Design, synthesis, and evaluation of a biomimetic artificial photolyase model

Article abstract of DOI:10.1021/jo0494329

Two new artificial photolyase models that recognize pyrimidine dimers in protic and aprotic organic solvents as well as in water through a combination of charge and hydrogen-bonding interactions and use a mimic of the flavine to achieve repair through red

Full text of DOI:10.1021/jo0494329

Design, Synthesis, and Evaluation of a Biomimetic Artificial
Photolyase Model
Olaf Wiest,*,† Christopher B. Harrison,† Nicolas J. Saettel,† Radek Cibulka,‡ Mirjam Sax,‡ and
Burkhard Ko¨nig*,‡
Department of Chemistry and Biochemistry, University of Notre Dame,
Notre Dame, Indiana 46556-5670, and Institut fu¨r Organische Chemie,
Universita¨t Regensburg, Universita¨tsstr. 31, D-93040 Regensburg, Germany
owiest@nd.edu; burkhard.koenig@chemie.uni-regensburg.de
Received April 6, 2004
Two new artificial photolyase models that recognize pyrimidine dimers in protic and aprotic organic
solvents as well as in water through a combination of charge and hydrogen-bonding interactions
and use a mimic of the flavine to achieve repair through reductive photoinduced electron transfer
are presented. Fluorescence and NMR titration studies show that it forms a 1:1 complex with
pyrimidine dimers with binding constants of ∼103 M-1 in acetonitrile or methanol, while binding
constants in water at pH 7.2 are slightly lower. Excitation of the complex with visible light leads
to clean and rapid cycloreversion of the pyrimidine dimer through photoinduced electron transfer
catalysis. The reaction in water is significantly faster than in organic solvents. The reaction slows
down at higher conversions due to product inhibition.
Introduction
where a flavine is covalently linked to a pyrimidine dimer
model system. Small peptides containing electron-donat-
ing groups were also found to induce a cycloreversion of
cyclobutane pyrimidine dimers.5 Several groups have
presented small-molecule recognition units that nonco-
valently bind to derivatives of thymine and uracil dimers
in organic solvents.6 By linking the 2,6-diacetaminopy-
ridine recognition unit to a redox active indole, Rose and
co-workers achieved repair of a uracil dimer through
reductive photoinduced electron transfer.7 Although Rose
observed binding constants of ∼200 M-1 in a methanol/
acetonitrile mixture, protic solvents were found to inter-
fere with the molecular recognition of the donor-acceptor-
donor hydrogen-bonding motif. A new strategy is thus
needed to achieve molecular recognition and repair in
water.
A major environmental damage to DNA is the forma-
tion of photolesions, which compromise the genetic
information and can lead to cell death or skin cancer. UV
radiation induces a [2 + 2] photocycloaddition between
two adjacent thymines on one strand of the DNA to
form a cis-syn thymine cyclobutane dimer as the major
product.1 DNA photolyase, which is present in many
organisms but not in humans, selectively recognizes the
thymine dimer in DNA single and double strands and
repairs it by photoinduced electron transfer using a non-
covalently bound, reduced flavine as the electron donor.2
The study of artificial DNA photolyases and enzyme
models yielded valuable insights into the mode of action
of the enzyme3 and offers the long-term prospect of
artificial DNA repair, which gained additional support
by the recent demonstration of xenobiotic repair of
thymine dimers in humans.4 Carell and co-workers
presented detailed studies of a series of model systems
Here, we report a functional photolyase mimic that
recognizes pyrimidines in both organic solvents and
water and, in analogy to the natural enzyme, uses a
flavine to achieve repair through reductive photoinduced
electron transfer. Figure 1 shows the function of the
† University of Notre Dame.
‡ Universita¨t Regensburg.
(1) (a) Begley, T. P. Comput. Nat. Prod. Chem. 1999, 5, 371. (b)
Heelis, P. F.; Hartman, R. F.; Rose, S. D. Chem. Soc. Rev. 1995, 24,
289-297. (c) Cadet, J.; Vigny, P. In Bioorganic Photochemistry Vol. I:
Photochemistry and Nucleic Acids; Morrison, H., Ed.; Wiley & Sons:
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Int. Ed. 1998, 37, 938. (d) Epple, R.; Carell, T. J. Am. Chem. Soc. 1999,
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A.; Burgdorf, L. T.; Carell, T. Angew. Chem., Int. Ed. 2000, 39, 3918.
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10.1021/jo0494329 CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/08/2004
J. Org. Chem. 2004, 69, 8183-8185
8183

Wiest et al.
SCHEME 1. Synthesis of Artificial Photolyases 2a
a Reagents and conditions: (a) BrCH2CO2H, DCC, CH2Cl2, 12
h, rt, 70%; (b) K2CO3, CH3CN, rt, 7 d, 75%; (c) TFA/CH2Cl2 1:1,
rt, 3 h, 94%; (d) Zn(ClO4)2‚6H2O, CH3OH, reflux, 1 h, 92%.
FIGURE 1. Function of artificial photolyases 2.
reductive artificial photolyases 2. Binding of 3a, a model
for thymine dimers,3,8 occurs via a zinc-cyclen moiety
covalently linked to a reduced flavine derivative. It was
shown that pyrimidines can be selectively recognized in
water as well as in the context of a DNA duplex by zinc-
cyclen complexes through a combination of hydrogen
bonding and complexation of the zinc dication to the
deprotonated N(3) nitrogen.9 Carell and co-workers3
studied a series of systems where flavine derivatives were
covalently linked to thymine dimer models and demon-
strated that electron transfer catalyzed (ETC) cyclo-
reversion can be achieved under physiologically relevant
conditions, including in a DNA duplex. Excitation of the
reduced flavine with visible light then induces electron
transfer (ET) to form 3a•-, which undergoes effectively
barrierless cycloreversion.10 Back-electron transfer (BET)
to the flavine closes the catalytic cycle and yields 2 equiv
of the monomer 1a.
FIGURE 2. HPLC traces for ETC cycloreversion catalyzed
by 2a.
similar systems. Due to the lower solubility of 3a in
methanol, the binding constant for 2a‚3a is determined
in acetonitrile as 2 × 103 M-1 by fluorescence titration.
The higher binding constant to thymine can be under-
stood when considering the binding mode of 2, which
binds to the deprotonated N(3) position.9 Since thymine
is approximately 0.6 pKa units more acidic than 3a, it is
reasonable that the binding to 2a will be stronger. It is
in any case gratifying to note that binding in both protic
and nonprotic organic solvents is strong, indicating
possible applications to aqueous solutions.
After formation of the reduced and deprotonated flavin
using the conditions developed by Carell,3 the electron-
transfer-catalyzed (ETC) cycloreversion of 3a is induced
in a 1:4 mixture of 2a and 3a in acetonitrile or methanol
by irradiation with a 450 W mercury lamp equipped with
a Pyrex filter. HPLC traces (Figure 2) reveal a fast and
clean conversion of 3a to 1a. Control experiments show
that no reaction takes place upon irradiation in the
absence of 2a.
Results and Discussion
Compound 2 is easily accessible by coupling of tri-Boc-
cyclen 49 to the flavine derivative 5,11 synthesized by the
classic Kuhn method, via a carboxymethyl linker as
shown in Scheme 1. By selecting the appropriate side
chain on the flavine, solubility in either organic solvents
or water can be ensured. Deprotection and complexation
of the zinc ion complete the synthesis. The stoichiometry
and binding constant of the complexes of 2 to thymine
and 3a are determined by fluorescence quenching titra-
tion of the oxidized flavine.12 A Job’s plot analysis shows
a 1:1 stoichiometry of the complex 2a‚thymine and 2a‚
3a. A binding constant of 6 × 103 M-1 for 2a‚thymine in
methanol is derived from fluorescence titration. The
value is in agreement with the results of Kimura9 on
(8) Butenandt, J.; Burgdorf, L. T.; Carell, T. Synthesis 1999, 1085.
(9) (a) Shionoya, M.; Kimura, E.; Shiro, M. J. Am. Chem. Soc. 1993,
115, 6730. (b) Shionoya, M.; Ikeda, T.; Kimura, E.; Shiro, M. J. Am.
Chem. Soc. 1994, 116, 3848. (c) Aoki, S.; Kimura, E. J. Am. Chem.
Soc. 2000, 122, 4542. (d) Aoki, S.; Sugimura, C.; Kimura, E. J. Am.
Chem. Soc. 1998, 120, 10094. (e) Kikuta, E.; Murata, M.; Katsube, N.;
Koike, T.; Kimura, E. J. Am. Chem. Soc. 1999, 121, 5426. (f) Kimura,
E.; Kikuchi, M.; Kitamura, H.; Koike, T. Chem. Eur. J. 1999, 5, 3113.
(g) Kimura, E.; Kitamura, H.; Ohtani, K.; Koike, T. J. Am. Chem. Soc.
2000, 122, 4668. (h) Kimura, E.; Aoki, S.; Koike, T.; Shiro, M. J. Am.
Chem. Soc. 1997, 119, 3068.
The conversions for the ETC cycloreversion of 3a
catalyzed by 2a as well as for the control reaction using
5a in acetonitrile and methanol are summarized in Table
1. It can be noted that the reaction is, with a conversion
of more than 25%, catalytic. The reaction in methanol is
faster, most likely due to the stabilization of the inter-
(12) Since the emission of the reduced flavine cannot be observed,
binding studies were performed with the oxidized form of the flavine,
assuming that the binding constant is independent of the redox state
of the flavine.
(10) Saettel, N. J.; Wiest, O. J. Am. Chem. Soc. 2001, 123, 2693.
(11) Ko¨nig, B.; Pelka, M.; Reichenbach-Klinke, R.; Schelter, J.; Daub,
J. Eur. J. Org. Chem. 2001, 2297.
8184 J. Org. Chem., Vol. 69, No. 24, 2004

Biomimetic Artificial Photolyase Model
TABLE 1. Cycloreversion of 3a to 1a Catalyzed by 2a or
5a
leads to 78% conversion after 10 s, compared to 20% for
the bimolecular background reaction catalyzed by 5b. The
very high rate compared to the reaction in methanol
cannot be rationalized only by the higher polarity and
hydrogen bonding ability of water, but might also be
affected by a hydrophobic effect that biases the confor-
mational equilibria toward a stacking of the flavine
toward 3b, which will facilitate the electron transfer. At
this conversion, the reaction is already subject to product
inhibition in analogy to the reaction of 2a discussed
above. This was confirmed by addition of one equivalent
of the product 1b, which significantly slows down the
reaction as shown in Figure 3. This is most likely due to
the slower diffusion of the complex 2b‚1b, which makes
it less efficient in the bimolecular repair reaction. Nev-
ertheless, the 4-fold acceleration at 10 s indicates that
the initial rate for repair in the complex is much higher
than for the bimolecular background reaction.
conversion (%)
solvent
time (min)
2a
5a
CH3CNa
15
30
45
5
10
15
55
67
75
74
80
84
38
60
75
59
69
75
CH3OHb
a [3a] ) 4 × 10-4 M-1, [2a] ) 1 × 10-4 M-1
.
b [3a] ) 1.8 × 10-4
M-1, [2a] ) 4.5 × 10-5 M-1
.
The product inhibition observed for these simple model
systems prevents the determination of the quantum yield
in our current experimental setup. However, the very
short reaction times in water qualitatively indicate that
the reaction is as efficient as the reaction of the covalently
linked model systems,3 but shows catalytic turnover.
Product inhibition will not be a problem in real DNA
since, similar to the case of DNA photolyase,13 the product
is removed from the equilibrium by reconstitution of the
Watson-Crick pairing. Since the application of our
artificial photolyase to a DNA duplex is a long-term goal
of the project, the problem of product inhibition was not
further pursued at this point.
FIGURE 3. ETC repair of 3b.
In summary, we present for the first time a small-
molecule photolyase model that binds to the model
dimers 3 with millimolar affinity in both protic and non-
protic solvents by reversible coordination. Upon irradia-
tion with visible light, 2 cleanly and efficiently cyclo-
reverts the cyclobutane pyrimidine dimers 3 to the mon-
omers 1. The reaction proceeds, analogous to the natural
DNA photolyase, through photoinduced reductive elec-
tron transfer using a reduced flavine as the redox active
cofactor. Based on the work by Carell3 and Kimura,9 it
can be anticipated that the water soluble derivative 2b
could be used to repair thymine dimers in a DNA duplex
under physiological conditions. These studies are cur-
rently underway and will be reported in due course.
mediate radical anion by hydrogen bonding in the protic
solvent, which was found previously to be important for
this reaction.10 The reaction can be run essentially to
completion by irradiation for 120 min in acetonitrile and
60 min in methanol (data not shown). It is interesting to
note that the repair of 3a, complexed to 2a, starts out
faster than the bimolecular reaction with (flavine) 5a but
then slows down at longer reaction times. This is
consistent with a product inhibition caused by the higher
binding constant of 2a to 1a formed over the course of
the reaction. Thus, the reaction is after short reaction
times (at larger than ∼15% conversion) effectively a
bimolecular reaction between 2a‚1a and 3a. The overall
acceleration observed at our first datapoint is therefore
only 2-fold, even though the rate constant in 2a‚3a at
very low conversions is expected to be significantly higher
than the bimolecular rate constant of 3a and 5a.
As the next step toward the biologically relevant
system, we investigated the repair of the thymine dimer
model 3b in aqueous solution using the water-soluble
artificial photolyase 2b. The binding of pyrimidines by
zinc-cyclen complexes in aqueous solution has been
studied extensively by Kimura et al., who found apparent
binding constants on the order of ∼103 M-1.9 Our own
results, obtained by NMR titrations in aqueous borate
buffer at pH 7.2, are consistent with these results and
the lower acidity of the pyrimidine dimers (see the
Supporting Information).
Acknowledgment. We gratefully acknowledge fi-
nancial support of this work by the NIH (Grant No.
CA073775 to O.W.), the Volkswagen Foundation for
support of B.K., and the Dreyfus Foundation Camille
Dreyfus Teacher-Scholar Award to O.W. as well as
collaboration with the Walther Cancer Research Center
at the University of Notre Dame.
Supporting Information Available: Experimental pro-
cedures for the synthesis and spectral characterization of new
compounds, details of the determination of the binding con-
stants, and a procedure for the repair assay used. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Figure 3 summarizes the results for the repair of 3b
in aqueous solution. It is noteworthy that the reaction
in water is much faster than in organic solvents. Even
at the shortest time that is practical with our current
experimental setup, irradiation of the complex of 2b‚3b
JO0494329
(13) (a) Christine, K. S.; MacFarlane IV, A. W.; Yang, K.; Stanley,
R. J. J. Biol. Chem. 2002, 277, 38229. (b) Vande Berg, B. J.; Sancar,
G. B. J. Biol. Chem. 1998, 273, 20276. (c) Butenandt, J., Burgdorf, L.
T.; Carell, T. Angew. Chem., Int. Ed. 1999, 38, 708.
J. Org. Chem, Vol. 69, No. 24, 2004 8185