Oxidative damage of thymidines by the atmospheric free-radical oxidant NO3 ?

Article abstract of DOI:10.1071/CH11102

Analysis of the products formed in the reaction of nitrate radicals, NO₃ ^?, with the N-and O-methylated and acetylated thymidines 1a and 1b revealed, for the first time, insight regarding how this important atmospheric free-radical oxidant can cause irreversible damage to DNA building blocks. Mechanistic studies indicated that the initial reaction step likely proceeds via NO₃ ^? induced electron transfer at the pyrimidine ring, followed by deprotonation of the methyl group at C5. The oxidation ultimately leads to formation of nitrates 2, aldehydes 4 and, in the case of high [NO₃ ^?], also to carboxylic acids 5. In addition to this, through a very minor pathway, loss of the methyl group at C5 also occurred to give the respective 2′-deoxyuridines 6. The nitrates 2 are highly labile compounds that undergo rapid hydrolysis during work-up and purification of the reaction mixtures, which could lead to serious misinterpretation of the experimental findings and reaction mechanism. Products resulting from NO₃ ^? addition to the C5≤C6 double bond in the pyrimidine ring were not observed. Also, no reaction of NO ₃ ^? with the 2′-deoxyribose moiety was detected.

Full text of DOI:10.1071/CH11102

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2011, 64, 833–842
Oxidative Damage of Thymidines by the Atmospheric
Free-Radical Oxidant NO3ꢀ
A B
,
A
Uta Wille
and Catrin Goeschen
A
ARC Centre of Excellence for Free Radical Chemistry and Biotechnology,
School of Chemistry and BIO21 Molecular Science and Biotechnology Institute,
The University of Melbourne, 30 Flemington Road, Parkville,
Vic. 3010, Australia.
B
Corresponding author. Email: uwille@unimelb.edu.au
Analysis of the products formed in the reaction of nitrate radicals, NOꢀ3, with the N- and O-methylated and acetylated
thymidines 1a and 1b revealed, for the first time, insight regarding how this important atmospheric free-radical oxidant
can cause irreversible damage to DNA building blocks. Mechanistic studies indicated that the initial reaction step likely
proceeds via NOꢀ3 induced electron transfer at the pyrimidine ring, followed by deprotonation of the methyl group at C5.
The oxidation ultimately leads to formation of nitrates 2, aldehydes 4 and, in the case of high [NOꢀ3], also to carboxylic
acids 5. In addition to this, through a very minor pathway, loss of the methyl group at C5 also occurred to give the
respective 20-deoxyuridines 6. The nitrates 2 are highly labile compounds that undergo rapid hydrolysis during work-up
and purification of the reaction mixtures, which could lead to serious misinterpretation of the experimental findings and
reaction mechanism. Products resulting from NOꢀ3 addition to the C5¼C6 double bond in the pyrimidine ring were not
observed. Also, no reaction of NOꢀ3 with the 20-deoxyribose moiety was detected.
Manuscript received: 7 March 2011.
Manuscript accepted: 31 March 2011.
reaction of HOꢀ with nucleosides usually occurs via initial
radical addition to double bonds, which in the case of thymidines
leads to products characterized by a saturated C5–C6
bond.[14–18] In contrast to this, the less reactive ROOꢀ are sug-
gested to react with thymidine by hydrogen abstraction at the C5
methyl group, which ultimately leads to formation of 5-formyl-
20-deoxyuridine.[12]
The nitrate radical (NOꢀ3) is the most important night-time
oxidant, which is formed in the atmosphere by reaction involv-
ing the environmental pollutants nitrogen dioxide, NOꢀ2, and
ozone, O3 (Eqn 1):
Introduction
Aging processes and several disease states such as cancer can
result from DNA damage, which could be induced by exposure
of DNA to a variety of oxidizing agents, leading to nucleotide
damage and strand breaks. The electron-rich purine and
pyrimidine heterocycles are prime targets for these oxidations,
which often involve free radical species. Although the initial
radical attack usually does not lead to direct DNA strand scis-
sion, it may lay the foundation for a site-specific cleavage in a
subsequent chemical step. The role of several O-centred oxi-
dizing radicals and radical ions, such as hydroxyl, alkoxyl,
peroxyl, superoxide, and sulfate radicals (e.g. HOꢀ, ROꢀ, ROOꢀ,
Oꢀ2ꢁ, and SOꢀ4ꢁ, respectively) in nucleobase damage has been
extensively studied.[1–13] It has also been shown that oxidation
products of the thymine base constitute the majority of DNA
damage resulting from ionizing radiation. For example, the
NOꢀ2 þ O3 ! NOꢀ3 þ O2
ð1Þ
Although numerous investigations have been performed to
obtain a fundamental understanding of the role of NOꢀ3 in the
Uta Wille graduated from her Ph.D. in Science at the University of Kiel, Germany, in 1993. Her Ph.D. thesis was performed in
the area of Atmospheric Chemistry. After this, she changed her research directions when she was offered a position for a
Habilitation in Organic Chemistry at the same institution, which was completed in 1999. In 1997/98 she undertook a
Postdoctoral Fellowship with Professor Bernd Giese at the University of Basel, Switzerland. In 1999, she was appointed as
Privatdozent at the University of Kiel and was invited in 2000 as a Visiting Fellow in the School of Chemistry at The University
of Melbourne. In January 2003, Uta moved permanently to Australia, where she was appointed as a Lecturer in the School of
Chemistry at The University of Melbourne, promoted to Senior Lecturer in 2006 and Associate Professor and Reader in 2011.
Uta Wille is a Chief Investigator in the ARC Centre of Excellence for Free Radical Chemistry and Biotechnology. Her research
program targets the chemistry of reactive intermediates by merging radicals of atmospheric importance with organic and bio-
organic chemistry.
Ó CSIRO 2011
10.1071/CH11102
0004-9425/11/060833

RESEARCH FRONT
834
U. Wille and C. Goeschen
tropospheric transformation processes,[19–22] there is only very
limited information available on the damage of biomolecules
caused by reaction with NOꢀ3. In contrast to other free radical
species, which generally react via one pathway only, NO3ꢀ
exhibits a multifaceted reaction pattern, e.g. (i) hydrogen atom
abstraction (HAT), (ii) addition to p systems, and (iii) electron
transfer (ET). Thus, every class of organic compounds is, in
principle, susceptible to attack by NOꢀ3. The oxidation power
of NOꢀ3, which is higher than that of any biochemical radical
and non-radical oxidant, has potentially serious implications
for biological systems. For example, we have recently shown
that aromatic amino acids are rapidly and irreversibly damaged
by NOꢀ3 induced oxidation, and from the products formed in
these reactions it might be suggested that atmospheric NO3ꢀ
could be the culprit in certain pollution-derived diseases of the
respiratory tract.[23,24] We have also demonstrated that NO3ꢀ
can initiate oxidative repair of pyrimidine cyclobutane dimers,
which are the most important reaction products in DNA caused
by UV irradiation.[25,26] Although it is not clear at the moment,
whether NOꢀ3 can actually enter biological systems beyond
biosurfaces exposed to the atmospheric environment, studies
of NOꢀ3 induced oxidative damage in DNA can reveal important
insight into the mechanism of these processes. Knowledge of the
latter is of fundamental importance since, in reactions of nucleo-
sides or DNA with various different oxidizing species, certain
intermediates appear to be common to many systems, and as
such the determination of one mechanistic pathway will help to
elucidate others.
In this paper, we wish to report the results of the first product
and mechanistic study of the reaction of NOꢀ3 with the N- and
O-methylated and acetylated thymidines 1a and 1b, respectively
(Fig. 1). These two pyrimidines were chosen to explore the role
of electron density at the heterocyclic base on the degree of
oxidative damage. Thus, whereas in thymidine 1a N-methyl-
ation leads to an increased electron density at the base, the
O-acetylated thymidine 1b features an unsubstituted imide
N–H bond as it occurs in DNA. The hydroxyl groups in the
20-deoxyribose moiety were protected by methylation or acetyl-
ation to mimic the diester linkage in DNA.
NOꢀ3 was generated in situ, in the presence of the respective
thymidines 1a/b, by photolyzing a solution of ammonium
cerium(IV)nitrate (CAN) in acetonitrile, which gives NO3ꢀ
through
a
photoinduced electron transfer reaction
(Eqn 2).[19,23,27,28]
ðNH4Þ2CeðNO3Þ6 þ hv ! ðNH4Þ2CeðNO3Þ5 þ NO3ꢀ
ð2Þ
Results and Discussion
Product Studies
Using CAN both in excess or as a minor compound, respec-
tively, the irradiations were usually performed until the char-
acteristic yellow-orange colour of CAN had disappeared,
indicating complete consumption of the radical precursor (typ-
ically 2–3.5 h in the reactions with excess CAN). Identification
of the products was performed by isolation and purification
through repeated column chromatography or by HPLC, fol-
lowed by spectroscopic characterization. Further details are
given in the Experimental section.
O
RN
5
3
6
1
N
O
RꢀO
Scheme 1 details the products obtained in the reaction of
CAN with 1a and 1b, respectively, under different experimental
conditions. The large variety of products is a clear indication
that the reactions are complex. Most importantly, we found that
the outcome was very sensitive to the method of reaction
analysis and purification of the products, which could lead
to serious misinterpretation of the reaction mechanism. These
findings will be discussed in detail in the section titled ‘Mecha-
nistic Considerations’ of this paper.
5ꢀ
O
1ꢀ
4ꢀ
3ꢀ
2ꢀ
ORꢀ
R ꢁ Rꢀ ꢁ Me
1a
1b R ꢁ H, Rꢀ ꢁ Ac
Fig. 1. Thymidines studied in this work.
O
O
O
O
O
O
O
CAN, h ν
RN 3
ONO2
RN 3
OH
RN 3
H
RN 3
OH
RN 3
5
6
5
6
5
6
5
6
5
6
ꢂ
ꢂ
ꢂ
1a/b
1
N
1
N
1
N
1
N
1
N
Ar or N2, MeCN
O
O
O
O
O
Rꢀ
Rꢀ
Rꢀ
Rꢀ
Rꢀ
2a/b
3a/b
4a/b
5a/b
δ ꢁ 1.99
6a/b
MeO
a R ꢁ Me, Rꢀ ꢁ dRibMe
AcO
b R ꢁ H, Rꢀ ꢁ dRibAc
δ ꢁ 2.79
O
O
O
O
O
H
H
H H
1ꢀ
1ꢀ
ꢁ
RN
N
Me
ꢂ
RN
N
Me
OMe
H
ꢂ
NO
O
N
O
N
O
Rꢀ
Rꢀ
ꢁ
7a
8a/b
8a δ ꢁ 4.77 ꢂ 4.58
δ ꢁ 4.16 ꢂ 4.09
OAc
Scheme 1.

RESEARCH FRONT
Radical-Induced Oxidative Damage of Thymidines
835
Table 1 compiles the products of the individual reactions,
which were performed with an excess of CAN ([CAN]/[1a or
1b]; 2/1). Under these conditions, consumption of the respective
thymidines was not complete, and the yields are therefore based
on consumed starting material.
results from hydrolysis of nitrate 2a during work-up (see below).
Indeed, 2a turned out to be highly labile and could not be
obtained as a pure compound. Therefore, identification and
spectroscopic characterization could only be performed from a
mixture containing also aldehyde 4a and by comparing the data
with those of the slightly more stable nitrate 2b, which was
formed in the reaction of thymidine 1b with NOꢀ3 and could be
isolated in pure form. As can be seen from Table 2, the most
characteristic and indicative signals in the 1H NMR spectrum of
nitrates 2a/b are the methylene protons of the C5 substituent,
which exhibit a strong downfield shift to d 5.29 ppm (2a and 2b)
and 5.21 ppm (2a) or 5.22 ppm (2b) ppm, respectively, resulting
from the electron-withdrawing nitrate substituent. For the reac-
tion involving thymidine 1a, it was further exemplarily shown
that with very high local NO3ꢀ concentrations, which could be
produced by using a focussed high-pressure mercury lamp,
oxidation to the carboxylic acid 5a occurred.
The yields were obtained from quantitative 1H NMR analysis
of the reaction mixture after filtration through a short silica gel
column to remove insoluble inorganic material, using a mea-
1
sured amount of benzene as internal standard. The H NMR
chemical shifts of benzene (d 7.36 ppm) and those of the starting
thymidines and the various products, showed some characteris-
tic differences, specifically the protons at the C5 side chain, the
vinyl proton H6, and the anomeric proton H10, which are listed
in Table 2. This enabled us to use these chemical shifts as a
marker to determine the absolute composition of the reaction
mixture from signal integration relative to that of the standard.
All reaction products were derived from oxidation of the
methyl group at C5, leading to formation of the respective
nitrates (2a/b), alcohols (3a/b), and aldehydes (4a/b) in varying
yields. A value of #3% indicates that yields were below the
detection limit of the 1H NꢀMR spectrometer. No products
arising from a reaction of NO3 with the 20-deoxyribose moiety
were found, which could only proceed via NOꢀ3 induced HAT
from a methylene group adjacent to an oxygen atom. Also, no
products resulting from radical addition to the C5¼C6 double
bond in the pyrimidine were formed.
The O-acetylated thymidine 1b which possessed a free imide
N–H bond, so that the pyrimidine ring system is less electron
rich compared with 1a, reacted with NOꢀ3 to form predominantly
nitrate 2b (59%), whereas the corresponding aldehyde 4b was
the minor product (19%). Alcohol 3b was not found in the
reaction mixture. In order to obtain spectroscopic data for 3b,
the latter was independently prepared from nitrate 2b through
acid-mediated hydrolysis (refer to Experimental section herein).
Interestingly, only after very careful purification of the
reaction mixtures by preparative HPLC, could very small
amounts of the 20-deoxyuridines 6a and 6b be isolated, which
likely result from oxidative demethylation at C5. The yield of
In the reaction of the permethylated thymidine 1a with NOꢀ3,
formation of aldehyde 4a with a yield of 44% was the major
pathway. Conversely, the nitrate 2a was obtained in 23% yield,
together with a minor amount of alcohol 3a, which we believe
1
these compounds was too low to be detected in the H NMR
Table 1. Products and yields of the reaction of NOꢀ3 with pyrimidines 1a and 1b under anaerobic conditions with [CAN]/[1a or 1b] ~2A
Entry
Pyrimidine
Products and yieldsA
1
2
1a
1b
2a: 23%
2b: 59%
3a: 6%B
3b: #3%C
4a: 44%
4b: 19%
5a: #3%C
5b: #3%C
6a: #3%C
6b: #3%C
ADetermined by quantitative 1H NMR analysis of the reaction mixture (see text).
BIsolated yield.
CYield below detection limit of 1H NMR spectrometer.
Table 2. Characteristic 1H NMR chemical shifts (d) in ppm of thymidines 1 and products 2–6 formed in the reaction with NO3ꢀ
n.d., not determined
Signal
Compound
1
2
3
4
5
6
(a) Reaction of NOꢀ3 with 1a
E
5-CH(3ꢁn)
X
1.92A
7.59
5.29, 5.21B
8.20
4.47, 4.35C
7.88
9.97D
8.81
–
5.77F
7.72
6.28
H6
H10
9.21
6.29
6.31
6.30
6.32
6.22
(b) Reaction of NOꢀ3 with 1b
5-CH(3ꢁn)X
H6
H10
1.93A
7.29
5.29, 5.22B
7.81
4.47, 4.39C
7.59
9.95D
8.47
n.d.
n.d.
n.d.
5.80F
7.53
6.26
6.32
6.26
6.31
6.28
An ¼ 0.
Bn ¼ 1, X ¼ ONO2.
Cn ¼ 1, X ¼ OH.
Dn ¼ 2, X ¼ (¼O).
En ¼ 3, X ¼ (¼O)OH.
FH5.

RESEARCH FRONT
836
U. Wille and C. Goeschen
spectra of the reaction mixtures. However, discussion of possi-
ble pathways for their formation will follow in ‘Mechanistic
Considerations’.
during HPLC analysis and is not directly formed in the reaction
of NOꢀ3 with 1a.
An analogous experiment performed with [CAN]/[1a] (2/1)
in the presence of oxygen showed that formation of both
aldehyde 4a and carboxylic acid 5a occurred much faster at
the expense of nitrate 2a (and alcohol 3a) production, compared
with the experiments in the absence of oxygen, despite the lower
concentration of NOꢀ3 (data not shown). This indicates that some
reaction intermediates are susceptible to trapping by oxygen.
Due to the high oxidation power of NOꢀ3 [E0 (NO3ꢀ/NO3ꢁ) ¼
2.3 V versus NHE],[28,30,31] we believe that the reaction is
initiated by ET at the pyrimidine ring, which likely proceeds
via an addition–elimination pathway, as has been suggested
for the reaction of NO3ꢀ with alkylaromatic compounds.[32]
Scheme 2 outlines the proposed mechanism for formation of
the nitrate 2a using the reaction of NOꢀ3 with thymidine 1a as
example.
The low stability of the nitrates 2a/b poses a serious problem
with regards to identification of the primary products formed in
the reaction of NOꢀ3 with thymidines 1a/b. We have found that
hydrolysis of 2a/b occurs quantitatively during separation of
the reaction products by preparative HPLC, if a non-buffered
solvent system is used. In those cases, we could only isolate the
respective alcohols 3 and the acetamides 7 and 8. The latter two
products result from a Ritter reaction involving the solvent
acetonitrile. The mechanism and implications of these second-
ary reactions will be discussed in Mechanistic Considerations.
In order to obtain insight into the influence of [NOꢀ3] on the
composition of the product mixture, the reaction of 1a and 1b
with NOꢀ3 was also explored with the pyrimidine in at least four-
fold excess. Qualitative 1H NMR analysis of the reaction
mixtures revealed that under these conditions only formation
of the respective nitrates 2a and 2b occurred, whereas higher
oxidation products, such as aldehydes, were not formed (data not
shown).
60
50
40
Mechanistic Considerations
The results of the product analysis have clearly shown that
reaction of NOꢀ3 with the pyrimidines 1a and 1b occurs exclu-
sively at the methyl substituent at C5. For the reaction of NO3ꢀ
with thymidine 1a, which was performed with excess NO3ꢀ
(e.g. [CAN]/[1a]; 6/1) under exclusion of oxygen, we have used
analytical HPLC to obtain qualitative concentration–time
profiles for the various reaction products (Fig. 2). The HPLC
runs were carried out using acetonitrile/water as solvent system,
which was buffered with 0.05 M triethylammonium acetate. The
data were obtained from the relative peak areas of the product
signals monitored at l 260 nm.[29] These experiments clearly
revealed that formation of 2a occurred first, whereas the alde-
hyde 4a and carboxylic acid 5a appeared only after induction
periods, indicative of secondary products. However, the lack of
an induction period for formation of the alcohol 3a supports our
assumption that 3a results only from hydrolysis of nitrate 2a
5a
4a
30
20
2a
3a
10
0
0
30
60
90
150
210
270
Reaction time [s]
Fig. 2. Qualitative concentration–time profiles for the products formed in
the reaction of NOꢀ3 with thymidine 1a under anaerobic conditions.
O
O
ꢂ
•
•
NO3•
NO3•
Hydrolysis
MeN
MeN
O
2a
O
3a
1a
ꢃNO3ꢃ
ꢃH ꢂ
O
N
N
NO3ꢃ
ET
ET
dRibMe
dRibMe
1a • ꢂ
CAN
or NO3•
9
ꢂ
MeN
O
N
During separation by preparative HPLC:
MeN
dRibMe
O
N
10
ꢂ
‘HNO2’
(from hydrolysis of 2a)
N
H2O
Hydrolysis
CH3CN
2a, 3a
10
7a
8a
O
dRibMe
11
Scheme 2.

RESEARCH FRONT
Radical-Induced Oxidative Damage of Thymidines
837
According to this, the initially formed radical cation 1aꢀþ
undergoes deprotonation to give benzyl-type radical 9,[9] in
analogy to the mechanism of the NOꢀ3 induced oxidation of
aromatic amino acids. The latter reactions have been shown to
proceed through an intermediate aryl radical cation, which can
be directly trapped if the reaction is performed in the presence
of NOꢀ2 to give nitroaromatic products.[24] In the absence of
NOꢀ2 stabilization through deprotonation occurs, which leads
to products with an oxidized benzylic site.[23] Principally, the
reaction of NOꢀ3 with 1a could also be initiated through HAT
from the C5 methyl group, which would give radical intermedi-
ate 9 in a single step. However, HAT reactions by NOꢀ3 are
usually much slower than ET.[19–22] The suggested ET is further
supported by the finding that reaction of 1a with CAN in the
absence of light (e.g. absence of NOꢀ3) leads to the same
products, but in a significantly slower reaction, which is in
agreement with the much lower redox potential of CAN
[E0 (Ce4þ/Ce3þ) ¼ 1.61 V versus NHE].[33]
expected from the initial reactant ratio [CAN]/[1a] for a stoi-
chiometric reaction.
Scheme 3 outlines the assumed mechanism for formation of
the aldehyde 4a from nitrate 2a, which could proceed without or
with involvement of oxygen.
In the absence of oxygen, nitrate 2a could be converted into
the corresponding benzylradical 12 through either a direct HAT
by NOꢀ3,[36] or through a coupled ET/deprotonation pathway,
analogous to the initial reaction step. The O–NO2 bond in 12
is very weak and can undergo rapid b-fragmentation to give
aldehyde 4a with release of NOꢀ2, which is too unreactive to
initiate a radical chain process.[37]
In the presence of oxygen, the radical intermediate 9 could be
trapped to give peroxyl radical 13, which could be transformed
into alkoxyl radical 15 through dimerization and release of
oxygen. Subsequent conversion of 15 into aldehyde 4a, could
occur through HAT by either NOꢀ3 or oxygen, respectively. The
closed-shell dimer 14 could principally also decompose directly
to the alcohol 3a and aldehyde 4a through a Russel mechanism
involving a cyclic transition state.[38] Thus, the latter pathway
could lead to formation of the alcohol 3a without hydrolysis of
the nitrate 2a. The suggested mechanism involving formation of
an intermediate peroxyl radical 13, which intercepts the pathway
leading to nitrate 2a, is in line with the qualitative finding that
less nitrate 2a and significantly more aldehyde 4a was obtained
when the reaction of NOꢀ3 with thymidine 1a was carried out in
the presence of oxygen (see above). In addition, formation of
peroxyl radical 13 is an additional support for our aforemen-
tioned assumption that the reaction of NOꢀ3 with thymidines does
not proceed via a cationic species 10, as the latter would not be
expected to react rapidly with molecular oxygen.
Scheme 4 shows the suggested mechanistic steps that lead to
formation of the carboxylic acid 5a. The mechanism is based on
findings from independent experiments, where we demonstrated
that aldehyde 4a can be oxidized to 5a by NOꢀ3. This reaction
likely proceeds via HAT,[39] where the resulting acyl radical 16
could subsequently be trapped by NOꢀ3 to give mixed anhydride
17, which is hydrolyzed to the acid 5a during the aqueous work-
up and/or purification by chromatography. The observed for-
mation of small amounts of the uridine 6a in the absence of
oxygen could be explained through a pathway involving dec-
arbonylation in 16,[40] followed by reduction of the resulting
highly reactive vinyl radical 18 through HAT (likely from the
solvent). The finding of a more rapid formation of carboxylic
acid 5a in the presence of oxygen could be rationalized by a
mechanism, where acyl radical 16 is trapped to give acylperoxyl
radical 19, which could subsequently be transformed into the
acyloxyl radical 20 through dimerization and expulsion of
oxygen. HAT from the solvent then leads to the acid 5a. It
cannot be excluded, however, that the radical intermediate 20
could also undergo decarboxylation to give vinyl radical 18,
which would represent an additional, oxygen-dependent path-
way to uridine 6a (not shown).
Formation of nitrate 2a could principally occur via two
different pathways, e.g. through direct trapping of 9 by NOꢀ3,
or in a two-step process, first by NOꢀ3 or CAN induced oxidation,
followed by quenching of the resulting benzyl-type cation 10
through ligand transfer from CAN. Subsequent hydrolysis of 2a
leads to alcohol 3a. Indeed, in light of the observed acetamides
7a and 8a (Scheme 1), it could be interpreted that the reaction
leading to nitrate 2a proceeds via an intermediate cation 10,
which is trapped by the solvent acetonitrile through a Ritter
reaction.[34,35] However, the acetamides 7/8 were only obtained,
when product isolation was performed by HPLC in an unbuf-
fered solvent system, in which case the nitrates were not
observed as products. On the other hand, HPLC analysis of
the reaction mixture under buffered conditions did not reveal
any acetamides. We conclude therefore, that the Ritter reaction
involving cation 10 occurs only during purification of the
reaction mixture through hydrolysis of nitrate 2a (and to some
extent possibly also the alcohol 3a), and that formation of a
cationic intermediate of type 10 in the actual reaction of NO3ꢀ
with thymidines is, if at all, only a very minor pathway.
Formation of the N-nitroso acetamide 8a is believed to proceed
also on the HPLC column through N-nitrosation of 7a by nitrous
acid, which could be intermediately formed during hydrolysis of
1
nitrate 2a. The H NMR spectrum of 8a is characterized by
significant downfield shifts of the methylene protons at C5 by
,0.5–0.6 ppm and of the acetyl protons by 0.8 ppm, compared
with the free acetamide 7a (see Scheme 1), which is in line with
the electron-withdrawing nature of the nitroso group.[24]
It is quite apparent from these findings that analysis of the
products formed in the reaction of NOꢀ3 (and potentially also
other oxidizing radicals) with thymidines must be carried out
with great care so that crucial details, which could lead to a
misinterpretation of the reaction mechanism, are not missed. It
is only because we have performed our analytical studies
under a variety of different conditions that we are able to
distinguish between direct reaction products and products
arising from post-reaction chemical modification of highly
labile compounds.
Conclusions
According to the proposed mechanism in Scheme 2, forma-
tion of the nitrate 2a would require two equivalents of NOꢀ3.
Indeed, our photochemical experiments with CAN as minor
compound confirmed this hypothesis by showing, that after
complete consumption of CAN, the ratio of nitrate 2a to
This work is the first product study of the reaction of the
atmospheric free radical oxidant NOꢀ3 with thymidines. Using
the N- and O-methylated and acetylated thymidines 1a and 1b as
model systems for thymidine nucleotides, it was found that the
reaction occurred exclusively at the methyl side chain at C5,
which led to formation of nitrates 2a/b and aldehydes 4a/b as
the major products. Using the reaction with thymidine 1a as
1
unreacted pyrimidine 1a, [2a]/[1a], in the H NMR spectrum
of the reaction mixture was significantly lower than would be

RESEARCH FRONT
838
U. Wille and C. Goeschen
In the absence of O2
O
N
H
C
•
HAT
MeN
ONO2
NO3•
4a
2a
or
ꢃNO2•
ꢃHNO3
O
dRibMe
12
ET
NO• /ꢃNOꢃ
(i)
(ii)
3
3
ꢂ
ꢃH
In the presence of O2
Me
N
O
O
O
O
CH2OO •
O2
MeN
ꢄ2
O
O
N
9
dRibMe
O
O
MeN
O
N
dRibMe
N
O
dRibMe
14
13
ꢃO2
ꢃO2
O
N
HAT
3a ꢂ 4a
CH2O •
NO3• /ꢃHNO3 MeN
4a
or
O
O2/ꢃHO2•
dRibMe
15
Scheme 3.
O
N
HAT
•
MeN
6a
O
dRibMe
ꢃCO
18
O
ONO2
O
O
N
HAT
NO3•
ꢃHNO3
•
NO3•
Hydrolysis
MeN
O
MeN
O
4a
5a
O
N
dRibMe
dRMe
HAT
16
17
O2
O
N
OO•
O
O•
ꢄ2
ꢃO2
MeN
O
O
MeN
O
O
N
dRibMe
dRibMe
19
20
Scheme 4.

RESEARCH FRONT
Radical-Induced Oxidative Damage of Thymidines
839
example, carboxylic acid 5a was also produced at high con-
centrations of NOꢀ3. In addition, also very small amounts of
20-deoxyuridines 6a/b were formed through demethylation at
C5. In contrast to the reaction of thymines and thymidines with
other O-centred radicals, we have no indications for products
arising from NOꢀ3 addition to the C5¼C6 double bond in the
pyrimidine. Also, no reaction of NOꢀ3 with the 20-deoxyribose
moiety in 1a/b was observed.
A thorough discussion of the reaction mechanism revealed
that the reaction is very likely initiated by NOꢀ3 induced ET,
which leads to the benzyl-type radical intermediate 9. The latter
has been suggested to be able to transfer damage from the
nucleobase to the deoxyribose moiety of an adjacent nucleotide
in the same oligonucleotide strand.[41]
The primary products in the reaction of NOꢀ3 with thymi-
dines, for example nitrates 2, are highly labile species, which
readily undergo hydrolysis to the significantly more stable
alcohols 3. Such a hydrolysis reaction could potentially provide
a source for reactive benzyl-type cations 10 under physiological
conditions, which is supported by the finding that formation of
10 readily occurs under certain conditions during isolation and
purification of the reaction mixtures by HPLC. In our system,
the cations 10 were trapped by the solvent acetonitrile to give
acetamides 7 and 8 through a Ritter reaction. Since our experi-
mental data clearly indicate that the NOꢀ3 induced oxidation of
thymidines 1 does not involve cation 10, it is important to note
that such a rapid post-reaction modification of products, as was
observed in this work, can lead to serious misinterpretation
of experimental data and reaction mechanisms, if it remains
undiscovered.
In contrast to the nitrates 2a/b, 5-formyl-20-deoxyuridines
of type 4a/b are reasonably stable species, which have been
frequently found during radical mediated oxidation of thymi-
dines[13,42,43] and are known to induce point mutations during
DNA replication.[44] Our experiments revealed that the rate of
NOꢀ3 induced formation of 5-formyluridine was dependent on
the electron density at the pyrimidine ring. In the case of the
more electron-rich permethylated thymidine 1a, the reasonably
stable formyluridine 4a was the major reaction product. Con-
versely, the reaction of thymidine 1b, in which the pyrimidine
ring has a free imide N–H group and therefore represents the
natural conditions of the base in DNA, gave the labile nitrate 2b
as major product under otherwise identical reaction conditions.
However, oxidation of thymidine 1 to 5-formyluridine 4 was
significantly accelerated in the presence of oxygen.
thymidines and can induce strand cleavage in DNA.[1–4,7,8]
Future work will be directed towards addressing details of the
mechanism of the oxidative damage of nucleosides by NOꢀ3,
as well as the secondary effects resulting from the presence of
residual water, by studying absolute rate constants and reac-
tion transients using nanosecond laser flash photolysis.
Experimental
1H NMR spectra were recorded on a Bruker AM 300, DRX 500,
DRX 600, and a Varian Unity Inova 500 spectrometer operating
at 300, 500, or 600 MHz, respectively. 13C NMR spectra were
obtained on the same instruments operating at 75.5, 125.8,
or 150.9 MHz, respectively. Mass spectra were recorded on a
Finnigan MAT 8200 instrument at 70 eV ionizing potential.
Isobutane was used for chemical ionization (CI). HR-MS was
conducted by ionizing the samples via electrospray ionization
into a Thermo-Finnigan LTQ FT-ICR hybrid mass spectrometer
or an Agilent 6520 LC/Q-TOF mass spectrometer with an
electrospray ionizing source coupled to an Agilent 1100 LC
system.
HPLC columns and conditions: (a) analytical: Merck
;
LiChrospher 100e, RP18, 5 mm, 250 ꢂ 4 mm, 1 mL minꢁ1
(b) semi-preparative: Machery-Nagel LiChrospher 100e,
RP18, 5 mm, 200 ꢂ 10 mm, 4 mL minꢁ1; solvents: acetonitrile
(A); water/acetonitrile (1%) (B); gradient: 0% A - 30% A in
40 min; (C) preparative: Phenomenex, C18, 5 mm, 150 ꢂ
21.2 mm, 8 mL minꢁ1; solvents: acetonitrile (A); water (B);
gradient: 0% A - 100% A in 2–3 h.
The irradiations were performed under a continuous gas flow
(nitrogen, argon, or oxygen) in either a Rayonet Photochemical
Reactor (l 300 or 350 nm) or with a mercury lamp, where UV
light below l 280 nm was eliminated either by a cut-off filter
or by conducting the reaction in duran glassware. Before the
irradiations, residual oxygen was removed from the reaction
mixture by bubbling argon or nitrogen through the solution.
Synthesis of 1a and 1b was performed according to standard
procedures.[47,48]
30,50-Di-O-methyl-N-methyl-20-desoxythymidine (1a): dH
(CDCl3, 500MHz) 7.59 (s, 1H), 6.31 (dd, J 7.5, 6.1 Hz, 1H),
4.11 (dd, J 5.6, 2.8 Hz, 1H), 3.98 (dt, J 5.9, 2.7Hz, 1H), 3.65 (dd, J
10.5, 2.8Hz, 1H), 3.56 (dd, J 10.5, 2.8Hz, 1H), 3.42 (s, 3H), 3.33
(s, 3H), 3.31 (s, 3H), 2.40 (ddd, J 13.7, 6.0, 2.7 Hz, 1H), 2.03
(m, 1H), 1.92 (s, 3H). dC (CDCl3, 125 MHz) 163.8, 151.2, 133.9,
109.8, 86.0, 83.8, 81.3, 73.1, 59.2, 57.0, 37.6, 27.9, 13.6. m/z (HR-
ESI). Calc. for C13H20N2O5þH: 285.1451. Found: 285.1656.
Calc. for C1213CH20N2O5þH: 286.1484. Found: 286.1497.
30,50-Di-O-acetyl-20-desoxythymidine (1b): dH (CDCl3,
500 MHz) 9.67 (s, 1H), 7.29 (s, 1H), 6.32 (dd, J 8.6, 5.7 Hz,
1H), 5.21 (dt, J 6.6, 2.2 Hz, 1H), 4.37 (dd, J 12.1, 4.2 Hz, 1H),
4.33 (dd, J 12.1, 3.3 Hz, 1H), 4.24 (dd, J 6.4, 3.4 Hz, 1H), 2.47
(ddd, J 14.2, 5.7, 1.9 Hz, 1H), 2.16 (m, 1H), 2.12 (s, 3H), 2.11 (s,
3H), 1.93 (s, 3H). dC (CDCl3, 125 MHz) 170.6, 170.3, 164.1,
150.5, 134.8, 111.7, 85.0, 82.3, 74.2, 64.0, 37.7, 21.0, 20.9, 12.8.
m/z (HR-ESI). Calc. for C14H18N2O7þH: 327.1192. Found:
327.1190. Calc. for C1313CH18N2O7þH: 328.1226. Found:
328.1222.
Clearly, our experimental conditions differed from the
typical conditions in biological systems, specifically with
regards to reaction solvent. However, it is important to note
that the aprotic acetonitrile, which was used as solvent in this
work, can reasonably mimic the hydrophobic environment of
the nucleobases in DNA.[45] In principle, residual water could
influence the reaction outcome in two ways, through (i)
trapping of intermediates produced in the reaction of NO3ꢀ
with thymidines, and through (ii) reaction with NOꢀ3 itself. The
first possibility can be excluded since our mechanistic con-
siderations revealed that no cationic intermediates are pro-
duced in the reaction of NOꢀ3 with thymidines, which could be
trapped by water acting as a nucleophile. Further, it has been
shown that the mechanism of NOꢀ3 reactions with organic
compounds in water is similar to that in organic solvents.[46]
Although the reaction of NOꢀ3 with water is not very fast
Reaction of NO3ꢀ with Thymidines. Typical Experimental
Procedure
Thymidine 1 (0.5 mmol) and CAN (1.0 mmol) were dissolved in
45 mL of absolute acetonitrile, and the mixture was degassed
with an argon stream and irradiated with argon continuously
M
(k ¼ 3 ꢂ 102 ꢁ1 sꢁ1 [46]); if it occurs, highly reactive HOꢀ
could be formed, which are known to react rapidly with

RESEARCH FRONT
840
U. Wille and C. Goeschen
flowing for ,2.5 h (until the orange-yellow colour of CAN had
disappeared). The reaction mixture was concentrated under
vacuum, the residue dissolved with water and extracted with
ethyl acetate. The combined organic fractions were dried
(MgSO4) and the solvent removed under vacuum.
2.5 Hz, 1H), 3.60 (dd, J 10.6, 2.1 Hz, 1H), 3.48 (s, 3H), 3.41 (s,
3H), 3.36 (s, 3H), 2.98 (br, 1H), 2.60 (ddd, J 13.8, 6.2, 2.7 Hz,
1H), 2.15 (ddd, J 13.9, 6.8, 5.8 Hz, 1H). dC (CDCl3, 150.9 MHz)
165.1, 163.3, 149.5, 147.3, 101.6, 88.2, 85.1, 81.3, 72.6, 59.5,
56.9, 38.8, 28.2. m/z (EI) 314 (4, Mþ), 145 (100), 126 (8), 11 (40).
m/z (CI) 315 (34, MþþH), 171 (79), 145 (100), 113 (58). m/z
(HR-EI). Calc. for C13H18N2O7: 314.11139. Found: 314.11110.
Calc. for C1213CH18N2O7: 315.11475. Found: 315.11450.
(e) 30,50-Di-O-methyl-N-methyl-20-desoxyuridine (6a).[49]
dH (CDCl3, 500 MHz) 7.72 (d, J 8.1 Hz, 1H), 6.28 (dd, J 7.6,
6.0 Hz, 1H), 5.77 (d, J 8.1 Hz, 1H), 4.15 (dd, J 5.6, 2.8 Hz, 1H),
3.99 (ddd, J 6.0, 2.7, 2.7 Hz, 1H), 3.66 (dd, J 10.5, 2.8 Hz, 1H),
3.55 (dd, J 10.5, 2.9 Hz, 1H), 3.42 (s, 3H), 3.35 (s, 3H), 3.32 (s,
3H), 2.47 (ddd, J 13.7, 6.0, 2.6 Hz, 1H), 2.03 (ddd, J 13.6, 7.6,
6.0 Hz, 1H). dC (CDCl3, 125 MHz) 163.0, 151.1, 137.7, 101.7,
86.3, 83.8, 81.2, 72.9, 59.3, 56.9, 37.7, 27.6.
1H NMR analysis of the reaction mixture was performed
after addition of a known amount of the standard benzene to the
crude reaction mixture.
The spectroscopical data of the products formed in the
reactions of NOꢀ3 with 1a and 1b, respectively, were obtained
from independent experiments through isolation and purifica-
tion of the compounds by column chromatography (SiO2, ethyl
acetate) or HPLC.
1. Reaction of NO3ꢀ with 30,50-di-O-methyl-N-
methylthymidine (1a)
(f)
5-Acetamidomethyl-30,50-di-O-methyl-N-methyl-20-
(a)
5-Nitratomethylene-30,50-di-O-methyl-N-methyl-20-
desoxyuridine (7a). dH (CDCl3, 500 MHz) 8.01 (s, 1H), 6.58
(s, 1H), 6.32 (dd, J 7.5, 6.1 Hz, 1H), 4.16 (m, 2H), 4.09 (dd,
J 14.3, 5.7 Hz, 1H), 4.01 (dt, J 5.5, 2.5 Hz, 1H), 3.69 (dd, J 10.5,
2.8 Hz, 1H), 3.58 (dd, J 10.5, 2.8 Hz, 1H), 3.49 (s, 3H), 3.35
(s, 3H), 3.34 (s, 3H), 2.44 (ddd, J 13.8, 6.0, 2.6 Hz, 1H), 2.08
(m, 1H), 1.99 (s, 3H). dC (CDCl3, 125 MHz) 170.8, 163.7, 150.6,
137.2, 109.7, 86.4, 84.0, 81.3, 72.9, 59.2, 56.9, 37.7, 37.6, 27.7,
23.2. m/z (HR-ESI). Calc. for C15H23N3O6þH: 342.1665.
Found: 342.1667. Calc. for C1413CH23 N3O6þH: 343.1699.
Found: 343.1694.
desoxyuridine (2a) (could not be obtained without impurities
from aldehyde 4a). dH (CDCl3, 300 MHz) 8.20 (s, 1H), 6.30 (dd,
J 6.0, 6.0 Hz, 1H), 5.29 (dd, J 12.5, 0.7 Hz, 1H), 5.21 (dd, J 12.5,
0.7 Hz, 1H), the further upfield signals could not be separated
from those of 4a. m/z (CI) 346 (19) [MþþH], 299 (100), 283
(32), 155 (92), 145 (80). m/z (HR-EI). Calc. for C13H19N3O8:
345.11722. Found: 345.11690.
(b)
5-Hydroxymethylene-30,50-di-O-methyl-N-methyl-20-
desoxyuridine (3a). RF ¼ 0.16 (SiO2, ethyl acetate): lmax/nm
(CH3CN) (lg e) 263 (4.08), 207 (4.01). dH (CDCl3, 500 MHz)
7.88 (s, 1H), 6.32 (dd, J 7.3, 6.1 Hz, 1H), 4.47 (dd, J 12.9, 0.8 Hz,
1H), 4.35 (dd, J 12.9, 0.6 Hz, 1H), 4.14 (dd, J 5.3, 2.6 Hz, 1H),
4.01 (dt, J 5.9, 2.9 Hz, 1H), 3.68 (dd, J 10.6, 2.7 Hz, 1H), 3.56
(dd, J 10.6, 2.7 Hz, 1H), 3.43 (s, 3H), 3.35 (s, 3H), 3.34 (s, 3H),
2.45 (ddd, J 13.7, 6.1, 2.9 Hz), 2.06 (ddd, J 13.5, 7.3, 6.0Hz, 1H).
dC (CDCl3, 125 MHz) 163.7, 150.9, 135.5, 112.7, 86.4, 84.1, 81.1,
73.0, 60.2, 59.3, 57.1, 37.9, 27.8. m/z (EI) 300 (11%, Mþ), 145
(100). m/z (HR-EI). Calc. for C13H20N2O6: 300.13214. Found:
300.13200. m/z (HR-ESI). Calc. for C13H20N2O6þNa: 323.1219.
Found: 323.1220. Calc. for C1213CH20N2O6þNa: 324.1219.
Found: 324.1239.
(c) 5-Formyl-30,50-di-O-methyl-N-methyl-20-desoxyuridine
(4a). lmax/nm (CH3CN) (lg e) 292 (3.17). dH (CDCl3,
500 MHz) 9.97 (s, 1H), 8.81 (s, 1H), 6.22 (t, J 6.5 Hz, 1H),
4.19 (q, J 2.3 Hz, 1H), 3.99 (dt, J 5.6, 2.7 Hz, 1H), 3.67 (dd, J
10.6, 2.5 Hz, 1H), 3.54 (dd, J 10.6, 2.2 Hz, 1H), 3.41 (s, 3H), 3.30
(s, 6H), 2.53 (ddd, J 13.8, 6.2, 2.9 Hz, 1H), 2.08 (m, 1H). dC
(CDCl3, 125 MHz) 187.0, 161.9, 150.2, 144.2, 110.3, 87.8, 84.9,
81.2, 72.6, 59.3, 56.9, 38.8, 27.6. m/z (EI) 298 (13, Mþ), 221
(26), 145 (100). m/z (HR-EI). Calc. for C13H18N2O6: 298.11649.
Found: 298.11630. m/z (HR-ESI). Calc. for C13H18N2O6þH:
299.1243. Found: 299.1247. Calc. for C1213CH18N2O6þH:
300.1277. Found: 300.1267.
(g)
5-(N-Nitroso)acetamidomethyl-30,50-di-O-methyl-N-
methyl-20-desoxyuridine (8a). dH (CDCl3, 500 MHz) 7.80
(s, 1H), 6.27 (dd, J 7.6, 6.0 Hz, 1H), 4.77 (d, J 14.6 Hz, 1H),
4.58 (d, J 14.6 Hz, 1H), 4.18 (q, J 2.7 Hz, 1H), 4.01 (dt, J 5.2,
2.5 Hz, 1H), 3.74 (dd, J 10.6, 2.7 Hz, 1H), 3.58 (dd, J 10.6,
2.7 Hz, 1H), 3.49 (s, 3H), 3.36 (s, 3H), 3.28 (s, 3H), 2.79 (s, 3H),
2.46 (ddd, J 13.7, 6.0, 2.5 Hz, 1H), 2.04 (m, 1H). dC (CDCl3,
125 MHz) 174.0, 162.0, 150.7, 138.4, 106.5, 86.6, 84.2, 81.5,
73.2, 59.5, 57.0, 37.9, 35.6, 27.9, 22.8. m/z (HR-ESI). Calc. for
C15H22N4O7þH: 371.1567. Found: 371.1568. Calc. for
C1413CH22 N4O7þH: 372.1600. Found: 372.1599.
2. Reaction of NOꢀ3 with 30,50-di-O-Acetylthymidine (1b)
(a) 5-Nitratomethylene-30,50-di-O-acetyl-20-desoxyuridine (2b).
l
max/nm (CH3CN) (lg e) 264 (4.08), 205 (4.17). dH (CDCl3,
300 MHz) 9.35 (br, 1H), 7.81 (d, J 0.7 Hz, 1H), 6.26 (dd, J 5.1,
8.6 Hz, 1H), 5.29 (dd, J 0.7, 12.6 Hz, 1H), 5.22 (dt, J 2.0, 4.3 Hz,
1H), 5.21 (dd, J 0.7, 12.6 Hz, 1H), 4.45 (dd, J 5.1, 12.7 Hz, 1H),
4.31 (m, 1H), 4.31 (dd, J 3.0, 12.7 Hz, 1H), 2.59 (ddd, J 1.8,
5.5, 14.3 Hz, 1H), 2.13 (s, 3H), 2.12 (s, 3H), 2.12 (m, 1H). dC
(CDCl3, 75.5 MHz) 170.5, 170.4, 161.8, 149.7, 141.3, 106.7,
85.8, 82.9, 74.1, 67.0, 63.7, 38.2, 20.9, 20.8. m/z (EI) 387
(,1, Mþ), 201 (15), 81 (100). m/z (CI) 388 (3, MþþH), 341 (7),
325 (8), 231 (37), 81 (100).
(d) 5-Carboxy-30,50-di-O-methyl-N-methyl-20-desoxyuridine
(5a). (NB independently synthesized to confirm spectroscopic
data): 10.34 mg (36.41 mmol) 1a and 40.42 mg (73.73 mmol)
CAN were dissolved in 2 mL of abs. acetonitrile and irradiated
under a continuous argon flow for 3 min at l $ 280 nm in a
quartz cuvette. For spectroscopical characterization the reaction
product was isolated by semi-preparative HPLC directly from
the reaction mixture.
(b) 5-Hydroxymethylene-30,50-di-O-acetyl-20-desoxyuridine
(3b) (NB independently synthesized to confirm spectroscopic
data): 50 mg (0.13 mmol) 2b and 32 mg (0.19 mmol) 4-toluene
sulfonic acid hydrate were dissolved in 15 mL of THF and
stirred for 15 h at room temperature. The reaction mixture
was quenched with saturated aqueous sodium bicarbonate and
filtered (SiO2, ethyl acetate). Column chromatography (SiO2,
ethyl acetate) yielded 7 mg (16%, RF ¼ 0.10) 3b as a colour-
less oil.
UV (CH3CN); lmax/nm (lg e) 279 (3.92), 218 (3.99). dH
(CDCl3, 600 MHz) 9.21 (s, 1H), 6.29 (t, J 6.5 Hz, 1H), 4.26 (dd, J
2.3, 2.3 Hz, 1H), 4.05 (dt, J 5.8, 2.7 Hz, 1H), 3.75 (dd, J 10.6,
dH (CDCl3, 500 MHz) 9.42 (s, 1H), 7.59 (s, 1H), 6.31 (dd, J
8.4, 5.6 Hz, 1H), 5.22 (dt, J 8.4, 5.6 Hz, 1H), 4.47 (d, J 13.5 Hz,

RESEARCH FRONT
Radical-Induced Oxidative Damage of Thymidines
841
1H), 4.39 (m, 2H), 4.32 (dd, J 12.2, 3.1 Hz, 1H), 4.27 (dt, J 4.2,
2.9 Hz, 1H), 2.51 (ddd, J 14.2, 5.6, 1.7 Hz, 1H), 2.20 (m, 1H),
2.13 (dd, J Hz, 3H), 2.11 (dd, J Hz, 3H). dC (CDCl3, 125 MHz)
170.8, 170.6, 163.5, 150.2, 136.6, 114.6, 85.5, 82.6, 74.3,
64.0, 58.4, 37.9, 21.0, 20.9. m/z (HR-ESI). Calc. for
C14H18N2O8þNa: 365.0961. Found: 365.0963. Calc. for
C1313CH18 N2O8þNa: 366.0994. Found: 366.0992.
[8] W. F. Ho, B. C. Gilbert, M. J. Davies, J. Chem. Soc., Perkin Trans. 2
1997, 2533. doi:10.1039/A702491F
[9] D. J. Deeble, M. N. Schuchmann, S. Steenken, C. von Sonntag, J. Phys.
Chem. 1990, 94, 8186. doi:10.1021/J100384A038
[10] K. Chabita, P. C. Mandal, S. N. Bhattacharyya, Bull. Chem. Soc. Jpn.
1994, 67, 2751. doi:10.1246/BCSJ.67.2751
[11] M. Faraggi, F. Broitman, J. B. Trent, M. H. Klapper, J. Phys. Chem.
1996, 100, 14751. doi:10.1021/JP960590G
[12] M. Martini, J. Termini, Chem. Res. Toxicol. 1997, 10, 234.
doi:10.1021/TX960154L
(c)
5-Formyl-30,50-di-O-acetyl-20-desoxyuridine
(4b).
l
max/nm (CH3CN) (lg e) 292 (3.49). dH (CDCl3, 500 MHz)
10.01 (s, 1H), 9.95 (s, 1H), 8.47 (s, 1H), 6.28 (dd, J 7.7,
5.9 Hz, 1H), 5.23 (dt, J 6.3, 2.2 Hz, 1H), 4.37 (dd, J 11.1,
2.2 Hz, 1H), 4.32 (m, 2H), 2.59 (ddd, J 14.3, 5.9, 2.3 Hz, 1H),
2.26 (m, 1H), 2.16 (s, 3H), 2.09 (s, 3H). dC (CDCl3, 125 MHz)
186.0, 170.8, 170.6, 162.3, 149.4, 144.9, 111.6, 86.3, 83.2, 74.1,
63.7, 38.7, 20.9, 20.7. m/z (EI) 340 (1, Mþ), 201 (11), 81 (100).
m/z (HR-ESI) Calc. for C14H16N2O8þH: 341.0985. Found:
341.0983. Calc. for C1313CH16N2O8þH: 342.1019. Found:
342.1013.
[13] T. Itahara, Y. Fujii, M. Tada, J. Org. Chem. 1988, 53, 3421.
doi:10.1021/JO00250A003
[14] J. R. Wagner, J. Cadet, G. J. Fisher, Photochem. Photobiol. 1984, 40,
589. doi:10.1111/J.1751-1097.1984.TB05346.X
[15] C. Lagercrantz, J. Am. Chem. Soc. 1973, 95, 220. doi:10.1021/
JA00782A039
[16] P. Neta, Radiat. Res. 1972, 49, 1. doi:10.2307/3573369
[17] S. Fujita, S. Steenken, J. Am. Chem. Soc. 1981, 103, 2540. doi:10.1021/
JA00400A009
[18] J. Cadet, R. Te´oule, Bull. Soc. Chim. Fr. 1975, 891.
[19] R. P. Wayne, I. Barnes, P. Biggs, J. P. Burrows, C. E. Canosa-Mas,
J. Hjorth, G. Le Bras, G. K. Moortgat, D. Perner, G. Restelli,
H. Sidebottom, Atmos. Environ. 1991, A25.
(d) 30,50-Di-O-acetyl-20-desoxyuridine (6b). dH (CDCl3,
500 MHz) 8.47 (s, 1H), 7.53 (d, J 8.2 Hz, 1H), 6.26 (dd, J 8.2,
7.5 Hz, 1H), 5.80 (d, J 8.2 Hz, 1H), 4.38 (dd, J 12.1, 4.2 Hz, 1H),
4.32 (dd, J 12.1, 3.3 Hz, 1H), 4.28 (dd, J 6.6, 3.4 Hz, 1H), 2.55
(ddd, J 14.2, 5.6, 2.1 Hz, 1H), 2.17 (ddd, J 14.2, 7.7, 5.6 Hz, 1H),
2.12 (s, 3H), 2.11 (s, 3H). dC (CDCl3, 125 MHz) 170.5, 170.4,
163.1, 149.9, 139.3, 102.8, 85.6, 82.6, 74.2, 63.9, 38.1, 21.0,
20.9. m/z (HR-ESI). Calc. for C13H16N2O7þH: 313.1036.
Found: 313.1033. Calc. for C1213CH16N2O7þH: 314.1069.
Found: 314.1066.
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30,50-Di-O-acetyl-5-(N-nitroso)acetamidomethyl-20-
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desoxyuridine (8b). dH (CDCl3, 500 MHz) 8.22 (s, 1H),
7.48 (s, 1H), 6.22 (dd, J 8.7, 5.4 Hz, 1H), 5.23 (dt, J 6.4,
1.8 Hz, 1H), 4.78 (d, J 14.8 Hz, 1H), 5.54 (d, J 14.8 Hz, 1H),
4.43 (dd, J 12.2, 4.5 Hz, 1H), 4.33 (dd, J 12.2, 3.1 Hz, 1H), 4.28
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C16H20N4O9þH: 413.1309. Found: 413.1309. Calc. for
C1513CH20N4O9þH: 414.1342.1600. Found: 414.1339.
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relative peak area in the HPLC chromatograms recorded at l 260 nm
cannot be used as a measure to determine absolute yields in depen-
dence of reaction time. The data shown in Figure 2 only provide a
qualitative, but nevertheless highly useful insight into formation and
consumption of the reaction products.
Acknowledgement
This work was supported by the Australian Research Council under the
Centre of Excellence Scheme and the Deutsche Forschungsgemeinschaft.
Support by the Institut fu¨r Organische Chemie of the University of Basel
and the Otto-Diels-Institut fu¨r Organische Chemie of the University of
Kiel is gratefully acknowledged. We thank Dr Oliver Kru¨ger for helpful
discussions.
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