(6-4)-Photolyase activity requires a charge shift reaction

Article abstract of DOI:10.1039/b503699b

A model compound containing a thymine oxetane moiety linked to a flavin chromophore was investigated regarding (6-4)-photolyase activity. The need for a charge shift reaction was demonstrated by a detailed pH-dependent kinetic analysis. The Royal Society

Full text of DOI:10.1039/b503699b

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(
6-4)-Photolyase activity requires a charge shift reaction{{
Thorsten Stafforst and Ulf Diederichsen*
Received (in Cambridge, UK) 14th March 2005, Accepted 3rd May 2005
First published as an Advance Article on the web 1st May 2005
DOI: 10.1039/b503699b
A model compound containing a thymine oxetane moiety
linked to a flavin chromophore was investigated regarding
oxidative process using several sensitisers and varying pH
conditions. Therefore, model compound 1 containing the thymine
oxetane covalently linked to a flavin moiety was synthesised and
(6-4)-photolyase activity. The need for a charge shift reaction
11
purified by HPLC. As shown in Fig. 1, the oxetane photo-
cleavage reaction was performed under reductive and oxidative
conditions in a fluorescence spectrometer by irradiation at 450 nm§
in methanol and varying buffers" in a 1:1 ratio. Reductive
conditions were realised by addition of 100 equivalents 0.2 M
sodium dithionite solution and controlled by fluorescence deple-
tion at 525 nm. During the irradiation, solutions (1 ml) were
bubbled with oxygen-free nitrogen. Oxidative conditions were
realised by bubbling with oxygen. The kinetic analysis was
performed by HPLC analysis at 450 nm with six to eight samples
was demonstrated by a detailed pH-dependent kinetic analysis.
Photodamage of DNA due to exposure to UV light is one of the
major environmental mutagens, which are associated with many
diseases like cancer, xeroderma pigmentosum, trichothiodystro-
1
phy, and the cockayne syndrome. The two most common forms
of photodamage are the cis, syn cyclobutane pyrimidine dimer
(
CPD) and the pyrimidine pyrimidone (6-4) photoproduct. The
6-4) photodamage can be repaired by (6-4)-photolyase enzymes,
the mechanism of which has attracted much attention in recent
(
2
3
years. Due to the high sequence homology with CPD-photo-
(25 ml) taken over a period of 75% conversion (Fig. 2). The peak
lyases a similar electron transfer repair mechanism involving an
4
oxetane intermediate was suggested by Kim. Falvey exemplified
area of starting material 1 was compared with the product signal
of thymine derivative 2. The constitutional integrity of the
photoproduct that has been obtained was proven by ESI-MS
and co-injection of independently prepared compound 2.
Quantum yields were obtained from the conversion rates after
calibration of the fluorescence spectrometer with potassium
this mechanism by a photolysis study on a thymine oxetane model
5
compound and photosensitisers under reductive conditions.
Carell observed the repair of the same thymine oxetane model
6
by covalently linked flavin exclusively under reductive conditions.
7
Although the reductive electron transfer is more plausible for the
12
ferrioxalate actinometry.
(
6-4)-photolyase, the oxidatively photosensitised cycloreversion
CR) of oxetanes is of high interest by means of artificial DNA
repair and electron transfer catalysed pericyclic reactions in
For the reductive conversions, all reactions showed first-order
(
kinetics.I As shown in Fig. 3 and Table 1, the reductive CR was
–7% efficient at pH values 5–11. The progression of the pH
4
8
9
general. In this regard, Barton proved the oxidative repair of
dependence of the reductive CR showed a maximum at pH 7 with
a strong decline to the acidic region and a slight decline to the basic
region. This is probably due to a change of the charge injection
from charge shift to charge separation. Around pH 7 the reduced
flavin is deprotonated (pK 5 6.2) and a charge shift reaction takes
10
CPD photodamage and Miranda studied the oxidative CR of
oxetanes.
In this study, we compare the strictly light-driven reductive CR
5
of the thymine oxetane described by Falvey with the respective
13
place. By changing to acidic conditions the flavin is protonated
and a charge separation reaction is enforced with a loss in
efficiency. With changing to basic conditions the thymine oxetane
(pK y 10) starts to become deprotonated, inhibiting the negative
{
Dedicated to Professor Albert Eschenmoser on the occasion of his 80th
birthday.
Electronic supplementary information (ESI) experimental section,
actinometry. See http://www.rsc.org/suppdata/cc/b5/b503699b/
{
*udieder@gwdg.de
14
charge donation from the flavin.
Fig. 1 Model compound 1 for simulation of (6-4)-photolyase activity by cycloreversion of the oxetane moiety forming 2.
3
430 | Chem. Commun., 2005, 3430–3432
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Fig. 4 Photolyase activity of carbazole containing model compound 3.
The reaction conditions were the same as for model compound 1 (buffer
10 mM phosphate (pH 7)/methanol 1:1, reductive conversion).
Fig. 2 Kinetic analysis of the photoconversion of model compound 1.
the thymine oxetane would change the mechanism again towards
charge shift.
Compared to the reductive conversion, the oxidative CR
showed the opposite progression with maximum efficiency strictly
at extreme pH values. This was reasoned by a maximum efficiency
only via charge shift reaction. In this context, Falvey observed the
reductive CR of thymine oxetanes with photosensitisers via charge
5
separation, but only in diffusion controlled contact pairs. We
synthesised model compound 3, which contained carbazole as
reductive sensitiser covalently linked to the thymine oxetane
(Fig. 4). This model compound showed no photolyase activity.
Obviously, CR cannot compete against charge back transfer. This
is in agreement with our observation that in a diffusion controlled
reaction, photolyase activity occurs when the sensitiser and the
thymine oxetane 5 are not covalently linked.
In conclusion, the CR of oxetanes in covalently linked systems
requires a charge shift reaction, since a charge separation reaction
is impeded by a fast charge back transfer. This was shown with
covalently linked flavin/thymine oxetanes and carbazole/thymine
oxetanes as model systems for (6-4)-photolyase activity.
This work was gratefully supported by the VolkswagenStiftung,
by the Fonds der Chemischen Industrie and the Deutsche
Forschungsgemeinschaft (GRK 782).
Fig. 3 Quantum yields of the photoconversion of model compound 1
depending on pH.
The oxidative CR showed a clean conversion to compound 2
obeying first-order kinetics with minimum efficiency at pH 5.
Interestingly, the oxidative CR was accelerated by one order of
magnitude on changing to acidic medium and by a factor of 6 by
changing to basic conditions (Fig. 3 and Table 1). This is again
consistent with a change in the charge injection mechanism. The
oxidised flavin is hardly basic (pK y 0), but the excited state is
Thorsten Stafforst and Ulf Diederichsen*
Institut f u¨ r Organische und Biomolekulare Chemie,
Georg-August-Universit a¨ t G o¨ ttingen, Tammannstrasse 2, D-37077
G o¨ ttingen,Germany.E-mail:udieder@gwdg.de;Fax:+49(0)551392944;
Tel: +49 (0)551 393221
1
3
much more basic (pK* y 2). Therefore, the excited state of the
flavin could be protonated and react with the oxetane in a charge
shift reaction, which is in accordance to oxidative CR of oxetanes
8,10
observed by Miranda with positively charged pyrylium salts. By
changing from neutral to basic conditions, the deprotonation of
Notes and references
§ Use of sodium dithionite as reducing agent in combination with
irradiation at 365 nm should be avoided due to the strong absorbance at
1
5
3
"
50 nm (lg(e) . 3) which leads to substantial errors in the kinetic analysis.
The following 10 mM buffer solutions containing 0.10 M NaCl were
Table 1 Kinetic data for light-driven conversion of 1
a
a
used: citric acid (pH 2–5), sodium hydrogenphosphate (pH 7), borax (pH
), and glycine (pH 11); pH 1 was realised with 1.0% HClO in water/
methanol 1:1.
I A strictly light-driven side reaction was observed and a side product with
two mass units higher than starting material 1 was formed in an overall
yield of 25%.
pH
W
ox/%
W
red/%
t
ox/min
tred/min
9
4
16
1
2
3
5
7
9
1.32
0.33
0.15
0.13
0.18
0.36
0.85
—
—
—
3.7
7.3
6.2
5.4
27 ¡ 1.5
—
—
—
112 ¡ 6
248 ¡ 14
277 ¡ 15
207 ¡ 11
102 ¡ 6
43 ¡ 2
115 ¡ 6
58 ¡ 3
68 ¡ 4
79 ¡ 4
1 D. L. Mitchell, in CRC Handbook of Organic Photochemistry and
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chapter 140, 1–7; O. D. Sch a¨ rer, Angew. Chem., 2003, 115, 3052–3082;
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11
a
No measurement for pH , 5 due to instability of dithionite.
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432 | Chem. Commun., 2005, 3430–3432
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