism of the reactions of ozone is
required. The determination of the rate of reaction of ozone with the nucleic acids
and their constituents by e.g. the stopped-flaw technique, is difficult because of the
strong overlap of the absorption spectra of the nucleobases with that of ozone (for the
data presently available see Table 5.1 ). This has led us to use another approach, the
application of competition kinetics.*'' Deprotonation or protonation of substrates has
a dramatic effect on the rate of ozone reactions. The most striking example is
phenol, whose ozone rate constant increases six orders of magnitude upon
deprotonation.302 As a consequence of this, the reaction of ozone with phenol i s
largely a reaction of ozone with the phenolate in equilibrium [pK,(phenol) = 101,
even in slightly acidic solutions. Similarly, arnines no longer react with ozone when
protonated. 2077303 Also in the reaction of ozone with the nucleobases, one has to take
their protonation/deprotonation equilibria into account. Thus, a detailed study
covering a large pH range is required for a better understanding of the kinetics of
ozone with the nucleobases and with DNA. Such data, together with some
additional information, will be presented in the present study.
~ l t h ~ ~ ~ h ,+here is a handfUl of rewrtsl 9.20.22.36.1 87.195-197.3M on the reactions of ozone
with nucleobase derivatives, some fine mechanistic aspects are still unresolved. Some
stable end products of the ozonation of uracil, thymidine, cytosine and
2'-deoxycytidine systems were rep~rted.'"~"~'"~ In order to study the fine
mechanistic details, we have selected thyminc and thymidine systems, which could
be applied for whole pyrimidine family.
Rate constunis und product s~ucldrs ... . -. 120
5.2. ~etermination of rate constants
In the present study, many rate constants have been determined by
competition kinetics. The theoretical background of the competition kinetics is
explained in section 2.4.2. I . Consider a competitor (C) and a nucleobase derivative
(N) react with ozone [reactions (5.1) and (5.211 and, products PI and P,. The product
of the reaction o f ozone with the competitor (P,) can be monitored while the
products of the reaction of ozone with the nucleobase derivative (P,) remain
unaffected.
C + 0, +PI (5.1)
N + 0, -+ P2 (5.2)
At a given initial ozone concentration ([OJ << [C] and [N]), the final
competition equation can be written as
IP, 1, - k, EC3 + ks.1 [Nl -- k5.2 LN1 = 1 +--- [pi I ks 1 LC1 k5.1 ECI
where k, , and k,, are the rate constants of the reaction (5.1) and (5.2). [P,], is the
concentration of the measured product in the absence of N and [P,] in the p m c e of N.
Plotting ([P,]J[P,] j 1 versus [Nj/[C] yields a straight line with the slope of
k, 2/k5,1. Since the rate constant of the competitor with ozone, k, ,, is known, the rate
constant of ozone with N, k,, can be calculated.
Buten-3-01 and nitrite were used as competitors, which yield one mol of
f~rrhaldehyde'~ and nitrate2"' per mol of ozone respectively. Solutions containing
Rate conslunfs and ~roduct studies ... . 121
substrate and competitor at varying ratios were mixed with an ozone solution, and
the competitor-derived product was quantified.
Around pH 7, all of the nucleobases are present largely in their uncharged
forms (cf; Table 5.1). In the case of uracil and guanine derivatives, protonation
occurs only at very low pH. On the other hand, cytosine, adenine and their
derivatives become protonated already at around pH 4. At high pH, the uracil
derivatives get deprotonated at pH -9.8, as long as one of the nitrogens remains
unsubstituted. In thymine, for example, the second nitrogen may also deprotanate at
very high pH. Most of the reported pK, values of 6-methyluracil were around 9.8.Tns
Because of the importance of pK, value for our study, we have remeasured it by
following the change of the ratio of the absorptions at 288 nm and 270 nm as a
function of pH (c:f.' inset in Figure 5.2), and our value of 9.8 is in agreement with
the rnajori ty of the literature data.
As can be seen from Table 5.1, the rate constants span more than five orders
of maptude. When the rate constant is small (c I d d d mol-' s-I), the indigo method is
most suited. For higher rate constants, usually buten-3-01 (k = 7.9 x 1 o4 d m h o l ' ' s-' ) Iu
has been wed as competitor. At pH > 10, the competition with butm-3-01 no longer yields
fully reliable values. For unknown reasons, the formaldehyde yiclds tend to be less
reproducible; they are typically lower than expected. In addition, when
tcrt-butylalcohol is added as 'OH scavenger, buten-3-01 can no longer be used as
competitor, since tert-butylalcohol also yields formaldehyde in 'OH-induced
Rate uonstunt.~ and aroduct studies ... . 122
reactions (-30%).2R9 Thus, under these conditions we used nitrite as competitor for
which rate constants of 3.7 x lo5 dm3 mol-' s-I, 3.3 x 1O" dm' mol-' s-' (at room
tempadture) and 1.6 x 1 05 dm3 mol-' s-' (at 1 1 O C ) are reported.?" The experiments were
done at 2 1 (f 1 ) "C, and at this temperature the rate constants of ozone with butene-3-01
and nitrite were redetermined using the stopped-flow technique under the conditions of
first-order kinetics (ten-fold excess of substrate) as 9.1 x 104 dm" mol-' s"' (c$ ref. 306) and
(3.5 - 4.0) x 10' dm3 mol-I s-', respectively, i.e, in agreement with the literature values.
To assure internal consistency, the competition of butene-3-01 vs NO, has been
studied. This can be carried out by measuring either fbddehyde or nitrate yields [cj.'
reactions (2.1 2) and (2.1 3)], e.g. as a a c t i o n of the [buten-3-ol]l[NO, 7 ratio.
The formaldehyde a s s a y is sensitive to elevated NO,- concentrations which
suppress the colow formation, and therefore the competition had to be done at low
concentrations of the competing substrates. Nitrate determination by HPIC is not affected
by the presence of the reactants. These two data sets yield k(N0,-jlk(buten-3-01) ratios of
5.4 and 5.7. Taking the literature data, k(N 0, -) = 3.7 x 1 O5 dm' mol-' s-' and ybuten-3 -01)
= 7.9 x lo4 dm mol-' s-' mentioned above, one anives at a ratio of 4.7. Considering the
inherent uncertainties of all these data, there is an acceptable agreement.
In their reactions with ozone, nitrogen-contain ing compounds, e.g, arnines,3"
can give rise to the formation of 'OH, and the ensuing ' 0 ~ - i n d u d reactions can distort
Rate constants und produd studies.. . . .,--- 123 -----
the kinetics considerably. For this reason, the b~ scavenger tert-butylalmhol has been
added to test, whether its presence has an influence on the rate of reaction.264 As will
be shown below, this precaution i s only necessary in the case of adenine and its
derivatives, i.e. also in DNA. However, the addition of tert-butylalcohol is essential
here. The rate constants determined in the present study are compiled in Table 5.1.
Table 5,l. Compilation of rate constants of ozone with nucleobases and related compounds (in units of dm3 mol-' s") at different stages of protonatiotl as determined in the present study. Literature values'" in brackets
(a) pK, values taken from ref.307, 5-~hlorouraci~'~%rotic acid and iso-orotic acid:" (b) rate constant determined at pH 7, where the carboxylate group is deprolvnated, but not yct une of the nitrogens; (c) n. d. - not determined; (d) in the presence o f 0.2 mol dmd3 ~vrt-butylalcohol; (e) see text; (f) the average rnr~lecular weight of the nucfeotides in DNA is taken as 350 Da.
Deprotonated
-3 x 10"
1.2 x 10"
-
9.2 x lo5 6.0 x lo5 1.3 x 10"
11. d. (Lj
n. d. 1.5 x 10'
-
1.3 x 10' (e) ( e )
4.0 x 10' n. d.
-
Substrate
Thymine
Thymidine 5'-dTMP 1,3-Dimethyluracil Uracil 6-Methyluracil 5-Chlorouracil Orotic acid(b' Iso-nrotic acid'b' Cytosine
2'-Deoxycytidine Cytidine 5'-dCMP ~ d e n i n e ' ~ ' 2'-~eoxyadenosine'~' ~d en~sine(~' 5'-dAMP Guanosine 2'-Deoxyguanosine 5'-dGM P DNA
Protonated
-
-
-
-
-
- - 18
44
40
5 5 5
< 300 n. d.
-
pK, values'"
9.9, >12
9.8 10.0 -
9.5, >I3 9.8 8.0
2.1,9.45 4.2, 8.9
4.6, 12.2
4.3 4.15 4.6
4.15, 9.8 3.8 3.5 4.4
2.5,9.2 2.5, 9.2 2.9,9.7
Neutral
4.2 x lo4 (2 .3 x 104) 3.0 x lo4
(1.6 x lo4) 2.8 lo3
650 140
4.3 x 10' 5.9 x 1 o3 3.7 x 10' 1.4 x 10'
(930) 3.5 x lo1 3.5 x lo7
( 1 . 4 ~ 10') 12 14 16
(200) 1.6 x 1 o4 1.9 x lo4 (5 x lo4) 4 1 Otd. CI
Rule constanis and product studies ... . 124 , . . - -. - - - -. - -
5.2.1. Uracil, thymine and their derivatives
The pH dependencies of the observed bimolecular rate constants of ozone
with thymine and 6-methyluracil are shown in Figures 5.1 and 5.2. Under these
conditions, where their neutral forms predominate, the reaction is relatively sIow,
but with increasing pH the rate of reaction increases. The position of the methyl
group at the C(5)-C(6) double bond has a noticeable effect on the rate of reaction.
Comparing the values at low pH, thymine reacts more than two orders of
magnitude faster than 6-rnethyluracil. The other uracil derivatives fa1 1 in between.
In contrast, the rate constants of the corresponding deprotonated forms are very
similar; they all center at around 1 Oh dm3 mol-' s-' (c,L Table 5.1).
Figure 5.1, Observed rate constant of the reaction of thymine with ozonc as a function of pH. The solid line is calculated on the basis of the rate constants measured at pH 2 , 1 1 and 13 and the established pK, values of 9.8 and 12.5. Inset: analogous data for thymidine. Competition with buten-3-01 (I) and nitrite in the absence (0) and presence (A) of tert- butylalcohol
Rate constants and product studies.. . . . -. -- 125
Upon raising the pH, the rate of reaction increases (Figures 5.1 and 5-21, and
in the case of thymine, experiments were extended into the pH range, where the second
nitrogen deprotonates, and a further increase in rate is observed (Figure 5.1 ) . At pH 1 3,
a value of 6.5 x 10' dm3 molL' s-' is found and this is attributed to the rate constant for
doubly deprotonated thymine. The determination of rate constant for the mono-
deprotonated thymine is associated with some ermr and can only be estirnatd h r n the
inflection point near pH 1 0, h m where we obtained a value of -3 x 1 0' dm3 moP1 s- ' .
Figure 5.2. Observed rate constant o f the reaction of 6-methyluracil with ozone as a function of pH. The solid line is calculated on the basis of the rate constants measured at pH 2 and 1 1 and the pK, value of 9.8 based on the data in the upper inset. Lower inset: a typical competition plot using buten-3-01 as competitor
As can be seen from Figurc 5.1 and 5.2, there is a considerable disagreement
between the measured pH dependencies (dotted lines) and the ones calculated on
the basis the pK, values and rate constants given in Table 5.1 (solid lines). 'The
Rate conslants and product studies .... 126
experimental curves suggwt that the reactions of compounds whch are noticeably more
acidic than represented by their pK, value would indicate. In the reaction of ozone with
olefins, there is evidence at low temperatures for the formation of a charge transfer
complex, which subsequently decays into products."ofi' If in the present case such a
charge bansfer complex is sufficiently long-lived it could turn more acidic than its
parent. As a consequence of this, the rate of reaction could be higher than the one
calculated on the basis of the pK, value of the parent. We are aware, that
spontaneous deprotonation of these compounds is slow due to their high pK, values
(-0.1 - 1 dl), but the presence of buffer that had to be added to keep the pH speeds
up the rate of deprotonation. Nevertheless, the ozone charge transfer complex
would have to be more than one unit more acidic and also quite long-livd to show this
effect. In this context, it is intriguing that only a very small, if any, deviation is tbund for
thymidine (inset in Figure 5.1), uracil and 5-chlorouracil (Figure 5.4) and also no such
deviation is found for the cytosine, adenine and guanine systen~s (see below).
Figure 5.3. A typical nwe obtained by the bleaching of indigo (15 x 10.' mol dm;') by ozone (8 x 1 o*' mol dm-3) in the case of 6-methyluracil (4.5 x 1 O3 mol dm-') at pH 2.1. Inset: logarithmic plot of the curve
0.24
- 5 0 0.20 0 w - V1 II
0.16
- 1
-
- o 20 40 60 80
Time (6%)
1 1 1 I I l l 1 0 20 40 60 80 100
Time (sec)
Rate constants undprr)Juet shdies ... . 127
The formation of *OH as the reason for this unexpected deviation has been
excluded for the thymine and 6-methyluracil systems. This has been done by
adding tert-butylalcohol in excess (in the absence of a competitor) at around the pH
where the deviations are most noticeable. No formaldehyde (due to 'OH-induced
tert-butylalcohol degradation) was detected. This is in agreement with the
observation that at high pH, addition of tert-butylalcohol had no effect on the rate
of reaction (nitrite as competitor).
Figure 5.4. Obsaved rate constant of the reaction of ozone with (a) 5-chlorouracil and (b) uracil as a function of pH. The solid line is calculated on the basis of the rate constants measured at pH 2.2 and 9.2 and the pK, value of 8.02 in the case of 5-chlorouracil and that in the case of uracil are at pH 4.5 and 1 1.3 and the pK, value of 9.5. Rate constants are measured using buten-3-01 (o), indigo (A) and nitrite (m) methods
It has been mentioned above that there is an increase in rate in the range
where the deprotonation of the second nitrogen of thymine occurs. Thc rate
constant of this doubly deprotonated species might have been underestimated
(experiments could not be extended beyond pH 13; the high salt load prevented
nitrate determinations by ion chromatography). Yet, the marked deviation of the data in
Rate constants and product studies ... . 128
the pH range 9- 12.5 from a slope of unity indicates that even the assumption of a much
higher rate constant for doubly charged thymine can not be explained the observed
deviation from the expected pH dependence.
It is remarkable that the position of the methyl group, i-a when the rate of reaction
with thymine is compared with that of 6-methyl uracil, has such a dramatic effect (a factor
of 300, c.f. Table 5.1). Considerable differences in the rate constants of isomers, e.g.
1 , 1 dichloroethene (k = 1 1 0 dmhmol" s"), cix- 1,2dichloroethene (k = 540 dm3 moi- ' s-' )
and trans-] 2-dichloroethene (6500 dm3 mol-' s-' ) have been noticed before,'" but there
the rate constants differed by a factor of 60, at the most. Even more surprising is the
observation that the rate constant of 6-methyl uracil is lower than that of uracil.
Typically, m additional methyl group at the reacting C=C double bond increases
the rate of reaction by a factor of -4.'" A very marked difference between these
uracil derivatives and simple olefins is also noticed when 6-methyl uracil is
compared with 5-chloro uracil. One would have expected that the former reacts
considerably faster with ozone than the latter, but the reverse has been observed
(cJ Table 5.1).
The observed increase in rate upon deprotonation of thyrmne is connected with a
change in the reaction mechanism. While the neutral nucleobase does not give rise to the
h a t i o n of singlet dioxygen (o,'AJ, de~rotonated thymine yields Q' A, in -8% y ~ e l d . ~ ~ ) ~
Possibly, it is formed via reactions (5.4H5.6) (Scheme 5.1 ).
Rate constmts andproduct studies .... . 129
Thymine deprotonates at N(1) as well as at N(3) [equilibrium (5.4)l. Dipolar
addition of ozone to thymine deprotonated at N(1) gves rise to an isopyrimidine
hydrotiioxide [reaction (5.6)]. Since the chemistry of these short-lived intermediates is
k n ~ w n , ~ ~ . ' ' * a product study which is on the way may help to elucidate mechanistic
details. Thus far, our mechanistic proposal is supported by the observation that
thymidine which can deprotonate only at N(3) does not afford any singlet dioxygen
at high h this context, it may be worth mentioning that 5-chloro uracil
shows this change in mechanism even more strongly; no singlet dioxygen (O,'A,) is
formed at pH -3.5, but when the 5-chloro uracilate ion predominates, it becomes a
major product (-42%).205
Scheme 5.1
5.2.2. Cytosine, cytidine and 2'-deoxycytidine
As can be seen from Figure 5.5, cytidine and 2'-deoxycytidine react
somewhat faster than the free base. This is most likely due to the electron-donating
Rate constants and product studies .... 130
property of the sugar moiety. Upon protonation, their rate of reaction drops by two
orders of magnitude. Whereas the nucleosides do not show an increase in rate at
high pH, cytosine does. At high pH, the latter deprotonates (pK, = 12.2). A similar p&
value (pK, = 12.5) is reported for cytidine, but here, the deprotonation is suggested to
occur at C(2')?I3 Apparently deprotonation of the sugar moiety hsts practically no effect on
the reactivity of these compounds with ozone.
Figure 5.5. Observed rate constant of the reaction of ozone with cytosine (m), cytidine (A) and 2'-dwxycytidine (A) as a function of pH. The solid line is calculated on the basis of the rate constants and the pK, values given in Table 5.1
5.2.3. Adenine, adenosine and 2'-deoxyadenosine
In the case of adenine and its derivatives, the addition of ted-butylalcohol on the
observed rate of reaction (Fibwe 5.6) has a dramatic effect (cj.' a1 so refs. I 9, 1 94). In its
absence, the rate constant with adenine became too fast for the indigo method, i.e.
the apparent bimolecular rate constant must have been fgstcr than -10' h' mol-I s-'.
Rute constants und aroduct studies ... . 131
This points to the intermediacy of 'OH at one stage. Indeed, when m-butylalcohol is
added in large excess (0.1 mol M3), we observed the formation of considerable amounts
of formaldehyde (-0.2 mol per rnol ozone) in the case of adenine as well as
2'deoxyadenosine. Formaldehyde is an important product of the *OH-induced oxidation
of terr- b~ty ia lcohd .~ The observed formaldehyde yield of -20% relates to -60% 'OH
(the use of ted-butylalcohol in the determination of *OH yield in ozmation reactions has
been understood recently).2bJ
Figure 5.6. Observed rate constant of the reaction of ozone with adenosine (a, Cl), and 2'-deoxyadenosine (A, A) as a function of pH; the solid line is calculated on the basis on an assumed limiting rate constant of 1.3 x 1 O5 dm3 mol-' s" as in adenine and the pK, values given in Table 5.1. Inset: adenine; the solid line i s calculatecl on the basis of the rate constants and pK, values given in Table 5.1. Solid symbols refer to experiments using the indigo method, open symbols to competition kinetics with nitrite as competitor. In all experiments, tert-butylalcohol (0.1 rnol dm-3) was added as 'OH scavenger
Rate conslunl.r. and product studicLs ... . -
132
The reduction potential of adenine and its derivatives15 is too high for an
electron transfer to occur in the ozone reaction (note that in the case of guanosine
which has a much lower reduction p ~ t e n t i a l ' ~ ? * ~ ~ no 'OH is formed, see below). Thus
there must be another reason for the intermediacy of 'OH in this system. The most
likely precursor for 'OH is considered to bc 0,". Here, it suffices to mention that
tetranitromethane added as 02' scavenger, is substantially degraded (via the
intermdacy of nitro form anions). Addition of tert-butylalcohol reduces the observed
rate constant by more than a factor of two. This indicates that the radicals that are
formed upon 'OH attack on adenine/2'-deoxyadenosine must induce a chain reaction.
The rea&on of ozone with the cyanide ion also proceeds via a chain reaction.'" 'fiere,
tert-butylalcohol cannot fully suppress it, i.e. there must be an additional chain carrier.
A similar situation may prevail here, but further work will be required to elucidate this
system in more detail.
In the case of adenine, the pH dependence of rate constant can be adquatcly
fitted by the rate constants and the pK, values given in Table 5.1 (cf. solid line in the inset
of Figure 5.6). The rate of reaction with denosine and 2'-deoxyadenosine also increasa
with increasing pH, and these data can he fitted (solid line in Fibwe 5.6) if the p& of 12.5
and the limiting rate constant of adenine are taken. It has been noted above that in thc case
of the cytosine nucleosides, no further i n m e of the rate of reaction has been observed in
the high pH range (c: f.' Fib- 5.5). This has been explained by the fact that the pK. value
at very high pH is athibutad to a pK, value of the sugar moiety and that these anions do
Rute constants and product studies ... . 133 - --
not react with a rate higher than 3.5 x lo3 drnho l - ' s-' given by neutral cytidind
2'-deoxycytidine themselves. At the highest pH that can be measured with some
confidence, the adenosind 2'-deoxyadenosine rate wnstant approaches - 10" dm3 mol-I s",
not much higher than the above value. There is no indication that this value is a l i~ni ting
one, and indeed it is impossible to fit the data with such a low value. The solid line
in Fibure 5.6 has been constructed by taking the pK, of 12.5 and the limiting value
of the adenine system (1.3 x 1 O5 dms mol" s-') . This is only acceptable as long as
the pK, at -12.5 is due to the deprotonation of the adenine moiety. We have
remeasured the absorption spectrum of 2'-deoxyadenosine at pH 14 and at the long-
wavelength band, no changes, neither E nor h,,,, with respect to its spectrum at pH 7 are
observed (below 230 nm, the high absorption of OH- prevents firm measurements). This
would be in agreement with the earlier conclusion that the pK, at -12.5 is due to a
deprotonation of the sugar moiety,"'"*~" '."' however not with the observed high
reactivity. Thus, the reason for this unexpected observation remains unresolved.
The earlier determination of the rate constant of an adenine derivative,
5'-dAMP,'Y4 deserves a comment. Its rate constant has been estimated at 2 x lo2 dm3
mol-' s-' (in thc absence of terf-butyldcohol) by measuring its degradation relative to that
of dCMP for which a rate constant of 1.4 x 1 O3 dm.' mol-' s" had bcen determined by the
stopped-flow technique under close to second-order conditions. In the presence of
teut-butylalwhol, the relative rate constant dropped by nearly an order of' magnitude. In
view of this, these data agree reasonably with our value of -15 dm' mol-' s-'. It has been
Rate constunts andproduct studieLv .... 134 --
mentioned above that in the absence of tert-butyldcohol, we estimate that the observed
rate constant of adenine and its derivatives is 2 10' dmbmoI"' s". This is hgher than the
value of 2 x 1 d dm3 mol-' s" obtained in the dCMP competition. Th~s is not surprising.
When 'OH radicals play a significant role in this system, they also attack dCMP
which is subsequently consumed, i.e, the estimate of the dAMP rate constant comes
out too low,
52.4. Guanosine and 2'-deoxyguanosine
Guanosine and 2'-deoxyguanosine react about equally fast with ozone
(Table 5.1). Upon deprotonation, the rate of reaction increases 250-fold. As can be
seen h m Figure 5.7, the experimental data can be adquately fitted using the rate
mmtants and pK, values given in Table 5.1 (experiments at very low pH indicate that
upon protonation the rate constant falls below 300 dm' mol-' s-', data not shown). Addition
of twt-butylalwhol had no effect on the rate of reaction nor was any formaldehyde formed
under these conditions. This excludes any electron tmnsfkr h i n the guanine moiety to
ozone, although guanine has the lowest reduction potential of all nucleoba~es"~~ and
mpecially in basic solutions where its anion predominates, it is readily oxidised by many
otherwise only weakly oxidising agents (c$ ref: 3 1 6).
Rate constants and product studies .... 135
Figure 5.7. Observed rate constant of the reaction of ozone with guanosine as a function of pH; the solid line is calculated on the basis of the rate constants and the pK, value given in Table 5.1. Competition with 3-butenol (a) and nitnte with (A) and without (A) toy-butylalcohol(0. I mol dm5
5.2.5. DNA
As has been noticed before," 'OH plays an important role in the reaction of ozone
with DNA. From ref, 1 9 and the present study, it is clear that 'OH radical formation must
be due to the reaction of ozone with the adenine moiety. For the determination of the
intrinsic ozone rate constant with DNA, tert-butylalcohol had to be added. Under
such conditions, the rate of reaction o f DNA is only 450 dm3 moi-' s-' (in the absence of
tcrt-butylalcmhol k, = 1 . I x 10' dm.' mol-' s-'), i.e. much lower than that of the weighted
average of the nucleubases. In the case of 'OH, which reacts with the nucleobases and
their derivatives at close to diffusion-controlled rates (k - 3 x 10' dm3 mol-' s"),& the rate
of reaction of 'OH with DNA is considerably lower (k = 2.5 x I I)s dm-' rnol-' s-'),"'~ since in
Rate comtants undproducl studies ... . .. - - - - 136
th~s non-homogeneous reaction with the macromolecule DNA, two terms, a diffusion
term (k,,,) and a reaction term (k) have to be wnsidmedNR The observed overall rate
constant (k&) is the harmonic mean of these two rate constants [equation (5.711.
Since, in contrast to 'OH, ozone reacts with the nucleobases at rates much
below the diffusion-controlled limit, the second term must fall away, and the rate of
reaction of ozone with the nucleic acids is only given by the first term, ie. it should
be close to that of the weighted average of the concentndtions of the various nuclmbaqes
in the nucleic acid times their rate constants with owne. This is not observed. Structural
effects such as hydrogen bonding between the nucleobases may he a reason lbr the
strong reduction in rate.
Corresponding experiments with RNA were not carried out, because it was
not guaranteed that the commercially available RNA is of similar purity and
sufficiently double-stranded to yield complementary data. From the rate constants
given in Table 5.1, one would assume that in RNA, the guanine moiety is the most
likely one to become degraded upon ozone treatment. This has been indeed observed.""
In DNA, the situation might be somewhat diffcrcnt. Besides guanine, thymine may be
the other preferred target.
Rate constants and producl studies .... -- - 137
5.3. Product studies of the reactions of ozone with thymine and thymidine
According to a detail4 rate constant and product study involving the
characterisation of short-lived hydroxyalkylhydroperoxides, it is known that there
is a considerable influence of the elecbon-donatindwithdrawing power of substituents
(D vs. A) on the final products [reactions (5.8)-(5.12)].IW An electron-donating group
D such as a methyl substituent favours the formation of the hydroxyhydroperoxide at its
position [reactions (5.9) and (5.1 l)]. Th~s has been rationalised by a stabilisation of the
carbocation formed in d o n (5.9).
I HO-C-
H e - 0
Scheme 5.2
In thymine and thymidine, ozone will add to the C(5)-C(6) double bond.
Due to the electron-donating properties of CH, group, the Criegee intermediate
formed as in reaction (5.8) will preferentially open according to reaction (5.9). It
Rate constunts and product studies ... . 138
will be shown that the substituents at N( I ) (H in thymine, Zdeoxyribosyl in thymidine)
have a pronounced effect on the subsequent reactions of the carbocation formed in
reachon (5.9). A detailed analysis of the ozonation products and intermediates of
thymine and thymidine are presented below.
5.3.1, Thymine
Ozone reacts with thymine at a rate constant of 4.2 x lo4 dm" mol-' s-I, and
upon deprotonation (pK, = 9.9), the rate constant increases to 3 x 10' dmmolL ' s-'.'
At pH 6.5, where the majority of experiments have been carried out, the
contribution of the anion in product formation can be neglected. Under typical
experimental conditions ([thymine] = 1 x 10" mol dm", [O,] = 1 x 1 OU4 in01 dm-j),
the half-life is 0.016 s, i.e. the reaction proceeds at the time scale of mixing the two
solutions.
5.3.1 .I. Conductometry and ion chromatography
When ozonolysis is carried out in a conductometric cell, the conductance
rises with a biphasic kinetics (Figure 5.8). The first step ("prompt") is too fast to be
resolved kinetically, the second one ("slow") is of first-order kinetics and occurs
with a rate constant of 1.1 x 10" s-' at 1 8°C. When the experiinents were carried out
at 3 'C, the conductance build-up becomes slower by approximately eight times.
The rate constant of the second increase in conductance at 3 "C is 1.3 x 1 0-4 s-'. The
rate constants relevant to this study are compiled in Table 5.2.
Rlite constanis und product studies.. . . 139
*.--- - a-- - - - m - - - -m ,..-* -
/,r- -. .. .
i o f ..- ...,-., -.. I . . 3 - 7 1 "D .,,,,,,
$1 ' " 1 .mh l
Y
Timeimin Figure 5.8. Ozonolysis of thymine in aqueous solution at 18 "C. Formation of acid
(expressed as mol acid per mol ozone consumed) as a function of time followed by conductance measurements. Inset: First-order kinetic plot of the slow part of conductance increase at 3 "C ($3) and 20 OC (m)
The calibration of the conductometnc set-up has been done with sulfuric acid. The
acids that are formed in this system are weak acids, the acidic hydroperoxide
6 ( 1 -Hydropaoxymethylen-3-(2-0x0-pmpanoyl) pK, = 4.0, see below) and formic
acid (p&=3.75) and are hence partially pmtonated ([ozone] = 2 x 1 o4 In01 dm-' in these
experiments). The "prompt" yield of acid (1 -Hydroperoxymethylm-3-(2-oxo-prop1oyl)-
urea} is calculated as -34% (with respect to consumed ozone). Further acid (formic acid)
is released in the slow process whereby its yield increases to -80% (Figure 5.8).
The formic acid formed during the reaction is determined by ion chromatography
(IC) and its yield was 75% after ozonolysis. Since the eluent is basic, the release of formic
acid speeded up in the column. When ozonated thymine solutions were treated with a base
(KOH, pH 10.8,90 min, I 8 "C), f i e r formic acid is released, and the yield is being now
100%. The yelds of various species are compiled in Table 5.3.
Rute constants and product studies .... 140
Table 5.2. Compilation of rate constants relevant to the ozonolysis of thymine
Reaction Thymine + 0, -+ products
Rate constant 3.4 x l 04 dm3 mol-' s-'
Thymine anion + 0, ---b products "Prompt" release of 6 "Slow" formic acid release, 18 "C
6-8 t 1- -+ products 43 dm3 mol-' s-'
19 + I - --+ products 7.5 dm3 mol-' s-' 6-8 + Fe(CN):- --b products 0.4 dm3 rnol-' s-' 19 + F ~ ( c N ) ~ - -b products 1 d m h o l - ' s-'
4.2 x 1 04 dm3 mol-' s-'* 3 x 1 O6 dm3 mol" s-" > 70 s-' 1.1 x 10"s- '
"Slow" formic acid release, 3 "C Decay of 6, 1 8°C Slow H,O, release, 1 8 O C
* b r n 113.6; for detailY about productslintermediatm see sation 5.3. I. h
1.3 S-I
1 .O 1 u3 s“ -1 x 10" s-'
HN H N OOH
0 0-OH H
H 6 7 8 14 19
In order to test whether the fast conductance increase can be resolved hetically,
stopped-flow experiments with conductometric detection were k c d out. As can be seen
from Figure 5.9, the build-up of conductance foliows pseudo f i s t order-kinetics, and the
rate of reaction depends on the thymine concentration. Thc rate constant derived from the
data shown in the inset of Figure 5.9 ( k = 3.4 x 1 o4 dm-' mol-' s-') is in agreement with
the value obtained by competition kinetics (4.2 x lo4 dm3 mol" s" ) .V t is thus
concluded that the rate-determining step in this fast conductance increase is the
reaction of ozone with thymine, i.e. these data do not give any further hint to the
nature of the intermediate that may be involved in the fast release of a proton.
Rulr ronsttmts and product stuu'le,~. . - . - - - -. -. 141 -
Figure 5.9. Ozonolysis of thymine ( I x 10.' rnol dm-') in aqueous solution at 18°C. Increase of conductance in the reaction of ozone with thymine as followed by stopped-flow with conductometric detection. Inset: k,,, as a function of thymine concentration
0.8
> 0.6 . ?L d 0.4
0.2
Table 5.3. Compilation of yields (with respect to ozone consumed) in the ozonolysis of thymine
-.. --- . . . .. .
-
-
-
- [Thymine] I mM
I
0.2 0.3 0.4 0.5 Time / s
'measured using ion chromatography, ' from ref. 205
Process "Prompt" acid formation (conductometry) "Slow" acid release (conductometry) Formic acid release* Total formic acid rebse at high pH* Total hydroperoxide (immediate) Total hydroperoxide (after 2 h) Hydrogen peroxide (immediate, catalase assay) Hydrogen peroxide (immediate, R,S assay) Hydrogen peroxide (after I h) Organic hydroperoxides (immediate) Organic hydroperoxides (after 1 h) 1 -Hydroperoxymethylen-3-(2-0x0-propanoyl-urea 6 5-Hydroxy-5-methylhydantoin,l4 (aRer R,S treatment)
5-Hydroxy-5-methylhydantoin, 14 (after R,S and OH- treatment) Singlet dioxygen (at high p ~ ) '
.-
Yield / % 34 50 75 100 100 93 25 2 5
40 75 53 34 h7
1 00
8 --
Rule constants an J product studies.. . . -. , 142 --
5.3.1~2. Formation and decay of hydroperoxides
The yield of total hydroperoxide is 1 000h as assayed with molybdate-activated
i d d e (Figure 5.10). Upon treatment of the ozonated solution with catalase, the
hydroperoxide yield is reduced to 75%. Catalase is well known for complete1 y degrading
H,O,. Thus, the yield of H,O, present in right after ozonolysis is 25%. However, we
have observed that formic peracid, a conceivable product in the present system, is
also rapidly degraded by catalasq2" and further experiments have been carried out
to ascertain that the hydroperoxide that is eliminated by catalase is indeed H,O,. Recent
experiments demonstrated that some organic sulfides can be successfully employed to
distinguish between highly reactive organic peroxides and ~ , 0 ~ ? ~ ~ * ~ ~ ~ For example,
bis(2-hydroxyethy1)mlfide rapidly reacts with formic peracid (k = 220 d m b o l " sL')2M
yielding formic acid [i.e. leading to an incrmse in conductance, pK,(HC(O)OOH) = 7.1,
pK(HC(0)OH = 3.81, whereas its reaction with H,O, is negbgible in comparison.
However, when bis(2-hydroxyethy1)sulfide ( 1 x 10'%01 dm") has been
added to the ozonated thymine solution, no additional increase of conductance i s
observed and the slow increase in conductance shown in Figure 5.13 is suppressed.
Instead, a dec,rease of conductance (k = 50.6 dm3 mol-' s") is observed. This can be
taken as the evidence for the fast increase of conductance is due to an acidic
peroxide 6. The slow increase of conductance also must have a hydroperoxide
precursor, and formic peracid is not a product of the ozonolysis of thymine.
Rate comfunt,~ undproduct studies ... . 143 ---.--
Therefore, it was of interest to evaluate the pK, value and optical features of this
intermediate, which is described in section 5.3.1 -3.
Figure 5.10. Ozone dependence in the formation of total hydroperoxide I.) and formic acid by HPIC (0) after the ozonolysis of thymine at natural pH. Inset: Ozone dependence in H,O, formation after the desttvction of organic peroxide by bis(2-hydroxyethy1)sulfide
Thc reaction of bis(2-hydroxyethy1)sulfide cannot be used to determine the
yield of reactive hydroperoxides, since its sulfoxide is difficult to detect by HPLC
due to its low absorption coefficient in the accessible wavelength regon. However,
methionine also undergoes the corresponding reaction with reactive hydroperoxides, and
its sulfoxide can be readily detected.zM Using this assay, the yicld of sulfidereactive
hydroperoxides is 75%. A oomplenlentary value (25%) has been determined by
measuring the remaining H,O, yield a h the destru&on of sulfide-reactive
hydroperoxides by bis(2-hydroxyethy1)sulfide (Figure 5.1 0) using UV measurements.
It has been shown that H,O, does not react with bis(2-hydroxyethy1)sulfide at an
appreciable rate.'M Based on the catalase and the sulfide assays, it is hence concludd that
Hare constants und oraduct studies ... . 144
H,O, is present in 25% yield right after ozonation. Thus, at least three kinds of
hydroperoxides arc formed immediately upon ozonation, an acidic hy droperoxide
(assigned to 6 , see below), neutral hydroperoxides (assigned to 7 and 8, see below)
and H,O,. The organic hydroperoxide rmaining after one hour is a further neutral
hydroperoxide (5-Hydropaoxy-5-methy lhydantoin 19, see section 5 -3.1 .h).
Figure 5.1 1. Normalised yields of total hydroperoxides (: 1) and hydrogen peroxide (a) as a function of time after the ozonolysis of thymine. Hydrogen peroxide yi&s were determined after the destruction of organic hydroperoxides by bis(2-hydroxyethyl) sulfide
As can be seen from Figure 5.1 1, the total hydroperoxide yield is not much
(-7%) derreased over time. A k t i o n of the sulfidsreactive p x i d e s decays yielding
mainly H,O,. This reaction proceeds with a half-life of - 12 min according to a co~nputer
fit through the data (m in Figure 5. L 1 ). Thus, it follows the wnc kinetics as the "slow"
conductance i n m e reported above. i.e. a new organic hydroperoxide (assigned to 19,
see below) appears during the decay of the initial peroxides, l 'he yield of H,O, during this
decay of the primarily tbrmed hydroperoxides (6-8) i s much lower (-1 5%) than that of
Rate constants und product studie.~ . . . . , -
145
formic acid (-75%), The loss of total hydmperoxide yield (-7%) also occurs at the same
time scale (c$ Figure 5.1 1).
Hydrogen peroxide does not react with iodide at an appreciable rate unless
activated by molybdate, but the more reactive hydroperoxides do. The
hydroperoxides present right after the ozonolysis (6-8) react with iodide more
rapidly (k = 43 dm" mol-' s-', determined by stopped flow) than the remaining aRer
one hour (19, k = 7.5 dm3 mol" s-'). Accordingly, the apparent k,, of the reaction
changes with time, since the rate constants of these hydroperoxides differ only by a
factor of -5.7, i.e, these two reactions are not well separated kinetically. The
change in k,,, with time gives a half-life of - 13 min, which is close to the half-life
of the intermediate 6a (deprotonated fbrm of 6), at 255 nm (c.J Figure 5.14).
The reactivity of the organic hydroperoxides (immediate 6-8 and delayed 19)
towards Fe(CN);- is very similar. Immediately after ozonolysis and destruction of
H,O, with catalase, the rate constant of the then prevailing hydropmoxides 6-8 was
k = 0.4 dm3 mol-' s-' and after keeping the samples for one hour to let 6-8 decay into
19, the rate constant was k = 1 dm%nolK' s '.
The presence of H,O, and other organic peroxides was observd by post
column derivatisation. A 25% yield of H,O, obtained at naturai pH by post coluinn
technique, which is well agreeable with other assays. Due to the lack of authentic
samples, the yield of other peroxides could not establish by this method.
5.3.1.3. HPLC and UV-spectrometry
Immediately after ozonolysis, a very prominent product (assigned to 6a)
with h,,, = 256 nm (c.f Figure 5.1 2) was ohsewed by HPLC on a rcversed-phase
column with retention time 7.6 rnin using water as eluent. There is also a second
product ( 1 -formyI-5-hydroxy-5-methylhydantoin 1 2) eluting at 8 .3 min. This has
only an end-absorption tailing towards 220 nm. Both products must be more polar
than thymine, since they elute much earlier than thymine ( 1 5.6 rnin).
h l n m k l nrn
Figure 5.12. Ozonolysis of thymine at natural pH: UV absorption spectra of the products obtained using HPLC with diode array detector. Frame A: a, anion of 1 -hydroperoxymethylen-3-(2-0x0-propanoy1)-urea 6a (7.6 rnin); ,:I, assigned to 5-hydroperoxy-5-methylhydantoin 19 (6 rnin). Frame B : A, assigned to I -formyl-5-hydroxy-5-methylhydantoin 12 (8.3 min); A, 5-hydroxy -5-methy I hydantoin 14 (5.4 min, reference ~naterial was avail able)
As will be discussed below, we are forced to account for further organic hydropxides
(assigned to 7 and 8). These products arc expected to have only little W-absorption as
compared to 6a and m y be masked if they have retention times very close to that of 6a.
Hate consfants and ~roduct studies ... . 147
The short retention time of 6a with water as eluent is due to the fact that under this
condition 6 is dqmtonated. When the pH of the eluent is decreased, 6 elutes sorriewhat
later with a concomitant shift of its absorption maximum towards 237 nrn (c:$ inset in
Fibye 5.13). From the data shown in the main graph of Figwe 5.13, the pK, value of 6 is
calculated as 4.0.
Figure 5.13. Percentage of undissociated 1 -hydroperoxyrnethylen-3-(2-ow- propanoy1)-urea 6 as a function of pH as obtained by HPLC with diode array detector (see text). UV spectra o f 6 (solid line: h,, =
237 nrn, eluent: water at pH 2.6) and its anion (6a, dotted line: h,,,,, = 256 nm, eluent: water at pH 7).
The hydroperoxide 6 decays by the same kinetics (Figure 5.1 4, inset: k = 1.0 x 1 0-3 S-I)
as found for the release of formic acid (c$ Figure 5.8). It gives rise to the hydroperoxide
19 which elutes at 6.0 min.
Ratr constants und product sludies... -- -- -. --.-- 148 - ---
20 40 60 Time / mln
- 20 60 100 140
Time / rnin
Figure 5.14. HPLC analysis of ozonatd thymine. Decay of 1 -hydropmxyrnethylene- 3-(2-oxepropanayl)-urea 6(A) and build-up uf 5-hydroperoxy -5- methylhydantoin 19 (m) as a hnction of time. Inset: first-order kinetic plots of the data
The hydroperoxides were wiped out upon the addition of bis(2-hydroxyethyl)
sulfide which shows that these products must be strongly oxidising hydroperoxides.
The hydroperoxide 6 (together with 7 and 8, see below) is rcducd to 12, while 19 is
reduced to 14. The yield of 5-hydroxy-5-methylhydantoin 14 after the completion of the
reduction by the sulfide is 67% of omnc consumd (reference materid for the
quantification of 14 was available). When this solution was treated with NaOH at pl4 10.5
for overnight, product 12 (1 -formyl-5-hydroxy-5-methylhydantoin) also d i s a p p d ,
and the yleld of 5-hydroxy-5-methy\hydantoin 14 has been increased to 100%.
Hydantoin 14 was also confirmed by GC-MS analysis. 0;conatd samples after
rotary evapratiun were subjected to silylation to I'MS ethers fbr betier volatility and
monitored the GC-MS. Three times silylation occurrod (MW - 346) and the prominent
peaks with m/z (%) are 33 1 (loo), 21 6(7), 147(46), 73(h3).
Rute constants and aroduct studies .... 149
HPLC was used to study the acid-base behaviour of the species 6. Thymine
was ozonated at varying pHs (from pH 1-7) and immediately injected into HPLC
with water (with same pH as the solution) as eluent. pH of the thymine solutions
and eluent were varied by the addition of appropriate amount of sulfuric acid or
sodium hydroxide. The spectrum of the first eluting peak was monitored at each
expenmental pH and it was strongly pH dependent. At pH 2, this hydroperoxide
intermediate has a maxima at 237 nm and at pH above 6 is 256 nm. Measuring the ratio
of the absorbances at 237 and 256 nrn, A2371A256, it was possible to calculate the
percentage of protonated form for each experimental pH point (see Figure 5.1 3) and
determined the pK, value of hydroperoxide as 4.0.
The molar absorption coefficient of the anion of 6 at 256 nm must be higher
than that of thymine at the same wavelength. When equal volumes of thymine
(4 x 1W4 mol dm-3) and ozone (2.25 x lou4 11-101 dm-') solutions were mixed, the
absorption at 256 nm rose by a factor of 1.18. Taking the dilution by ozone solution
into account and subsequent decay again (k = 1.0 x 1 0-3 s- ' , ses above) to a level which is
compatible with the expected remaining thymine mncentmtion and no significant
absorption of the remaining products at 256 nm. Taklng the yield of h c species absorbing
at 256 nm as 34% (based on the imnediate conductance formation, c,f: Figure 5.81, its
molar absorption coefficient is calculated to he 2.5 x 1 o4 dm3 mol-' cm-' , i.c. more than
twice of that of thymine at its maximum at 265 nrn (E = 7.9 x 10' dm3 cm-' s-I).
Ku!r com~tants ur~dproduut studies. . . -- 1.50
.- - - --
5.3.1.4. Liquid Chromatography-Mass Spectrometry
At 3"C, the decay of 6 is much slower (1.3 x lo4 s-', c.j: inset it] Figure 5.8)
than at roonl temperature. This eight-fold pmlongtd 11fe time enabled us to carry out
LC-MS measurements with a sample that had been ozonated at 3OC using
acetonitrilelwater (1: 1) as eluent in the positive mode of electrospray ionisation
(Figure 5.15). Smng m/z values of 175 (M+ 1)' and 197 (M-tNa)' was observed
indicating that the molecular weight of 6 is 174 Da. Product 12 does not gve rise to a
mass spectrum under these expenmental conditions. When 6 has decayed, the mass
spectrum of 19 could be taken md this showed a pronounced i / z = 147(M+l)', and
169 (M+Na)' . Also, it gives two strong dimer peaks with m/z values 293 (2M-t 1 )'
and 315 (2M+Na)' i.e. its molecular weight is 146 Da. Similar to product 12, the
final product 5-hydroxy-5-methylhydantoin 14 (authentic reference material was
available) also did not give a mass spectrum under these conditions.
As mentioned above, 6 and 19 are hydroperoxides and are eliminated by the
addition of bis-(2-hydroxyethy1)-sulfide. As a consequence of this, after the sulfidc
beatment, compounds having an LC-MS response are no longer present except for the
resulting sulfoxide (MW = 138 Da) which shows a pronounced signal at mdz = 139,
(M-t I ) ' . The parent sulfide does not give an LC-MS signal.
Rate auastunts and product sfudics ... . 151
l o o (b)
4 A
Timelmin
Figure 5. 15. LC-ES-MS analaysis of ~zonated thymine at natural pH (a) ES-MS spectra of I - hydroperoxymethylen-3-(2-0x0-propanoy1)-urea 6 (b) ES-MS spectra of 5-hydroperoxy-5-methyl hydantoin 19 and (c) chrornat&ram at 230 nm
5.3.1.5. Assignment of the products
Product 6 has a molecular weight of' 174 Da. i . ~ . it contains three oxygen
atoms more than thymine. It is a strongly oxidising hydroperoxide, i.e. the
hydroperoxide funchon must be activated by electron-withdrawing groups. It is fairly a
strong acid IpK, = 4.0). The spectral shift k r n 237 nm to 256 nm upon deprotonation
requires a considerable conjugation of the n system. Its structure is assigned to
I -hydropemxymethylen-3-(2-0x0-propanoy1)-um 6. Product 12 is not peroxide. Upon
basic hydrolysis, it is converted into 5-hydroxy-5-methylhy dan toin 1 4 and formic acid.
So product 12 is assigned to 1 -formyl-5-hydroxy -5-methylhydantoin.
Product 19 is a hydroperoxide with the molecular weight of 146 Da. Its
precursor is 6 (and 7 plus 8). Upon reduction of 19 with sulfide, 5-hydroxy-5-
lnethylhydantoin 14 is formed. Hence 19 is assigned to S-hydropmxy-5-
methylhydantoin.
5.3.1.6. Mechanistic aspects
The release of formic acid and other conducting species at various steps has
considerable importance in the proposed reaction mechanism. It is observed that fonnic
acid is released in two steps, "slow" and "very slaw" (hydrolysis at high pH). The
"prompt" increase in condu-ce is due to the formation of an unstable species 6 , which
has a pK, 4.0, The formation and decay of this acidic organic hydroperoxide,
which shows a very strong UV absorption, have to be accounted.
Rat< conslants an Jproduut studies ... . -- 153
Ozone is a strong electrophilic agent and will hence preferentially add to the
C(5)-position of thymine 1 [reaction (5.14)). The zwitter ion 2 thus formed will
close the ring thereby forming the Criegee intermediate 3 [reaction (5.1 5)l.
Scheme 5.3
The Criegee intermediate 3 opens according to reaction (5.16) yielding
zwitter ion 4 (Scheme 5.4). The highly absorbing peroxide 6 is formed by losing a
H at N(1) position of 4 [reaction (5.18)]. Formation of this peroxidic intermediate is
responsible for the initial fast conductance ("prompt") increase. Hydroperoxides
have generally high pK, values [c..f: p&(H,O,) = 1 1.8; pK(HC(0)UOH) = 7. I], and
the hydroperoxide hnction of 6 will thus be protonated at around pH 7. In
competition with reaction (5.1 81, the zwitterion 4 may react with water yielding thc
a-hydroxyhydroperoxide 7 [reaction (5.19)]. One may also consider that the
zwitterion 4 may form 8 upon losing the acidic proton at N(3) [reaction (5.20)].
However, the strain exerted by the four-membered ring may not be much in favour
of this reaction. The hydroperoxide 6 has a conjugated x system and shows a strong
absorption at 237 nm (,c$ Figure 5.13). The proton at the N(3) is acidic
[equilibrium (5.23), pK,(6) = 4.01, and the absorption maximum is shifted to 256
mn upon deprotonation. The fast build-up of conductivity that occurs at the same
Rate consfants and product studies .... 154
time scale as the reaction of thymine 1 with ozone (Figure 5.9) is due to the
formation of 6. In the sequence of reactions that lead to 6a and H', the rate-limiting
step is the reaction of ozone with 1 [reaction (5.14)]. After correcting for some
undissociated 6 due to its pK, value of 4.0 calculated from the conductivity data
shown in Figure 5.8, the yield of 6 is close to 34% of ozone consumed.
Subsequent hydrolytic process of 6 lead to the release of formic acid
(t,:, 13 min at room temperature) monitored by the "slow" increase in
conductivity (Figure 5.8). These conductivity data suggest that the combined yields
of the formic-acid-releasing hydroperoxides (attributed to 6-8) is close to 80%, I.e.
the yield of the sum of 7 and 8 is around 46%.
The kinetics of this "slow" rise in conductivity is mirrored by a very similar
kinetics of the disappearance of the optical absorption of 6 . The fact that 6 has a
pK, value of 4.0 and that of formic acid is 3.75 warrants the presence of additional
precursors of the fbrmic acid that is released in the "slow" process. It is suggested
that 7 and 8 contribute here. The evaluation of build-up kinetics are fraught with
considerable errors, especially when more than one species is involved.720 When the
individual rate constants are not dmqtically different, they cannot be disentangled. The
plots shown in the inset of Fibwre 5.8 only indicates the marked reduction in rate of
hydrolysis upon reducing the temperature to 3OC. This enabled us to run the mass spectra
of the hydroperoxides by HPLC/MS. The molecular wei@t of 6 and 8 is 1 74 Da, agreeing
with mass spectral data. There is no mass-spectral evidence for the formation of' 7, but in
Rate constunts und product studies. .. . - . - - - 15s
the view of the absence of any mass-spectral response in the case of 12 and 14, this lack of
confirmation does not carry much weight and does not rule out the formati011 of 7. ARm
hydrolysis, a hydroperoxide 19 with the molecular weight 1 46 Da remains.
Scheme 5.4
In competition with reaction (5.1 61, the Criegee intermediate 3 can decay
according to reaction (5.17). The zwitter ion 5 may give rise to the hydroperoxides 9
and 10 [reactions (5.2 1 )-(522)]. The hydroperoxide 10 is an hydroxyhydropnnxide.
Such hydroperoxides often eliminate H,O, very rapidly and it is not possiMe to determine
their lifetime. A case in point is the h y d r ~ x y h e ~ l h ~ d ~ ~ p a ~ x i d e . ' " On the other hand,
Rule constants andproducl studies .... -- 15h .- -. -----A. --
there are also hydroxyhydropaoxides such as the hydroxym et h y 1 hydroperoxide which
have very long lifetimes in neutral solution and only release H?O1 rapidly at high pH.'"
The structural parameters that determine the stability of h ydroxyhy droperoxides is as
yet not known. We believe that hydroperoxide 10 belongs to the unstable ones and that
the H,O, observed right after ozonolysis is due to reaction (5.25). The resulting product
1 1 will convert to 1 -fomyl-5-hydroxy-5-methylhydantoin 12. This product is
rather stable and releases formic acid only upon treatment with base at high pH.
The yields of immediate H,O, and formic acid released at high pH are both -25%.
It is thus suggested that reaction (5.22) dominates over reaction (5.21) and that
reaction (5.24) is slow compared to reaction (5.25). However, the ozonolysis of
thymidine which will be discussed below takes a very different route, and acetic
acid is released from an intermediate analogous to 10. No acetic acid is formed in the
thymine system that we are concerned here. This requires that in the thymine system there
must be a much faster process competing with potentially possible acetic acid releasing
process. These two systems differ in the substitution at N( 1). The rapid release of H?02 in
the thymine system may thus involve the N(1) proton. We thus suggest that H,O, is
released in the concerted pathway (5.26) which may well proceed with a relay of water
molecules to accommodate a good transition state for the reacdiun to take place. In
thymidine, the rate of acetic acid release is 60 s-' (see below). To compete with such a
process effectively, the rate of reaction (5.26) must be considerably h t c r .
The reduction of the hydroperoxide 6 by the sulfide is depicted in reactions
(5.28)-(5.30), and since l -forrnyl-5-hydroxy-5-methylhy dantoin I 2 hydroly ses
Halt consirtnts und product studie.~. . . . . -. . . . . - - . - -. - - . -. - - .- , - - -. -. .
157
on1 y at high pH [reachon (5.31)], the mnductance observed right after the addition of
ozone disappears upon the addition of the sulfide. After hydrolysis at high pH, the only
remaining product i s 5-hydroxy-5-rnethylhyd.dntoin 1 4 ( 1 OOO!, reference material wa.,
available). The pathway depicted here is, of course, not the only one, but the other
intermediates are also hally converted into this product.
The "slow" release of formic acid with concomitant decay of the hydropero~ide 6
and the other related hydroperoxides, 7 and 8, may be rationali sod by assuming that these
hydropaoxides form hydroxyendopaoxides such as 1 5 [quilibriwn (5.3 2 )I.
Hydroperoxides readily undergo such a reaction with carbony1 compounds. Oftcm, the
equilibrium constants are quite hgh, and the hydrolysis of hydroxyhydroperoxjdes is
observed. Addition of water to 15 leads 16 [reachon (5.3311. It is noted that upon
endoperoxide formation, 7 immediately lead to 16. It is irnpurtant that in the next step no
N-formyl compound is formed. This would not hydrolyse as readily ( t , , = 13 rrlin) as
observed. Instead, we suggest reaction (5.34) which leads to the amide 17. This may then
undergo ring closure forming the hydantoin 18 [reaction (5.3 S)] which subsequently
hydrolysa [reaction (5.3611 and release formic acid and hydroperoxide hydantoin.
Mass spectral evidence for the formation of the hydroperoxide hydantoin t 9
has been given above. Its reduction by sulfide to 5-hydroxy-5-methylhydantoin
14 has also been ascertained well. It has been mentioned above that the decay
kinetics of 6 and the build-up of fbrmic acid are very close to one another and do
not deviate too much firom a first-order rate law (c,f.' inset in Figure 5.14). 'l'his
Rate c o n s t ~ n l ~ ~ ondproducl studies .... -. - --. 158
could be accounted for if the rate determining step in this sequence of reactions is
the formation of the endoperoxide 15 (or 16 fiom 7).
Scheme 5.5
The observation that no singlet dioxygen i s formed in ncutral solution
requires that ring closure of the zwitterion 2 [reaction (5.15)] is much faster than its
deprntonation at N(1). This is not surprising since recombination of oppositc
charges can be very fast (at every encounter) but the deprotonation is comparatively
slow (the rate depending on the pK;, value).
Rate constants and produel sludies.. . . -. 159
5.3.1.7. Ozonolysis at high p H
Thymine deprotonates at high pH CpK, = 9.9), and the rate of' reaction with
ozone becomes considerably faster (k = 3 x 10' dm3 mol-' s-'). Concomitantly, the
reaction mechanism must be partially altered, since now the formation of singlet
dioxygen, o,('A,) is observed in 8% yield.205 Thymine glycol is a conceivable
product at high pH. Its presence is characterised by the GC-MS spectrum after
silylating the dried sample obtained by rotary evaporating the ozonated thymine
solution (Figure 5.16). Four times silylated thymine glycol obtained with MW 448.
The major m/z (%) peaks are 433 (lo), 405(8), 33 1 (75), 147(25), 73(100).
i 0 - N' ."
I TMS
- m.w. 448
Figure 5.16. Mass spectrum of the thymine glycol obtained at pH 11.6 aRm silylation of the ozonated thymine solution
Since the acidity of N(1) and N(3) i s very similar, the anions l a and I b are
both present at comparable concentrations. The higher electron density of la in the
C(5)-C(6) double bond will favour reaction (5.5). The hydrotrioxide anion 20b has
then no positive charge at C ( 6 ) for a rapid ring closure to the Criegce intermediate.
Rate constants and product studies ... . .- -
160
This will enhance the life time of this hydrotnoxide, i.e. i t stands a chance to
decompose into 21 and singlet dioxygen, O?('AJ [reaction (5.6)]. The yield of singlet
dioxygen is a lower limit of this process. Spin conversion and elimination of
ground-state triplet dioxygen, which is energetically favoured by 105 kJ mol-' can
compete. 'This effect is most prominent in the case of the reaction of I- and B r with
ozone.2w In these systems, spin-orbit coupling via the heavy-atom effect favours the
singlet triplet conversion, and singlet dioxygen formation is dramatically reduced.
Also, when a hydrotrioxide anion has a long lifetime and the energetics of singlet
dioxygen elimination disfavour the reaction to proceed ei'fectively, loss of ground-
state triplet dioxygen may proceed with quite some efiiciency.
Scheme 5.6
Rute constclnts undproduet studies ... . . . I h l
Isopynmidines are well-established intermediates in the fiesradical chemistry of
pynmidines.m"2 They react quite readily with water. In the present system, this reaction
leads to the formation of thymine glycol 22. Reactions (5.37) and (5.38) followed by a
reprotonation at N(3) would yield the Criegee intermediate 3. This has been shown above
to yield the strongly UV-absorbing hydroperoxide 616a. This is not formed under present
conditions. Although 6a is not very stable in basic solutions, its formation should not have
escaped the detection. This poses the qumtion as to whether in basic solution the (Jriegee
intermediate 3 is formed at all. If the formation of 20b were not the only prominent but the
only primary process, further reactions of this hydrohioxide would have to be considered.
A detailed product study is still missing.
5.3.2. Thymidine
Ozone reacts with thymidine at a rate constant of 3 x 1 O4 dm7 mol-' s-' and with its
anion at a rate constant of 1 -2 x I o6 dm' mol-' s-' . In contrast to thymine, no singlet
dioxygen is formed at high pH?'' f i e products that have been reported in an carlier
study1* are also markedly different h r n those reported above for the thyrninc reaction. So
it is worthwhile to have a closer look on the mechanistic details.
5.3.2.1. Conductometry and ion chromatography
Similar to thymine, there is a fast and slow build-up o f conductance upon
the addition of omne to the thymidine solution. The ratc of the slow component is four
times faster (k = 0.005 1 s-') than the corresponding process in the m e of thyrninc (Figure
5.17). The fast component in th~s system was resolved by stopped-flow conductance
Huge constants und product studies. ,. . - - 162 . ---
(Figure 5.17, inset). Using ion chromatography, 75% yield of formate was determined.
This yield was increased to 100% by keeping the solution at high pH (base hydrolysis).
L- L
0 3 Time /set
Figure 5.1 7. Ozonolysis of thymidine in aqueous solution. Furmation of acids (proposal as acetic and formic: acid) in the reaction of omne with thymidine as followed by steady state conductance. Inset: Stopped flow conductance analysis of the formation of prompt release of acetic acid
When the reaction between thymidine and ozone has been followed by stop@
flow technique with conductivity detection, a conduc~ance increase was observd, which
was completed within 10 seconds. The rate of h s proton release step (k = 0.55 s-I) is two
orders of magnitude slower than the calculated k,, for the reaction of thymidine with
ozone (at 2 mM thymidine, k = 60 s- ' ) .~ This indicates that protons are released due to
decomposition of primary intermediate. Regarding the nature of acidic product
responsible for the fast conductance increase, k, h r this increase is much lowm than thc
first conductance step for thymine. And most importwtly, such a reaction sequence is
leading to the loss of total peroxide. The yield of the acetic acid determined by ion
chromatogaphy was equal to 18%, which fits well with the percentage o f decoimposed
Rulc L.on.srants nnd prndur,/ studies. .. . - . -- lh3
. - - -
peroxide. The yeld af acetic acid remains same at high pH. 'l'hmefore, we suggest that acctic
acid is responsible fbr the observed condu~;tancc pic~re. Such kind of tramfirmation tor
hy~~xyhydroperoxide was recently reported for the reaction of glyoxylic acid with ~ ~ 0 ~ ~ ' ~
w h ~ h prOcOedS at a rate consmt of the same order (k > 1 s-'1.
Table 5.4. Compilation of yields (with respect to ozone consumed) in the ozonolysis of thyrnidine - -
* measured using ion chromatography
Species
Acetic acid*
Slow acid release (acetic and formic acids, conductoinetry)
Formic acid release'
Total formic acid release at high p ~ *
Total hydroperoxide (immediate) Total hydroperoxide(afier 25 s - 1.5h)
Hydrogen peroxide (immediate, catalase assay)
Hydrogen peroxide (immediate, R,S assay)
Hydrogen peroxide (after 1 h) Organic hydropcroxides (after 1 h)
1 -(2'-deoxyribosy1)-5-hydroxy-5-methylhydtin 35 (after R,S treatment)
1 -(2'-deoxyribosy1)-5-Hydroxy-5-methyl hydantoin 35 (after R,S and OH ' treatment)
Singlet dioxygen (at high pH) A- - . - - -
Simultaneously with acetic acid, carbon dioxide and N-(2'-dmxyribosyl)
fbrmylurea are expect& as by-products. The latter one (N-(2'deoxynbosyl) formylurea)
was detectad by us and determined by Cadet et a1 with 1 8% yield.'" Its f~rmzition as
the product in the ozonolysis o f thyrnidine is indirectly proved by its hydrolysis
(N-(2'-deoxyribosyl) furmylurea undergoes hydrolysis leading to an increase of
Yield I %
I8 -40-45
76
1 00
100
78
8
8
8
70
45
7 5
0 - - . .
Rule constants find product studies.. . . 164
formic acid for about 20%), when ozonated thymidine solution was kept for 2 hours at
pH 11.6. Exactly, at the same experimental conditions it was found that reference
N-formylurea (without sugar moiety) is fully hydro1 ysed giving the stoichiometric
amounts of formic acid. The yields of acidic and peroxidic materials are given in
Table 5.4.
5.3.2.2. Formation and decay of hydroperoxides
To investigate the nature of the peroxide, the Allen's reagent was added to
the ozonated thymidine solution at 2, 10 and 25 s after the ozonation. The yield of
instant total peroxide (at 2 s) was 100% while in the other cases, it was only 80%
(20% loss of total peroxide). Hence there must he 20% very reactive organic
hydroperoxide. The addition of catalase reduces this yield by 8%, 1. e. only 8% H,O,
is formed. The total hydroperoxide yield remains practically stable over time.
However, no further increase of H,O, was observed.
Since, catalase datroys not only H,O,, but some reactive organic peroxides as
well, experiments with non-activated Allen's reagent was carried out to analyse the nature
of the peroxides in the ozonated thymidine solution. Under these conditions, reactive
organic peroxides react with non-activated 1- as fast as with Mo-activated iodide
(k = 100 dm3 mol-' s-' as measured by stopped tlow technique), but H,O, reacts much
slower (2.1 dm7 mol-' s-' for activated iodide and 1 .# x 1 0"dm3 mol-' s-' for non-
activated iodide, r e ~ ~ e c t i v e l ~ ) . ~ " ~ SO, the formation of 1, in this case i s biphasic:
fast initial step (could not be resolved by conventional UV-spectroscopy) and the
Rate cunstants and product studies .... 165
slow one, which is nicely resolved. The kinetics of slow component is matching
with experimentally determined rate constant for H,O, reaction with non-activated
Allen's reagent.
To investigate the nature of long-lived intemediates that can account for
more stable organic peroxides formed in 70% yield, the reaction W e e n thymidine
and ozone has been followed by conventional UV-spectroscopy. As can be seen from the
Figure 5.18, 30 set afia the ozonation, an intermediate is f o m d with absorption
maximum at 2110 run with a small absorption at 270 nm. Within 12 min, this
intermediate has been totally disappeared and a product with h,, = 227 run is formed.
Figure 5.18, OzonoIysis of thymidine at natural pH. UV-absorption spectra obtained at 0.5 (m), 1.5 (A) and 7.5 min (A) after omolysis. Inset: Decay of 240 species (A) and build up of the 220 species (a)
The kinetics of the intermediates at different wavelengths has been analysed using
conventional UV spectrophotometer. The decay of the spedcs at 240 nm and the
formation at 220 nrn resulted an identical rate constant of 0.0062 s". These rate constants
art: close to the rate constant of the slow increasc of conductance (0.005 1 s-I j.
K t r ~ r constunts undprodurt studies. .. . . --- - . - -- --- -.
Ihh -
5.3.3.3. HPLC and LC-MS analysis
In contrast to thymine, the HPLC chromatogram of thy~nidine contains a nuinbcr
of product peaks. The retention times of the product peaks are 2.9, 1 1 37 , 12.83, 14.93 and
24.96 min. All of these species have h,, around 227 nrn except in the case of the first
eluting peak, which absorb at 256 and a tailing to~vards 220. All of these initial peks were
disappeared by the duction with bis(2-hydroxyethy1)sulfide. Upon post column
derivatisation with molybdate activated iodide, five hydroperoxides were detected. The
hydantoins were eluted at 12.74 and 14.25 min with A,, i 220. Because of its low yield
and the presence of peroxide peaks at close prox~mity, it is not well separated in the
immediate injection. Except the first unstable peroxide, other peroxides are stable with
time. Thymidine was eluted at 60 min at this condition of the reversed phase column with
water as eluent. The larger number of products as compared to thymine is largely due to
the fact that now rneso/d,l isomers are formed.
Figure 5.1 9. ES-MS spectrum (positive mode) of the separated I 42'- deox~~ribosyl)-5-hydroxy-5-methy I hydantoin 35 Iiaction obtained after the ozonation of thymidine
257 I I I I . tP I l l n r ' " ' = ' ~ ' ' ' ' ' ' ~ ~ ' ~ ~ ~ ' ~ ~ ' ~ ~ ~ ~ r ~ ~ ~ ~ ~ c l ~ ~ ~ ~ ~ ~ ~
a o o 253 300 3 50 uao 451: son 5 5 0 609 1; 5 0
miz
A few mg of hydantoins were isolated by piAeparative HPLC as reference
material. The material was confirmed by ES-MS (Figtrre 5.19). Afta rotary evaporation,
thts material was remained oily and HPLC chromatogram still showed some trace amount
o f impurities. Using h s material for calibration, the total hydantoin yeld was determined
to he 45%. 'The yield of hydantoins was increased to 75% by base hydrolysis.
Considering that the reference material contained some trace mounts of impurities, we
have also calculated their yield based on the assumption that they have the same molar
absorption coefficient like their parent, 5-hydroxy-5-methylhydantoin for which retiable
material was available and obtained a close value. In any case, their yields wert much
higher than the reported value of only 1 9%. In the latter study, the hydropaoxides
were subjected without prior reduction to a laborious work-up, and possibly only a
fraction underwent H,O, elimination, while the rest was degraded.
Upon LC-ES-MS analysis, four products were detected in the experimental
condition. The ozonation was carried out at 3". The first peak gave a molecular
weight 176 Da with m/z 1 77 (M+1) and 353 (2Mt- 1). The next three peaks gave the
molecular weight 1 6 1,262 and 290 Da with m/z 1 62,263 and 29 1 (M+ 1 ) respectively in
positive mode of electrospray ionisation (Figure 5.20). The pmks with molecular weights
262 and 290 are considered to be peroxide peaks. For these experimcnts, the
conversion of thymidine had to be very high, and it as yet not ascertained whether
the first two decomposition products (M W 16 1 and 1 76) are really primary ones.
'I'he peroxide 26 is considered to be the precurser of 290 pcak with MW 308 (see
section 5.3.3 -4). After the treatment with sulfidc the two hydroperoxide peaks werc
Kart, r.on,stun/e and product stud~es ... - - - - -. . - - - - - -- IhX
-. -
disappeared. The sulfoxide (138 Da) that is formed in [his reaction was also
detected. So it is assigned that product with M W 262 i s 34 and product with M W
1 76 is 29 (see Scheme 5 .8 ) . At present, other products are not fully assigned.
Figure 5.20, LC-ES-MS spectra of (a) 1 -(2'-deoxyribosyl)-5-hydroperoxy-5- methyl hydantoin 34 and (b) N-(2'-deoxyribosyl) urea 29 obtained by the ozonolysis of thymidine
5.3.3.4. Mechanistic aspects
Similar to thymine, the mechanism of thymidine ozonation is bascd on the
release of acidic materials, acetic and formic acid and the reduction of peroxides
with sulfide. Thc initial fast increase of conductance is because uf the I-elease of
acetic acid. The initial formation of acetic acid can be represented as in Scheme 5.7.
We propuse that initially formed hydroxyhydroperoxide 25 is undergoing C-C: bond
cleavage and release acetic acid [reaction (5.42)] to form 27.
27 Scheme 5.7
Hydrolysis of the urea derivative 28 can bc considered as the very slow
release of formic acid af ia base hydrolysis. ' lhs initially t'ormed hydroxy
hydroperoxide 25 can eliminate formic acid by the hydrolysis of formyl group
attached to the N(1) position and cyclisation of 33 leads to the formation of'
N-(2'-deoxyribosyl)-5-hydroperoxy-5-hydntin 34. The R,S reduction of 34 yield
N-(2'-deoxynbosyl)-5-hydroxy-5-methylhydntoin 35. The slow release of formic
acid can be schematised as follows.
Rate constants and product studies.. -- . . .------ 170
-%Q R2Sl- R2S0 OOH _I__)
15.49)
1 0 0
Scheme 5.8
The hydropemxide 26 would have a molecular weight of 308 Da, but the highest
observed molecular weight in LC-MS analysis is 290 Da, i.e the hydroperoxide either has
lost one molecule of water upon detection by LCMS or effectively contains one water
less. The latter could be accounted for if reactions (5.51) and (5.52) would have taken
place prior to the LC-MS analysis (MW(37) = 290 Da). A hydroperoxide that has a
noticeable absorption at h > 260 m has been detected (see below), and this could
be due to the extended chromophore in 37.
Rate conslants and u ~ ~ d u c t studies .... 17 1
Scheme 5.9
5.4. Summary and conclusions
In this chapter, a detailed study covering the determination of the rate
constants of the reactions of ozone with nucleobases and their related compounds
and the pH dependence on the rate constant have been carried out. A detailed
investigation of the products and intermediate species formed by the reaction of
ozone with thymine and thymidine is alm presented. The mechanism and product
analysis of the ozonolysis of thymine and thymidine have been undertaken as
model systems for pyrimidine nucleobases.
The pH dependent rate constants of the reaction of ozone with DNA, i t s
constituents and related compounds have been determined using competition kinetics
(nitrite andlor butcn-3-01} and the indigo bleaching method. The rate constants vary
drastically with the degree of protonation of the nucleobase derivatives (in units of
dm' mol" s-'), c.g. in the case of cytosine, k = 1 8 (protonated), k - 1 -4 x 1 o3 (neutral) and
k = 1.5 x 1 Oh (deprotonatd). All the of selected nucleubases have shown similar results.
Upon deprotonation, the mechanism of the ozone reaction may also change; e.g. no
singlet dioxygen ( 0 , ' ~ ~ ) is formed in its reaction with 5-chlorouracil, but when the
Kate consrunt.7 rrnd product studre.5 . 172 -- -.- - - .-- ---- -
5-chlorouracilate ion predominates it becomes a major product (-42%). Rate
constants f i r the neutral compounds are: thymine (4.2 x 1 07, thy rnidinu (3 -0 x 1 04),
1,3-dimethyluracil (2.8 x 10'1, uracil (6501, 6-mcthyluraci l ( 1 40), 5-chlorouracil
(4.3 x I#), orotic acid (5.9 x 10'), isoorotic acid (3.7 x 1 O'), 2'-deoxycytidine
(3 -5 x 1 0->), cytidine (3.5 x 1 O'), adenine (1 2), 2 '-deoxyadenosine ( 1 4), adenosine
(1 6), guanosine (1.6 x 1 04), 2'-deoxyguanosine (1.9 x 1 0 ~ ) and DNA (4 10). In the
case of adenine and its derivatives, and thus also in the case of DNA, 'OH is
produced (via 02*- as an intermediate). For the determination of their intrinsic
ozone rate constants, tert-butylalcohol was hence added as 'OH scavenger.
The yield of instant total peroxide in thymine and thymidine is 100% with
respect to the consumed ozone. In the case of thymine, a very little decay of the
total peroxide is observed (-7% loss), while in the case of thymidine, a 20% loss of
peroxide is observed very rapidly. In thymine, a 25% H,O, is observed by catalase and
R,S assay and it was increased up to 40% with time with a half life -1 2min. The total
organic peroxide has been decreased from 75% to 53 % with the same rate of increase of
H,O,. It is tbund that a highiy UV absorbing acidic peroxide (pKa = 4.0) is released
promptly with v a y high rate (3.4x104 dm3molL1s-'1. The subsequent hydrolysis of the
above paoxide releases formic acid along with other formic acid rcleaqing precursors. The
two organic peroxide identified are 1 -hydmperoxymethyl en-3-(2-oxo-propanoy 1 f-urm and
5-hybpxy-5-methyIhydantoin. After the reduction with bis(2-hydroxyethyl)suIfidc,
67% 5-hydroxy -5-methyhydantoin was ohserverl and after base treatment its yield
H L ~ ~ P cuns!unn and product studies .. . - --A .
17.3 -- -
went up to 100%. The very slow release of formic acid is considered to be fi-oin the
hydrolysis of N(1)-fomyl-5-hydroxy-5methylhydantoin. At high pH, thymine
glycol and singlet oxygen are formed during ozonation.
In the case of thymidine, only a very little H,O, was observed (-8%) ,and
there was no change in the concentration of H,O, with time. The fast conductance
increase is due to the release of acetic acid (18% was measured using ion
chromatography). HPIC measurements showed a 75 % formic acid yield which has
been increased to 100% after base hydrolysis. TotaI yields of the two isomers of
1-(2'-deoxyribosyl)-5-rnethyl-5-hydroxy hydantoin were observed as 45% after
reduction with R,S, while the yield was increased upto 75% after base hydrolysis.
A tentative mechanism to satisfy ail these features is proposed.
In brief, the reactions of ozone with nucleobases proceeds via a cyclic
ozonide which cleaved into peroxyl and carbonyl compounds. The formation of
peroxidic materials gives much impact in biological systems due to the presence of
trace amounts of transition metal ions in physiological condition which can produce
highly reactive radicals. The formation of acidic materials in the present systems clearly
demonstrate that the reaction proceeds via the release of acids and H,O, against the simple
H,02 elimination of ozonide as proposed in some earlier reprts.'"~'"S