Dissociative electron transfer to and from pyrimidine cyclobutane dimers: an electrochemical study.

Article abstract of DOI:10.1039/b406923d

Cyclic voltammetry was used to study the reduction and oxidation behaviour of several pyrimidine cyclobutane dimers mimicking UV induced lesion in DNA strands in polar solvents (N,N-dimethylformamide and acetonitrile). Both electron injection and removal to and from the dimers, respectively, lead to their cleavage and reformation of the monomeric base. The influence of stereochemistry and substitution pattern at the cyclobutane motif on the reactivity has been studied. It appears that the repair process always proceeds in a sequential fashion with initial formation of a dimer ion radical intermediate, which then undergoes ring opening by homolytic cleavage of the two C-C bonds. Standard redox potentials for the formation of both radical anion and radical cation state of the dimers were determined. Quantum calculations on simplified model compounds reveal the reason for the finding that the exergonic homolytic cleavages of the carbon-carbon bonds are endowed with sizeable activation barriers. The consequences of these mechanistic studies on the natural enzymatic repair by photolyase enzyme are discussed. Copyright 2004 The Royal Society of Chemistry

Full text of DOI:10.1039/b406923d

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A R T I C L E
Dissociative electron transfer to and from pyrimidine cyclobutane
dimers: An electrochemical study
Fabien Boussicault,a Oliver Krüger,b Marc Robert*a and Uta Wille*c
a
Laboratoire d’Electrochimie Moléculaire, UMR - CNRS No 7591,
Université de Paris 7 - Denis Diderot, 2 place Jussieu, 75251 Paris Cedex 05,
France. E-mail: robert@paris7.jussieu.fr; Fax: + 33 1 44 27 76 25
Institut für Organische Chemie, Universität Kiel, Olshausenstr. 40, 24098 Kiel, Germany
b
c
School of Chemistry, The University of Melbourne, Victoria 3010, Australia.
E-mail: uwille@unimelb.edu.au
Received 10th May 2004, Accepted 15th July 2004
First published as an Advance Article on the web 31 August 2004
Cyclic voltammetry was used to study the reduction and oxidation behaviour of several pyrimidine cyclobutane dimers
mimicking UV induced lesion in DNA strands in polar solvents (N,N-dimethylformamide and acetonitrile). Both electron
injection and removal to and from the dimers, respectively, lead to their cleavage and reformation of the monomeric base.
The influence of stereochemistry and substitution pattern at the cyclobutane motif on the reactivity has been studied.
It appears that the repair process always proceeds in a sequential fashion with initial formation of a dimer ion radical
intermediate, which then undergoes ring opening by homolytic cleavage of the two C–C bonds. Standard redox potentials
for the formation of both radical anion and radical cation state of the dimers were determined. Quantum calculations on
simplified model compounds reveal the reason for the finding that the exergonic homolytic cleavages of the carbon–carbon
bonds are endowed with sizeable activation barriers. The consequences of these mechanistic studies on the natural
enzymatic repair by photolyase enzyme are discussed.
Introduction
Major DNA lesions caused by UV (A or B) irradiation are
bipyrimidine products (Scheme 1). Among them, the cis–syn
thymine–thymine cyclobutane dimer, resulting from 2 + 2 cyclo-
addition of two adjacent bases in the same oligonucleotide strand,
appears as the main photoproduct.1,2 In addition to the classical
excision repair mechanism, redox photolyase enzymes are also able
to repair these damages in bacteria in a catalytic electron transfer
cycle. Inside the enzymatic pocket, fully reduced flavine cofactor
(FADH−) is excited by a photon to its singlet state, which transfers
/
\
Scheme 2 Catalytic cycle for photoreparation of a thymine dimer (T \/ T)
by a flavin cofactor (FADH−) in a photolyase enzyme.
/
\
one electron to the dimer, T \/ T (Scheme 2). Splitting ensues, and
the two DNA bases are finally restored after reoxidation of the radi-
− 2
of the cofactor inside the enzyme pocket relative to the lesion, the
microscopic aspects of the reaction are still not fully understood.
In particular, is the charge transfer and the breaking of the dimer
(which implies two homolytic C–C bond cleavages) concerted or
not (Scheme 3)?4 If not, how does the stereochemistry of the dimer
(cis vs. trans, syn vs. anti, see below) affect the cleavage rate? And
what are the reasons why these homolytic breakages may possess
sizable activation barriers? Whereas photolyase type enzymes are
able to reductively repair DNA photoproducts, it has also been
shown that pyrimidine cyclobutane dimer splitting could be induced
by oxidation.5a For example, the nitrate radical NO3 is a powerful
oxidant able to irreversibly cleave N,N-dimethyl-substituted uracil
cal anion of the monomer, T . This general picture is also supposed
to be valid for the repair of 6–4 photoproducts, which are believed to
be converted to oxetane or azetidine cyclic structures, prior to their
photoreduction by 1FADH−*.2c,3
Beside the questions of the ability of the enzyme to recognize
and bind to modifed DNA sequences and the exact configuration
cyclobutane dimers, DMU \/ DMU.5b The same questions arise con-
cerning the possible simultaneousness of the electron removal from
the dimer and the cleavage of the cyclobutane ring, as well as on the
role of the stereochemistry at the ring. It should be noted that such
charge induced cleavage of cyclobutane containing small organic
molecules have already been reported, both under reductive5c and
oxidative conditions.5d–f
/
\
Scheme 3 Reductive cleavage of a molecule AB (the electron donor
could be either an electrode, the excited state of an aromatic molecule or an
homogeneous donor).
Scheme 1 Main photoproducts obtained from UV induced dimerization
of pyrimidine bases in the DNA.
2 7 4 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 4 2 – 2 7 5 0
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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Beyond these mechanistic questions, bipyrimidine dimers are
interesting structures for studying excess electron transfer in DNA,
i.e. the hopping of a negative charge through the bases over long
distances.6 Carell et al. pioneered this emerging field by studying
charge transfer in DNAhairpins containing a cis–syn thymine dimer
and a flavin, covalently attached to the head of the hairpin at a con-
trolled distance from the lesion.7 The redox reaction is triggered
from UV irradiation and its efficiency could be quantified through
measurement of cleaved DNA strands as a function of time.
In an effort to better understand the mechanism of the chemical
repair of bipyrimidine lesions, we have performed direct electro-
chemistry (cyclic voltammetry) on model lesion compounds pos-
sessing different substitution pattern and stereochemistry at the
cyclobutane ring (e.g. N,N-dimethylated uracil, 6-methyl uracil and
thymine cyclobutane dimers, see Scheme 4) as well as on selected
pyrimidine monomers (e.g. N,N-dimethylated and non methylated
uracil and thymine) at glassy carbon electrodes. The mechanisms
have been studied in N,N-dimethylformamide (DMF) in the case of
the reductive processes and in acetonitrile (ACN) in the case of the
oxidative repair.
The observation that the position of Ep is slightly sensitive to the
substrate concentration (∂Ep/∂log C = +8 mV at low scan rates) is
an indication for a second order reaction.
Fig. 1 Cyclic voltammetry of DMU (1.76 mM) in DMF + 0.1 M n-
Bu4NBF4. Scan rate: 0.1 V s−1. Temperature: 22 °C.
Electron stoichiometry could be obtained from comparison with
the fully reversible one electron wave of benzoquinone, for which
the peak current is given by9
ip = 0.446 FSC0D1/2(Fv/RT)1/2
(1)
(ip: peak current, S: electrode surface area, C0: bulk concentration,
D: diffusion coefficient, v: scan rate). For DMU, the current equals
ip = npFSC0D1/2(Fv/RT)1/2
(2)
The non-dimensional peak current p varies from 0.527 to 0.351 as
the kinetic control progressively passes from the chemical reaction
(pure kinetic conditions) to the charge transfer and was estimated
at each scan rate from the peak-width and ∂Ep/∂ log v values. From
this, a value of n ≈ 1 was obtained, corresponding to a monoelec-
tronic reduction wave. It is likely that the radical anion produced
−
after one electron consumption, DMU , dimerizes at the C(6)
position, since B3LYP calculations performed for the uracil radical
−
anion, U , in the gas phase revealed that the spin density is mainly
localized on this position. (Fig. 2).10 Moreover, semi-empirical cal-
culations have shown that dimerization at C(6) gives a more stable
(less constrained) structure than a coupling involving the two C(4)
atoms (Fig. 2). This behaviour is in sharp contrast with that of the
non methylated uracil. With this latter compound, we experimen-
tally found that the radical anion formed by injection of one electron
protonates from the neutral substrate, taking advantage of the acidic
proton at the nitrogen atom N(1).8 The resulting neutral radical then
dimerizes. The stoichiometry of the wave concomitantly drops to
half an electron per substrate molecule. Addition of a strong acid
to a solution of uracil leads to the increase of the wave height by a
factor two as predicted by such a mechanism. It’s noteworthy that
Elving et al. proposed a similar mechanism for uracil reduction in
dimethylsulfoxide.8a
Scheme 4 N,N′-methylated pyrimidine monomers and dimers investi-
gated.
In a first part, we’ll briefly describe the reduction and oxidation
mechanism of the monobases. Elving et al. carefully studied the
electrochemistry of DNA bases in polar aprotic solvent.8a,b We get
back to these studies by looking at the effect of methyl substitution
onto the two nitrogen atoms. From there, we’ll turn to the electro-
chemical activity of the model dimers. We’ll take advantage of a
careful analysis of the voltammograms and of their characteristics as
a function of the scan rate in order to decipher the exact mechanism
by which these compounds are reduced and oxidized. In addition,
density functional theoretical calculations (B3LYP) for simplified
model compounds were performed to theoretically describe the
potential energy surfaces involved. These calculations will prove to
be useful for underlying the reasons why such exergonic homolytic
cleavages may be endowed with non zero activation barriers.
Experimental results
All substrates were studied by cyclic voltammetry at 3 mm diameter
glassy carbon electrodes in N,N′-dimethyl formamide (DMF, reduc-
tion) or acetonitrile (ACN, oxidation). For details, see Experimental
section.
Electron transfer to and from DNA thymine and uracil bases
and N,N′-methylated bases at carbon electrodes
N,N-dimethyl uracil (DMU) is reduced at very negative potentials,
in an irreversible process. As shown in Fig. 1, the wave is thin (the
peak-width, Ep/2–Ep, is close to 55 mV at low scan rates). The peak
potential (−2.32 V vs. SCE at 0.1 V s−1) moves toward negative
values at the rate of 31 mV per decade log v between 50 mV s−1 and
3 V s−1. This behaviour is typical of an E + C mechanism, where
the charge transfer, E, is followed by a fast chemical reaction, C.
Fig. 2 Top, spin density (bold numbers) on C(4) and C(6) atoms for uracil
−
radical anion, U (B3LYP/6–31G*, gas phase). Bottom, dimers obtained
from C–C coupling (left: C(4)–C(4)′ coupling; right: C(6)–C(6)′ coupling,
favoured).
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 4 2 – 2 7 5 0
2 7 4 3

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The reduction behaviour of N,N-dimethyl thymine (DMT) is
very similar to that of DMU, with a very negative reduction wave at
about −2.41 V vs SCE (0.1 V s−1). The peak-width was 52 mV at the
same scan rate and an Ep variation with scan rate and substrate con-
centration of about −26 mV and 12 mV per decade log, respectively.
The number of electrons exchanged per reduced starting molecule is
n = 1.05, suggesting a similar mechanism as with DMU. Since the
dimerization step may change only marginally (due to the presence
of a methyl group adjacent to the C(4) atom) and since the rate of
electron transfer should be very close to that of DMU, it may be
concluded that the reduction is intrinsically more difficult, which is
reflected by the one hundred mV difference in peak potential values
between DMU and DMT.Atypical voltammetric response is shown
on Fig. 3 (upper left curve).
compared to DMU, with 1.81 V vs. SCE at 0.1 V s−1. The wave is
thin (Ep–Ep/2 is close to 65 mV at 0.1 V s−1) and drives smoothly to
positive potentials with increasing scan rates (∂Ep/∂log v ≈ 25 mV).
The process involves the abstraction of two electrons per substrate
molecule (n = 2.07).
The 200 mV difference in peak potential values between DMT
and DMU certainly reflects the fact that the spin density of the
radical cation formed after the loss of the first electron is mainly
concentrated on the C(5) atom (about 60% of the total density), as
+
was confirmed by quantum calculations. Thus, in DMT the radical
+
is tertiary and secondary in DMU , leading to a thermodynamic ad-
vantage for the formation of the former over the latter. The oxidation
mechanisms for both DMU and DMT are summarized in Scheme 5.
+
+
After formation of DMT and DMU , respectively, deprotonation
+
is likely to occur; in the case of DMT at the C(5)-methyl side chain
+
and in the case of DMU presumably at the methyl group at N(1),
which is then followed by a second oxidation step.
Dissociative electron transfer to and from cyclobutane
pyrimidine dimers at carbon electrodes
The next step consists of a detailed study of the cleavage pathways
for various DNA photolesion model compounds (Scheme 4). The
methodology and experimental conditions were the same as for the
preceeding bases.
/
\
c,s-DMU \/ DMU. This compound represents a typical example
for the behaviour of pyrimidine cyclobutane dimer upon electron
injection or removal. It gives rise to two irreversible oxidation
waves, located at 1.68 V vs. SCE and at 2.02 V vs. SCE (scan rate of
0.1 V s−1), respectively, as is illustrated in Fig. 4. The second wave
is identical to that observed during DMU oxidation, which indicates
that the first wave leads to the clevage of the dimer and formation
of DMU. The first peak shifts 52 mV to more positive values per
decade log v, while the peak-width amounts to 80 mV at low scan
rates. Thus, during this first oxidation process, the charge transfer
from the substrate to the electrode surface is the rate limiting step (at
least when the scan rate is increased to a few hundred mV s−1) and
is coupled to a fast and irreversible subsequent chemical reaction.
Since Ep is insensitive to the substrate concentration, this reaction
is first order. Application of the methodology mentioned above11
revealed that the first wave is bielectronic (n = 2.25).
Fig. 3 Top: Cyclic voltammetry of DMT (1.67 mM) in DMF + 0.1 M n-
Bu4NBF4 (left); DMT (1.96 mM) in ACN + 0.1 M n-Bu4NBF4 (right). Bot-
tom: Cyclic voltammetry of DMU (2.06 mM) in ACN + 0.1 M n-Bu4NBF4.
Scan rate: 0.1 V s−1. Temperature: 22 °C.
Upon oxidation, both DMU and DMT show anodic waves. Oxi-
dation of DMU is difficult, as is reflected by the very positive peak
potentials (Fig. 3, Ep = 2.01 V vs. SCE at 0.1 V s−1). The peak-width,
Ep − Ep/2, is close to 55 mV at low scan rates, and the peak potential
shifts to more positive values with 24 mV per decade log v. This
is the signature of a mixed kinetic control between the first charge
transfer and a following chemical reaction. Since Ep does not
change with varying substrate concentrations, the chemical reaction
should be a first order or pseudo first order reaction. Comparison
of the wave height with the reversible one electron reduction of
benzoquinone in the same solvent leads to the conclusion that the
oxidation of DMU is a bielectronic process with n = 2.1. A likely
mechanism involves the removal of an electron, followed by rapid
loss of a proton from the methyl group linked to the nitrogen N(1)
atom and a second oxidation (Scheme 5).5 The product (DMU+)
may slowly hydrolyze, a reaction not observed on the time scale of
cyclic voltammetry.
/
\
Fig. 4 Cyclic voltammetry of c,s-DMU \/ DMU (3.46 mM) in ACN + 0.1
The C(5) methylated base, DMT, follows a similar mechanism
upon oxidation (Fig. 3). The potential is markedly less positive,
M n-Bu4NBF4. Scan rate: 0.1 V s−1. Temperature: 22 °C.
On the basis of these observations, a mechanism for c,s-
/
\
DMU \/ DMU oxidation is proposed in Scheme 6. The first
electron removal leads to the formation of a cation radical, c,s-
/
\
+
DMU \/ DMU . Then, the C(6)–C(6′) bond next to the oxidized
nitrogen atom homolytically breaks, a step endowed with a sizable
activation barrier, immediately followed by the homolytic scission
of the C(5)–C(5′) bond, which is endowed with a small, if any, ac-
tivation barrier (see the Discussion section for a justification of the
+
order of bond cleavage). The radical cation DMU loses one proton
and is subsequently oxidized to an iminium cation DMU+, which
slowly hydrolyses to the mono-methylated uracil MU, as already
described for the monobase. The neutral DMU monomer produced
by the scission of c,s-DMU+–DMU is in turn oxidized along the
second oxidation wave. Characteristics of this oxidative process
Scheme 5 Oxidation mechanisms of DMU and DMT.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 4 2 – 2 7 5 0
2 7 4 4

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/
\
Fig. 5 Cyclic voltammetry of c,s-DMU \/ DMU (1.70 mM) in DMF + 0.1
M n-Bu4NBF4. Scan rate: 0.1 V s−1. Temperature: 22 °C.
is taken up by the carbonyl group at C(4) (* like orbital),12 before
being transferred into the *C(5)–C(5)′ orbital. This intramolecular
charge transfer is at the origin of the activation barrier.4c The C(6)–
C(6′) bond then homolytically breaks, concertedly or not with the
/
\
Scheme 6 Oxidation mechanism of c,s-DMU \/ DMU under electro-
chemical conditions.
−
first cleavage step, to produce both DMU and DMU. The first may
unambiguously show that cleavage of the dimer is a sequential pro-
cess, charge removing and cleavage step are not concerted.
dimerize to yield a C6–C6-dimer, as previously shown, whereas
DMU takes up the second electron before dimerizing likewise. If
the two successive C–C cleavages are fast enough, the kinetics
of the reaction may well be jointly controlled by the first charge
/
\
+
It was not possible to “trap” the radical cation (c,s-DMU \/ DMU )
resulting from the first charge transfer because of the quickness of the
bond-breaking steps and the slow electron transfer rate. Nevertheless,
since the location of the peak is kinetically controlled by the charge
transfer above 0.5 V s−1, the following equation may be applied,
which relates the peak potential value to the electron transfer rate9
−
transfer and dimerization of DMU . This seems to be the case here,
as indicated by the slight sensitivity of the peak potential to dimer
concentration.




app 

1/ 2


RT RT
RT




Ep = E0
+ 0.78
−
ln k
(3)





s
dimeri+ /dimer





αF αF
αFvD






E0
is the standard potential for dimer oxidation,  is the
+
dimer /dimer
transfer coefficient which is close to 0.5 and ksapp is the standard
heterogeneous rate constant for electron flowing. ksapp may vary
between 0.15 and 0.30 cm/s (by comparison with molecules of
similar size bearing the same functional groups), and the diffu-
sion coefficient D could be evaluated through application of the
Stokes–Einstein equation.11 This leads to a standard potential for the
/
\
oxidation of c,s-DMU \/ DMU of
E0dimer /dimer = 1.825 ± 0.025 V vs. SCE
+
/
\
/
\
It is worth noting that c,s-DMU \/ DMU is substantially more easily
oxidizable than DMU on thermodynamic grounds. Indeed the oxi-
dation peak of DMU is on one hand 200 mV more positive than the
Scheme 7 Reduction mechanism of c,s-DMU \/ DMU under electro-
chemical conditions.
/
\
/ \
standard potential estimated for c,s-DMU \/ DMU, and on the other
hand is less positive than the standard potential of DMU, meaning
that there’s an important stabilization of the radical cation state in
Other pyrimidine cyclobutane dimers. c,a-DMU6Me \/ DMU6Me
provides an illustrative example of the modification of both reduc-
tive and oxidative voltammetric waves, when the configuration at
the cyclobutane motif changes from syn to anti. Oxidation of c,a-
/
\
+
c,s-DMU \/ DMU by the neighbouring base.
/
\
/ \
Electron injection into c,s-DMU \/ DMU leads to a reductive
wave at very negative potentials (peak potential equals −2.34 V vs.
SCE at 0.1 V s−1, see Fig. 5). The wave is thin with a peak-width
being close to 45 mV at low scan rates and ∂Ep/∂log v ≈ −42 mV.
All these observations indicate an E + C mechanism, in which a first
electron transfer is followed by a fast chemical reaction and a mixed
kinetic control between the E and the C step. Surprisingly, the peak
potential is moderately sensitive to the dimer concentration, indicat-
ing the interference of a second order reaction. That the reduction
process does lead to dimer cleavage and formation of DMU could
not be directly confirmed as in the case of oxidation, since DMU is
reduced at the same potential than the dimer. However, an electroly-
sis performed at controlled potential (−2.3 V vs. SCE), followed by
an oxidative scan revealed the disappearance of the first oxidation
wave, which can be explained by a reductive cleavage of the dimer
to form DMU. Additionally, the electron stoichiometry, determined
from the peak-width and peak shifting with scan rate, indicates a
two electron wave (n = 1.85), one electron being used to cleave the
dimer, and one electron to reduce the monomer formed. The reduc-
tive cleavage is thus two steps, electron transfer and bond breaking
are not concerted.
DMU6Me \/ DMU6Me gives rise to only one irreversible wave, located
at 2.02 V vs SCE (scan rate 0.1 V s−1), as illustrated in Fig. 6. The
peak shifts 45 mV positively per decade log v while the peak-width
amounts to 65 mV at low scan rates. Charge transfer from the sub-
strate to the electrode surface is coupled to a fast and irreversible
subsequent chemical reaction in a two-step sequence (E + C), which
jointly control the kinetics of the reaction. Since Ep is insensitive to
the substrate concentration, this reaction is of first order. Calcula-
tion of the electron stoichiometry revealed release of four electrons
per substrate molecule (n = 3.75). This outcome is in contrast to the
results of the cleavage of the c,s- and t,s-configurated dimers, where
only two electrons per substrate molecule are exchanged. The rea-
son for this difference is the higher potential required to oxidize c,a-
DMU6Me DMU6Me. One electron is consumed during the actual
/
\
\/
cleavage of the dimer to form back a neutral monomeric base and a
monomeric radical cation, whereas the three others are used to oxi-
dize these products, since they are formed at a potential where they
are oxidizable. It is remarkable that c,a-DMU6Me \/ DMU6Me is oxi-
/
\
/
\
dized at a potential 340 mV more positive than c,s-DMU \/ DMU.
Not only is the oxidation more difficult on thermodynamic grounds,
but it can also be concluded that the splitting of the C(6)–C(6′) bond
is slower. Indeed, while intrinsic heterogeneous electron transfer
At this point and based on the preceeding analysis, the follow-
ing mechanism could be proposed (Scheme 7). The first electron
/
\
rates are likely to be very close for both c,s-DMU \/ DMU and
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 4 2 – 2 7 5 0
2 7 4 5

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Table 1 Voltammetric data for reduction and oxidation of various N,N-dimethyl thymine and uracil cyclobutane dimers (at 295 K)
Reduction a
Oxidation b
Compound
Ep
Ec
∂Ep/∂logv
nd
Ep
Ec
∂Ep/∂logv
nd
/
\
c,s-DMU \/ DMU
−2.34
−2.45
−2.34
−2.58
−2.61
−2.52
45
80
42
80
80
51
−42
−62
−34
−50
−50
−39
1.85
1.75
1.50
1.85
2.00
1.95
1.68
1.67
1.70
2.05
1.99
2.02
80
80
80
60
75
65
52
52
60
42
54
45
2.25
1.95
2.10
3.50
4.50
3.75
/
\
c,s-DMT \/ DMT
/
\
t,s-DMU \/ DMU
/
\
c,a-DMU \/ DMU
/
\
c,a-DMT \/ DMT
/
\
c,a-DMU6Me \/ DMU6Me
a In DMF. b In ACN. c Peak width (|Ep/2 − Ep|) at 0.1 V s−1. d Electron stoichiometry.
c,a-DMU6Me \/ DMU6Me, the peak widths at low scan rates indicate
that the kinetics is already controlled by the charge transfer for c,s-
dation wave obtained at low scan rate is shown in Fig. 7. The first
peak leads to the breaking of the dimer through a sequential path-
way and involves the exchange of two electrons (n = 1.95), whereas
the second peak corresponds to the oxidation of DMT produced
/
\
/
\
DMU \/ DMU but is still jointly determined by charge transfer and
bond breaking for c,a-DMU6Me \/ DMU6Me, meaning the chemical
reaction is faster for the splitting of c,s-DMU \/ DMU .
/
\
/
\
+
/ \
during the cleavage process. Reduction of c,s-DMT \/ DMT occurs
/ \
at a potential slightly more negative than that of c,s-DMU \/ DMU,
Ep = −2.45 V vs. SCE, and the wave is also slightly larger
(Ep/2 − Ep = 80 mV at 0.1 V s−1). This likely reflects the more nega-
tive value of E0
for c,s-DMT \/ DMT.
/
\
−
dimer/dimer
/
\
Fig. 7 Cyclic voltammetry of c,s-DMT \/ DMT (2 mM) in ACN + 0.1 M
n-Bu4NBF4. Scan rate: 0.1 V s−1. Temperature: 22 °C.
Discussion and calculations
Thermodynamical parameters from reduction and oxidation
of the dimers
/
\
Fig. 6 Cyclic voltammetry of c,a-DMU6Me \/ DMU6Me. Top: C = 1.50 mM,
ACN + 0.1 M n-Bu4NBF4. Scan rate: 0.5 V s−1. Temperature: 22 °C. Bottom:
C = 1.01 mM in DMF + 0.1 M n-Bu4NBF4. Scan rate: 0.1 V s−1. Tempera-
ture: 22 °C.
Due to the slow electron transfer and the fast following chemical
reaction, it was not possible to obtain standard potentials and cleav-
age rate constants for the dimer cleavage for both reduction and
oxidation processes, neither from direct methods (high scan rate
voltammetry) nor from indirect methods, such as redox catalysis.
The reduction of c,a-DMU6Me \/ DMU6Me involves a single irre-
versible wave, observed at −2.52 V vs SCE (0.1 V s−1). The wave
is thin (the peak-width amounts to 55 mV at low scan rates) while
Ep shifts towards negative potentials at a rate of 39 mV per decade.
This is again typical for a two-step process, with a mixed kinetic
control between electron transfer and chemical reaction. That the
peak potential smoothly changes to positive values while increasing
the substrate concentration indicates the interference of a second
order reaction. With an electron stoichiometry of n = 1.95, it can be
/
\
/
\
However, using the same strategy as applied for c,s-DMU \/ DMU,
one may take advantage of the finding that for several compounds,
an increase of the scan rate quickly leads to kinetic control by
charge transfer only. In these cases, the peak potential for an
oxidative wave could be related to the standard potential and to the
standard heterogeneous rate constant according to eqn. (3) and for a
reductive wave according to eqn. (4).9
assumed that the reduction mechanism of c,a-DMU6Me \/ DMU6Me
/
\
1/ 2
/
\


app 





is similar to that of c,s-DMU \/ DMU. It is difficult to compare the
RT
RT
RT
Ep = Ed0imer/dimeri− −0.78
+
ln k
(4)





cleavage rate of c,s-DMU \/ DMU and c,a-DMU6Me \/ DMU6Me −
since both peak width and ∂Ep/∂log v are quite similar. It can never-

/
\
−
/ \
s







αF αF
αFvD




theless be concluded that c,a-DMU6Me \/ DMU6Me is more difficult
E0
is the standard potential for dimer reduction and 
/
\
−
dimer/dimer
is the transfer coefficient (close to 0.5). ksapp was estimated by
comparison with molecules of similar size to be 0.15–0.3 cm s−1,
The value for the diffusion coefficient D was estimated using the
Stokes–Einstein equation to be on average 1.25 × 10−5 cm2 s−1 in
ACN and 6.6 × 10−6 cm2 s−1 in DMF, since the hard sphere radii for
all dimers under investigation are almost identical.11
/
\
to reduce than c,s-DMU \/ DMU on thermodynamic grounds, since
the reduction wave of this latter compound is 180 mV more posi-
tive than that of c,a-DMU6Me \/ DMU6Me. A voltammetric response
is shown in Fig. 6.
The characteristics of both oxidative and reductive cleavages for
all six dimers are compiled in Table 1. Comparison of the results
obtained for t,s-DMU \/ DMU and c,s-DMU \/ DMU clearly shows
that a cis or trans stereochemistry at the cyclobutane ring of the
dimer has only marginal effects on both thermodynamics and kinet-
ics of reduction and oxidation processes, since both wave shape
(location, width, changes with the scan rate) and stoichiometry
(n = 2) are very similar.
/
\
/
\
/ \
With these data, the standard potential for dimer radical anion
/
\
/ \
formation was calculated for c,s-DMT \/ DMT, c,a-DMU \/ DMU
/
\
and c,a-DMT \/ DMT, since in these cases complete kinetic control
by charge transfer was achieved above 1 V s−1 (Table 2). Compari-
/
\
son of the standard potentials obtained for c,s-DMT \/ DMT and
/
\
c,a-DMT \/ DMT indicates that a syn configuration stabilizes the
radical anion by 150 mV over an anti configuration.
/
\
Comparison of the cleavage process of c,s-DMT \/ DMT and
c,s-DMU \/ DMU (Table 1) reveals that changing from a uracil to
thymine does not have a profund impact on the mechanism. An oxi-
/
\
The standard potential for the formation of a dimer radical cation
/
\
/ \
was calculated for c,s-DMT \/ DMT and t,s-DMU \/ DMU, using
2 7 4 6
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 4 2 – 2 7 5 0

View Article Online
Table 2 Estimation of standard potentials for reduction and oxidation
of N,N-dimethyl thymine and uracil dimers, and for DMU and DMT
monomers
Table 3 Gas phase reduction of a model compound of cyclobutane pyrimi-
dine dimer (A + 1e−). Spin repartition along the reaction pathway
C(4)
O(4)
C(5)
C(5)′
C(6)
−a
a
−ab
+
Compound
E°dimer/dimer
E°dimer /dimer
E°mono/mono
1
TS1
2
TS2
3
0.52
0.29
0
0.03
0.13
0.26
0.22
0.02
0.06
0.12
0
0.05
0.385
0.97
0.58
0
0.01
0.02
0.01
0.19
0.57
/
\
c,s-DMU \/ DMU
—
1.825 ± 0.025
1.815 ± 0.025
1.850 ± 0.025
≈2.195
≈ −2.39
≈ −2.46
0.04
0.06
0.14
0.09
/
\
c,s-DMT \/ DMT
−2.620 ± 0.025
—
−2.735 ± 0.025
−2.770 ± 0.025
/
\
t,s-DMU \/ DMU
/
\
c,a-DMU \/ DMU
/
\
c,a-DMT \/ DMT
—
a In V vs. SCE. b Standard potential for the formation of monomer radical
ing a second barrier (TS2, Fig. 9), which corresponds to the break-
ing of the C(6)–C(6′) bond, a step endowed with a smaller barrier of
G‡ = 0.11 eV. From structure 2 to TS2, the C(6)–C(6′) bond length
increases from 1.585 to 2.049 Å.At the same time, the spin on C(5′),
which is close to 1 in state 2, decreases to 0.58 in TS2 and finally
drops to zero as the enamine leaving group is formed (CH2CHNH2).
At the end of the cleavage process, the spin is mainly localized on
C(6), which corresponds to the radical anion state of uracil. The
spin density repartition on the various atoms all along the reactive
pathway is summarized in Table 3.
anion.13
eqn. (3) and taking into account that a slow limiting charge trans-
fer is fully achieved around 1 V s−1 (Table 2). As already noticed,
it appears that neither a cis or a trans configuration at the cyclo-
butane motif nor the presence or absence of a methyl group at the
C(5)/C(5′) positions have a significant influence on the standard
/
\
/ \
potential. The radical cations of c,s-DMU \/ DMU, c,s-DMT \/ DMT
/
\
and t,s-DMU \/ DMU should also cleave at comparable rates.
These calculations confirm our suggestion made for the reductive
/
\
Interestingly, the anti dimer c,a-DMU \/ DMU is the only com-
/
\
cleavage of c,s-DMU \/ DMU that the C(5)–C(5′) bond breaks first
pound (except c,a-DMU6Me \/ DMU6Me), where, upon oxidation, the
kinetics are jointly controlled by charge transfer and breaking of the
C–C bond at low and moderate scan rates. In these cases, variation
of peak width E (Ep − Ep/2) and peak potential location Ep with
/
\
log v depends on three parameters, e.g. E°dimer /dimer, ksapp and kc, the
+
cleavage rate of the C–C bond. Using an estimated value for ksapp of
0.25 cm s−1 (mean value) and the experimentally determined values
of E and Ep, simulations of cyclovoltammetric curves were per-
formed (Digisim, BioAnalytical System) to determine the standard
/
\
−
potential and the cleavage rate constant. For c,a-DMU \/ DMU we
Fig. 8 Model compound (A ) sketching cyclobutane pyrimidine dimer
+
obtained E°(dimer /dimer) ≈ 2.195 V vs. SCE and kc ≈ 4 × 106 s−1.
reduction. Optimized radical anion geometry (right: top view).
Since the cleavage rate constant is quite sensitive to the exact value
of ksapp, kc should be considered in terms of order of magnitude rather
than an accurate absolute value. Nevertheless it can be concluded
that anti dimers are substantially more difficult to oxidize than their
/
\
syn homologues. As an example, in the case of c,a-DMU \/ DMU
the standard potential for radical cation formation is 370 mV more
/
\
positive than that of c,s-DMU \/ DMU (Table 2), indicating a desta-
bilization of the radical cation state of anti dimers compare to syn
compounds, as was already suggested by Pac et al.14
Modelling calculations
Mapping of the potential energy surface for a model compound15
may give a more accurate view of the cleavage process in terms of
electron reorganization, and may thus lead to a fundamental under-
standing of the origin of the activation barrier which is endowed,
as shown experimentally, for both reductive and oxidative cleavage
processes. To keep the required computation times at a reasonable
level, density functional calculations (using the B3LYP/6–31G*
method) were performed for simplified cyclobutane adduct models
in order to get a refined qualitative description of the pyrimidine
reformation, rather than a quantitative estimation of the size of the
cleavage barriers.
The minimum energy pathway for the homolytic cleavage of
−
A (Fig. 8) was calculated in mass-weighted internal coordinates
(IRC), revealing that the C(5)–C(5′) bond cleaves first with a free
enthalpy of activation of G‡ = 0.21 eV (Fig. 9) (it was verified that
C(6)–C(6′) bond breaking is far less favorable). It should be noted
that the activation barrier in solution is certainly slightly higher,
since a small percentage (10%) of the negative charge located onto
the C(4)O(4) group is transfered to the C(5′) atom when reaching
the transition state, thus leading to an additional solvent reorganiza-
tion energy. At the transition state (TS1, Fig. 9), C(5)–C(5′) elon-
gates from 1.569 to 1.937 Å, and almost 40% of spin density locates
Fig. 9 Gas phase reduction of pyrimidine dimer adduct model A. Top:
structures at minima (1, 2) and at transition states (TS1, TS2). Bottom:
Potential energy at the B3LYP/6–31G* level (left) and C–C bond distances
(right) as a function of the mass-weighted IRC coordinate (reaction coordi-
nate in u−1/2 a0).
−
onto the C(5′) atom [in the ground state geometry of A only 5% of
the spin is located on C(5′)]. This indicates that an intramolecular
electron transfer occurs during this first homolytic cleavage. The
pathway then leads to a second minimum (2, Fig. 9), before reach-
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 4 2 – 2 7 5 0
2 7 4 7

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with a sizable activation barrier. In a second step, the C(6)–C(6′)
elongates to lead to the formation of the two monomeric DMU.
It was not possible to locate transition states for the homolytic
that the canonical DNA structure is lost locally at the catalytic site,18
so that the dimer is certainly under considerable strain upon binding
to photolyase. It is therefore possible that the *C(5)–C(5)′ orbital is suf-
ficiently stabilized (through elongation) to render the concerted path
more favorable than the stepwise. Thus, the concerted or sequential
nature of the first electron transfer/bond breaking reaction of the re-
ductive repair sequence could not be infered only from model stud-
ies. Nevertheless, a qualitative description of the repair reaction in
terms of reaction pathway is possible, as is illustrated in Fig. 12 for
+
cleavage of of the cyclobutane ring in A . However, calculations
+
performed for model compound B (Fig. 10) revealed that the oxi-
dative cleavage goes through an activation barrier, corresponding
to the splitting of the C(6)–C(6′) bond firstly. The free enthalpy of
activation is calculated to be G‡ = 0.03 eV, whereas the alternative
cleavage of the C(5)–C(5′) bond is much less favorable. From the
optimized radical cation geometry to the transition state, C(6)–C(6′)
elongates from 1.791 to 1.95 Å, while the spin density on C(6′)
increases from 35% to 50%. At the same time, the spin density on
N(1) decreases from 42% to 31%, thus indicating an intramolecular
electron transfer to the *C(6)–C(6)′ orbital. In a subsequent step the
C(5)–C(5′) bond breaks, but it was not possible to characterize a
transition state for this second cleavage.
the reduction of a thymine dimer (T \/ T) by excited FADH−.
/
\
+
Fig. 12 Diabatic potential energy profiles (zero order) for the redox reac-
tion between the flavin cofactor (FADH−) and a thymine dimer (TT). a:
Concerted pathway. b: Sequential pathway going through the radical anion
state TT . The inset on diagram b details the avoided crossing intersection
(ACI zone) between the ground state reactants and products energy curves
(dotted lines: zero order profile, full lines: first order profile).
Fig. 10 Model compound (B ) used for simulating cyclobutane pyrimi-
dine dimer oxidation. Optimized radical cation geometry (upper right: top
view).
−
Consequences for the enzymatic repair mechanism
It is remarkable that monomers and dimers have comparable stan-
dard redox potentials (see Table 2, reduction reaction), stressing the
importance of the structure of the damaged DNA/photolyase com-
plex and the position of dimer motif inside the catalytic site, which
should be brought close to the cofactor.2c,16
A sequential pathway is outlined in diagram (b). After photon
absorption (formation of 2) and electron transfer to the dimer via a
small activation barrier (2 → 3), the short lived radical anion (3) then
breaks through a second activation barrier, to give the fragmented
dimer 4. The restored bases resulting from fragmention of 4 (cleav-
age of a second C–C bond) and subsequent rapid oxidation are not
sketched in the diagram (this process is highly exergonic and goes
through a small, if any, activation barrier). Before reaching 4, the
system crosses the intersection between the potential energy surface
of the fragmented state and that of the initial ground state (ACI).
There, it partitions between the two surfaces thus concurrently head-
ing for the fragments and for the initial ground state.19 The degree of
partitioning is a function of the degree of mixing of the two states,
i.e. of the degree of avoided crossing, at the intersection.
This avoided crossing return electron transfer is responsible for
a lowering of the quantum yield, which may asymptotically reach a
value of 0.5 upon increasing the avoided crossing energy.19a,b This
should be the main reason why the quantum yield for repair is less
than one. The non concerted character of the process should have
no influence on the repair yield because back electron transfer at
the level of the radical anion (3 → 1) is lying deep in the Marcus in-
verted region and is thus not competitive with the rapid C(6)–C(6′)
fragmentation. In fact, the driving force for the charge transfer
reaction 2 → 3 is −0.62 eV, as indicated from our experimentally
determined standard potential values for the two redox couples
We have shown that the reductive cleavage of model dimers
follows a two steps sequential pathway at an electrode. Since driv-
ing forces in photoinduced electron transfer reactions are higher,
it is tempting to conclude that the enzymatic repair by photolyase
enzymes should also proceed through intermediate formation of a
dimer radical anion. Indeed, the reducing power of the excited fla-
1
vin cofactor FADH−* amounts to −3.24 V vs SCE, since the first
excited state energy is 2.76 eV2c and the standard redox potential for
FADH− oxidation equals −0.48 V vs. SCE, as was estimated from
the reversible voltamograms obtained in aqueous buffered solutions
of riboflavin (Fig. 11).
However, in a recent thymidine repair study by DNA photolyase
in real time (picosecond range), the data shown by Stanley et al.
strongly suggest that the first carbon–carbon bond cleavage and elec-
tron transfer are concerted processes.17 Crystal structures have shown
FADH/FADH− and c,s-DMT \/ DMT/c,s-DMT \/ DMT . The back
electron transfer 3 → 1 is thus exergonic with a driving force of
about −2.14 eV (−2.76 + 0.62), to be compared to a reorganization
energy around a likely value of 1 eV.
/
\
/
\
−
The second possible pathway, a concerted electron transfer/bond
breaking reaction (2 → 4), is depicted on the left part of Fig. 12 (dia-
gram a). This is a highly exergonic process, which is endowed with
a large intrinsic barrier, due to the contribution of the bond being
broken during the process. The strain at the dimer motif bound to the
enzyme may lower the barrier for this pathway, leading to a kinetic
Fig. 11 Cyclic voltammetry of riboflavin (1.48 mM) + dithionite (1 eq.)
in aqueous buffered solution (Buffer Tris, C = 0.05 M, pH = 7.3). Scan rate:
0.1 V s−1. Temperature: 22 °C.
2 7 4 8
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 4 2 – 2 7 5 0

View Article Online
/
\
advantage over the sequential pathway. In this case, the system,
en route to 4, again crosses the intersection between the potential
energy surface of the fragmented state and that of the initial ground
state (ACI). This means that the quantum yield for the repair would
also be controlled by the partitioning at the intersection.19a
DMU \/ DMU. In a pyrex reactor 520 mg (3.71 mmol) DMU was
dissolved in 100 mL of acetone and deaerated in an ultrasonic bath
by bubbling argon through the solution. The reaction mixture was
irradiated under argon for 10 h using a medium-pressure mercury
lamp.After evaporation of the solvent in vacuo the residue was puri-
/
\
It is therefore not surprising that the quantum yields () for repair
by the enzyme containing FADH− are 0.7–1.2c Whatever may be the
pathway followed (concerted or sequential), and even if this path-
way may be different from one dimer stereochemistry to an other,
 mainly depends from the crossing of the ground state reactant and
products curves (ACI region, Fig. 12). The high values of  reflect
the fact that the crossing is moderately adiabatic and weakly sensi-
tive to the dimer structure and stereochemistry.
fied by chromatography to yield the various DMU \/ DMU isomers
in 75% combined yield. The stereoconfiguration of the isomers was
assigned by comparison with literature data.21b
/
\
DMT \/ DMT. In a pyrex reactor 680 mg (4.42 mmol) DMT was
dissolved in 100 mL of acetone and deaerated in an ultrasonic bath
by bubbling argon through the solution. The reaction mixture was
irradiated under argon for 3 d using a medium-pressure mercury
lamp.After evaporation of the solvent in vacuo the residue was puri-
/
\
fied by chromatography to yield the various DMT \/ DMT isomers
in 55% combined yield. The stereoconfiguration of the isomers was
assigned by comparison with literature data.21c
Conclusions
Pyrimidine cyclobutane dimers are cleaved under both reductive
and oxidative conditions at a carbon electrode. En route to cleavage,
ion radical intermediates are formed prior to two successive homo-
lytic C–C bond cleavages, leading to the formation of the neutral
monomeric base and the ion radical of the monobase. The former
species undergoes further electron exchange, since it’s formed at a
potential beyond it’s standard redox potential. Syn dimers are more
easily oxidized and reduced than anti structures, while only mar-
ginal changes are observed upon alkyl substitution at the cyclobu-
tane motif. The cleavage rate constants of the dimers were too fast to
be measured, but a careful analysis of the shape of the voltammetric
curves allows the estimation of the standard potential for the forma-
DMU6Me \/ DMU6Me. In a pyrex reactor 1.01 g (6.56 mmol)
DMU6Me was dissolved in 100 mL of acetone and deaerated in
an ultrasonic bath by bubbling argon through the solution. The
reaction mixture was irradiated under argon for 4 d using a me-
dium-pressure mercury lamp. After evaporation of the solvent in
vacuo the residue was purified by chromatography to yield the
/
\
various DMU6Me \/ DMU6Me isomers in 95% combined yield. The
/
\
stereoconfiguration of c,a-DMU6Me DMU6Me was assigned by
/
\
\/
X-ray analysis.21d Yield: 69%; Rf = 0.19 (SiO2, ethyl acetate); mp.
206–207 °C (colourless solid); max (KBr)/cm−1: 2980, 1698, 1664,
1458, 1342; H (CDCl3, 300 MHz):3.22[6H, s, N(3)CH3, N(3′)CH3],
3.18 (2H, s, 5-H, 5′-H), 2.83 [6H, s, N(1)CH3, N(1′)CH3], 1.66 (6H,
s, 6-CH3, 6′-CH3); C (CDCl3, 75.5 MHz): 165.1 (s, C-4, C-4′),
151.9 (s, C-2, C-2′), 57.9 (s, C-6, C-6′), 53.4 (d, C-5, C-5′), 31.0 [q,
N(1)CH3, N(1′)CH3], 28.1 [q, N(3)CH3, N(3′)CH3], 27.7 (6-CH3,
6′-CH3); m/z (EI): 154 (100), 97 (49), 82 (25); m/z (CI): 155 (100);
C14H20N4O4·0.1 C4H8O2: calcd. C 54.33, H 6.59, N 17.60; found C
54.39, H 6.61, N 17.72.
/
\
/ \
tion of the radical anion for c,s-DMT \/ DMT, c,a-DMU \/ DMU,
/
\
c,a-DMT \/ DMT, while the standard potential for the formation
/
\
/ \
\/
of the radical cation for c,s-DMU DMU, c,s-DMT DMT,
\/
/
\
/ \
\/
t,s-DMU DMU and c,a-DMU DMU were obtained. Radical
\/
anions of syn dimers are stabilized over those of anti configuration
by ca. 150 mV while radical cation of syn compounds are stabilized
over those of anti configuration by ca. 350 mV. The barrier for
cleavage of the intermediate mainly originates in an intramolecular
electron transfer from the orbital where the unpaired electron tem-
porarily sites to the * of the carbon–carbon bond being broken,
a contribution to which solvent reorganization energy should be
added. The standard potential for the formation of the radical anion
of the monobases (DMU and DMT) are more positive than those of
all the dimers investigated. In the catalytic site where photolyase
enzyme repair such dimer adducts on DNA strands, the lesion is
flipped out the double strand, and is thus brought in close contact
to the cofactor. The strain at the dimer motif probably facilitates the
reductive cleavage and may even render the fully concerted path-
way more favorable than the stepwise mechanism observed under
our electrochemical conditions for model compounds. But whatever
may be the pathway followed inside the enzyme, the repair quantum
yield is likely to be controlled by the crossing of the two surfaces
associated to fragmented dimer and the reactants respectively,
which should be moderately adiabatic, in accordance with the high
efficiency of the enzymatic repair.
Electrochemical experiments
All substrates were studied by cyclic voltammetry in N,N′-dimethyl
formamide (DMF, reduction) or acetonitrile (ACN, oxidation). The
working electrode was a 3 mm-diameter glassy carbon electrode
disk (Tokai) carefully polished and ultrasonically rinsed in absolute
ethanol before use. The counter-electrode was a platinum wire and
the reference electrode an aqueous SCE electrode. The potentiostat,
equipped with a positive feedback compensation and current mea-
surer was the same as previously described.22 All experiments have
been done at 22 °C, the double-wall jacket cell being thermostated
by circulation of water.
Molecular modelling
All the calculations were performed with the Gaussian 98 series of
programs.23 DFT (B3LYP) method and 6-31G* basis set were used.
Minimum energy structures were fully optimized. Frequency cal-
culations were made to verify that the structures were minima (no
imaginary frequencies) or saddle point (one imaginary frequency)
and to evaluate thermodynamical functions.
The nature of the reactant and products linked to transition states
was assigned by the intrinsic reaction coordinate (IRC) method at
the same level of calculation as used for saddle point characteriza-
tion. IRC’s have been determined in mass-weighted internal coor-
dinates with a step size of 0.05 or 0.1 in atomic units. The activation
barriers were calculated as the free enthalpy difference between the
saddle point and the minimum structures.
Experimental
Chemicals
N,N′-dimethyl formamide (Fluka, > 99.8%, stored over molecular
sieves and under argon atmosphere), acetonitrile (Fluka, > 99.5%,
stored over molecular sieves and under argon atmosphere), acetone
(Acros, HPLC grade, 99.8%), the supporting electrolyte n-Bu4NBF4
(Fluka, > 99%), uracil (Sigma), thymine (Sigma), 1,3-dimethyl-
uracil (Aldrich), riboflavin (Sigma) and sodium dithionite (Aldrich)
were used as received.
Synthesis of the pyrimidine cyclobutane dimers
Notes and references
1,3-Dimethylthymine (DMT) was synthesized following the proce-
dure described by Falvey and coworkers.20
1,3,6-Trimethyluracil (DMU6Me) was prepared according to
reference 21a.
1 T. Douki, A. Reynaud-Angelin, J. Cadet and E. Sage, Biochemistry,
2003, 42, 9221–9226.
2 (a) P. F. Heelis, R. F. Hartman and S. D. Rose, Chem. Soc. Rev., 1995,
289–297; (b) T. Carell, L. T. Burgdorf, L. M. Kundu and M. Cichon,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 4 2 – 2 7 5 0
2 7 4 9

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Curr. Opin. Chem. Biol., 2001, 5, 491–498; (c) A. Sancar, Chem. Rev.,
2003, 103, 2203–2237, and references cited theirein.
only marginally from one dimer to an other. Thus D was taken equal
to 1.25 10−5 cm2 s−1 in ACN and to 6.6 10−6 cm2 s−1 in DMF for all com-
pounds.
3 (a) G. Prakash and D. E. Falvey, J. Am. Chem. Soc., 1995, 117,
11375–11376; (b) X. Zhao, J. Liu, D. S. Hsu, S. Zhao, J. S. Taylor
and A. Sancar, J. Biol. Chem., 1997, 272, 32580–32590; (c) J. Arul,
G. Prakash and D. E. Falvey, J. Am. Chem. Soc., 2000, 122,
11219–11225; (d) K. Hitomi, H. Nakamura, S. T. Kim, T. Mizukoshi,
T. Ishikawa, S. Iwai and T. Todo, J. Biol. Chem., 2001, 276,
10103–10109.
4 (a) J.-M. Savéant, in Advances in Physical Organic Chemistry, ed.
T. T. Tidwell, Academic Press, New York, 2000, vol. 35, 117–192;
(b) L. Pause, M. Robert and J.-M. Savéant, J. Am. Chem. Soc., 2001,
123, 4886–4895; (c) C. Costentin, M. Robert and J. M. Savéant, J. Am.
Chem. Soc., 2003, 125, 105–112.
12 S. T. Kim and A. Sancar, Biochemistry, 1991, 30, 8623–8630.
13 Simulation of the experimental curves with Digisim (BioAnalytical
System) leads to the reported values. Three parameters control the shape
and the position of the wave: the standard potential, the rate constant for
radical anion dimerization and the heterogenous electron transfer rate
constant, ksapp. The two last parameters were estimated to 109 M−1 s−1
and 0.1 cm s−1 respectively, leading to the sole variation of the standard
potential for adjustment with the experimental voltammetric curves. The
uncertainty on the E°’s obtained is estimated to equal 25 mV.
14 C. Pac, J. Kubo, T. Majima and H. Sakurai, Photochem. Photobiol.,
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10 It is known that vinylarylsulfones and vinyl pyridines undergo cyclodi-
merization at the level of the radical anion within catalytic amounts
of charge (see for example J. F. Bergamini, J. Delaunay, P. Hapiot,
M. Hillebrand, C. Lagrost, J. Simonet and E. Volanschi, J. Electro-
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J. H. P. Utley, Chem. Commun., 1998, 539–540). This process involves a
neutral molecule and its radical anion and is accompanied with a signifi-
cant activation barrier. Our observation that the electron stoichiometry
for pyrimidine monomer reduction remains unchanged even at very low
scan rates and the excellent reproducibility of the reduction wave dur-
ing the cycling experiments indicate that a cyclodimerization process as
described for vinylarylsulfones and vinyl pyridines did not occur in our
case. In contrast, dimerization of the pyrimidine monomer radical anions
at the C(6) position only involves minor electronic reorganization and
is likely endowed with only a negligible if any activation barrier, thus
making this step nearly diffusion controlled.
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11 The diffusion coefficient D was estimated through application of the
Stokes–Einstein equation D = kBT/(6a),  being the solvent viscosity
and a the hard sphere radius equivalent to the diffusing molecule. Radii
were obtained from a quantum calculation at the B3LYP level (key-
word Volume). Typically, values close to 4.7 Å were found and vary
2 7 5 0
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 7 4 2 – 2 7 5 0