Efficient photosensitized splitting of thymine dimer by a covalently linked tryptophan in solvents of high polarity

Article abstract of DOI:10.1002/ejoc.200400631

Tryptophan-thymine dimer model compounds used to mimic the repair reaction of DNA photolyase have been synthesized. The photosensitized cleavage of the dimer by the covalently linked tryptophan is strongly solvent-dependent with the reaction rates increasing in increasingly polar solvents, for example, the quantum yield π = 0.004 in THF/hexane (5:95) and 0.093 in water. The fluorescence of the tryptophan residue is quenched by the dimer moiety by electron transfer from the excited tryptophan to the dimer. Fluorescence- quenching studies indicated that the electron transfer was efficient in polar solvents. The splitting efficiency of the dimer radical anion within the tryptophan^.+-dimer^.- species is also remarkably solvent-dependent and increases with the polarity of the solvents. The back-electron-transfer reaction in the charge-separated species, which competes with cleavage, was suppressed in polar solvents. These results are in contrast to those of earlier solvent-dependent studies of indole-dimer systems, but they can be rationalized in terms of the differences in the distances between the chromophore unit and the attached dimer. The pH-dependent measurements of the splitting reaction and the deuterium isotope effect showed that the tryptophan radical cation within the charge-separated species does not deprotonate prior to the cleavage of the dimer radical anion. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005.

Full text of DOI:10.1002/ejoc.200400631

FULL PAPER
Efficient Photosensitized Splitting of Thymine Dimer by a Covalently Linked
Tryptophan in Solvents of High Polarity
[
a]
[a]
[a]
[a]
Qin-Hua Song,* Wen-Jian Tang, Xiao-Ming Hei, Hong-Bo Wang,
[a]
[b]
Qing-Xiang Guo, and Shu-Qin Yu
Keywords: DNA photorepair / Electron transfer / Pyrimidine dimer / Tryptophan / Solvent effects
Tryptophan-thymine dimer model compounds used to mimic
the repair reaction of DNA photolyase have been synthe-
sized. The photosensitized cleavage of the dimer by the co-
valently linked tryptophan is strongly solvent-dependent
with the reaction rates increasing in increasingly polar sol-
polarity of the solvents. The back-electron-transfer reaction
in the charge-separated species, which competes with cleav-
age, was suppressed in polar solvents. These results are in
contrast to those of earlier solvent-dependent studies of in-
dole-dimer systems, but they can be rationalized in terms of
vents, for example, the quantum yield Φ = 0.004 in THF/hex- the differences in the distances between the chromophore
ane (5:95) and 0.093 in water. The fluorescence of the trypto-
phan residue is quenched by the dimer moiety by electron
transfer from the excited tryptophan to the dimer. Fluores-
cence-quenching studies indicated that the electron transfer
was efficient in polar solvents. The splitting efficiency of the
unit and the attached dimer. The pH-dependent measure-
ments of the splitting reaction and the deuterium isotope ef-
fect showed that the tryptophan radical cation within the
charge-separated species does not deprotonate prior to the
cleavage of the dimer radical anion.
·+
·–
dimer radical anion within the tryptophan –dimer species
is also remarkably solvent-dependent and increases with the
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
Introduction
stores the dipyrimidine and the functional form of the fla-
vin ready for a new catalytic cycle.
DNA repair has received heightened attention in recent
years as ozone depletion threatens to significantly increase
DNA damage by UVB radiation (280–320 nm).[ One of
the major effects on DNA of this type of radiation is the
formation of cyclobutane pyrimidine dimers (CPD). DNA
photolyases are flavin-containing repair enzymes that cata-
lyze the efficient repair of cis-syn cyclobutane pyrimidine
dimers by utilizing the energy of visible light to break the
[4–6]
Several model compounds
that mimic the action of
photolyase have been designed, for example, a chromophore
attached to a pyrimidine dimer. Studies performed with
these compounds have helped to unravel the mechanisms
described in detail above. However, many questions con-
1]
cerning the physical-chemical mechanisms involved in the
[3,7]
whole process remain unanswered.
In particular, it is un-
clear what factors cause the enzyme to mediate with a very
cyclobutane ring of the dimer. The mechanisms of CPD
[3]
high efficiency (Φ = 0.7–0.98 ) in DNA photoreactivation
photolyases have been thoroughly investigated.[
2,3]
The en-
including the energy and electron transfer. In addition, un-
der excitation with 280 nm light, Trp277, as a substrate
binding site, can also directly repair CPD with a relatively
zymes contain a redox-active cofactor, flavin adenine dinu-
cleotide (FAD), which operates in the fully reduced and de-
protonated form (FADH ), and an auxiliary antenna chro-
–
[8,9]
high quantum yield (Φ = 0.56) by electron transfer.
mophore. A second cofactor activates the process of repair
by absorbing near UV/Vis radiation (300–500 nm) and
transferring the energy to the flavin. Subsequently, the ex-
Model systems appeared to be poorly efficient at re-
pairing the lesion by intramolecular electron transfer, for
[10]
example, Φ = 0.06–0.40 for indole-dimer systems
0
and
–
cited FADH transfers one electron to the cyclobutane pyr-
[11]
.062–0.016 for flavin-dimer systems in various polar sol-
imidine dimer to form the dimer radical anion, which
cleaves spontaneously, and then back-electron-transfer re-
vents such as water and 1,4-dioxane. This can be explained
by the fact that model systems cannot stabilize the charge-
separated species in which the electron is on the acceptor
[
a] Department of Chemistry, University of Science and Technol- dimer before cleavage. Hence, competition between an un-
ogy of China,
productive back-electron-transfer reaction and the cleavage
of the dimer radical anion is an important factor that leads
to the low efficiency. In fact, rapid splitting of the CPD
plays an important role in the highly efficient repair medi-
ated by photolyase since it competes efficiently with the
Hefei 230026, P. R. China
Fax: +86-551-3601592
E-mail: qhsong@ustc.edu.cn
b] Department of Chemical physics, University of Science and
Technology of China,
[
Hefei 230026, P. R. China
Eur. J. Org. Chem. 2005, 1097–1106
DOI: 10.1002/ejoc.200400631
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1097

Q.-H. Song, W.-J. Tang, X.-M. Hei, H.-B. Wang, Q.-X. Guo, S.-Q. Yu
FULL PAPER
back-electron-transfer reaction. The splitting and back elec- the photosensitized splitting of the thymine dimer by the
tron-transfer from a thymine radical anion to the flavin rad- attached tryptophan in various solvents (Scheme 1). The
[
12]
ical are expected to be complete within 0.5–2 ns,
whilst quantum yield for the splitting of the dimer is very sensitive
[
13]
[14]
it takes 200
and 556 ns
for only the splitting process to the polarity of the solvents. Remarkably high splitting
in solution. It is important to understand what factors can efficiencies were observed in solvents of high polarity. Inter-
accelerate forward electron transfer from an excited chro- estingly, this solvent effect contrasts strongly with the re-
[
10,15,16]
mophore to the linked dimer and suppress the back elec- sults
tron-transfer because these processes are undoubtedly tems, but is in agreement with the results[
counterparts in the enzymatic repair process. flavin-dimer systems. Furthermore, these results together
In the chromophore-dimer systems, the photosensitized with data from fluorescence quenching experiments and
obtained previously with non-flavin-dimer sys-
11,18]
obtained with
[10]
cleavage of the dimer by the attached indole,
aryl- studies of the effect of pH on splitting of the dimer, some
was strongly solvent-de- new insights into the intramolecular electron-transfer pro-
[
15]
[16]
amine
or methoxybenzene
pendent with efficient splitting taking place in solvents of cess in the tryptophan-dimer system were gained.
low polarity. This unusual solvent effect can be explained
by the fact that fast and unproductive charge recombina-
Results
tion by back electron-transfer within the charge-separated
species is suppressed in solvents of low polarity. The back
electron-transfer in low-polar media would become so exo-
thermic as to lie in the Marcus inverted region, which would
Synthesis of the Model Compounds 1 and 2
The preparation of the cis-syn thymine dimer (Scheme 2)
is a prerequisite for the synthesis of the model compounds.
[
10,15,16]
significantly reduce the rate of the transfer reaction.
The results seem to support previous findings that the pho- The ethyl ester 6 of 1-(2-carboxyethyl)thymine was prepared
[
19]
tolyase may provide a hydrophobic environment for the from thymine. In the presence of acetone, an acetonitrile
[
8,17]
substrate (dimer).
solution of 6 was placed in a Pyrex photoreactor (λ Ͼ
However, an inverse solvent dependence was observed in 290 nm); pure N was bubbled through the solution which
2
covalently linked flavin-dimer systems by Carell and co- was irradiated with a 300-W high-pressure Hg lamp to give
[
11,18]
workers.
By taking two sets of measurements in water/ a mixture of all four possible isomers. The cis-syn thymine
ethylene glycol mixtures and various organic solvents, they dimer 7 was separated by chromatography on silica gel-H.
observed increased splitting efficiencies in polar solvents. In X-ray analysis of the cis-syn isomer allowed the correct
contrast to the non-flavin-containing systems, which yield structural assignment (Figure 1). Subsequent hydro-
a zwitterionic intermediate after photoinduced electron genolytic cleavage of the ethyl ester gave the cis-syn thymine
transfer, the reduced and deprotonated flavin systems result dimer dicarboxylic acid 11 as a white powder in excellent
in a non-zwitterionic intermediate as a result of a shift of yield. The tryptophan methyl ester 5 was prepared from
the negative charge from the flavin moiety to the dimer. tryptophan and methanol in 91% yield.
Since a much smaller driving force can be expected for the
The model compounds 1 and 2 were prepared by con-
unproductive back charge transfer, it shouldn’t lie in the densation of tryptophan methyl ester (5) and the thymine
Marcus inverted region. The results suggested that an un- dimer dicarboxylic acid 11 with (benzotriazol-1-yl)oxytris(di-
usually polar flavin-containing pocket, observed in the X- methylamino)phosphonium hexafluorophosphate (BOP)[
20]
ray crystal structures of the E. coli photolyase, may be re- activation of the carboxylic acids in DMF (Scheme 3).
quired to increase the catalytic repair efficiency.[
11]
After addition of one equivalent of the tryptophan methyl
In this work, we have prepared covalently linked trypto- ester, the mixture was stirred for 2 h. A large excess of n-
phan-dimer model compounds 1 and 2, and investigated pentylamine was then added and allowed to react for 5 h
Scheme 1.
1098
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2005, 1097–1106

Photosensitized Splitting of Thymine Dimer by a Covalently Linked Tryptophan
FULL PAPER
Scheme 2. Reagents and conditions: (a) ethyl acrylate, hydroquinone, NaOH, EtOH, reflux, 20 h; (b) hv (Ͼ 290 nm), acetone/MeCN,
room temp., 20 h; (c) 5 m HCl, reflux, 1 h.
Scheme 3.
photoproducts 3 and 4 were isolated and identified by
Figure 1. X-ray crystal structure of the cis-syn thymine photodimer NMR spectroscopy. A representative set of HPLC chroma-
7
shown with 30 % ellipsoids. Crystal data for 7: colorless needles,
tograms showing the simultaneous splitting of compound 1
into 3 and 4 is depicted in Figure 2. Clearly, the reaction of
compound 1, with a retention time of 5.3 min, to the pho-
tosplit products 3 and 4, with retention times of 3.3 and
C
20 28 4 8 c
H N O , M = 452.46, monoclinic, space group P21/n, D =
–1
1
.342 gcm , Z = 4, a = 8.427(3), b = 12.346(5), c = 21.542(8) Å,
3
β = 92.104(7)°, V = 2239.7(15) Å , T = 293(2) K, µ(MoK
0
0
α
) =
.105 mm , 11202 reflections collected, 3935 unique (Rint
.0543), R = 0.0563, wR = 0.1241 [I Ͼσ2(I)]. CCDC-253225 con-
–1
=
1
2
3.5 min, respectively, is a clean conversion as no other prod-
tains the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
ucts could be detected.
to yield compound 1. More than two equivalents of the
tryptophan methyl ester were added and allowed to react
for 5 h to afford compound 2. Both the model compounds
1
and 2 were extracted with chloroform and then purified
by chromatography on silica gel-H to give white powers in
1 and 25% yields, respectively.
2
Photosplitting Properties of the Model Compounds
The model compound 1 or 2 in aqueous solution was
irradiated with a 290 nm light beam from a fluorescence
spectrometer containing a 125-W Xe lamp. Analysis of the Figure 2. Typical HPLC chromatograms obtained after 0, 3, 6, 9,
–
5
1
2
2 and 15 min of irradiation of 1 in water (3 mL, 5×10 m) with
photolyzed mixture by reversed-phase HPLC and co-injec-
tion of the synthesized expected photoproducts 3 and 4
confirmed that the model compounds react cleanly to give
90 nm light. Assay conditions: C18 reversed-phase column, 70%
methanol and 30% water as eluent, detection at 270 nm. Retention
times: 3.3 min (product 3), 3.5 min (product 4), 5.3 min (model
only 3 from 2 and only 3 and 4 from 1. Furthermore, the compound 1).
Eur. J. Org. Chem. 2005, 1097–1106
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Q.-H. Song, W.-J. Tang, X.-M. Hei, H.-B. Wang, Q.-X. Guo, S.-Q. Yu
FULL PAPER
The splitting reaction occurs by intramolecular electron duction in solvent polarity results in a significant decrease
transfer since the quantum yield is not concentration-de- in the quantum yield for the splitting reaction of compound
pendent at 0.01, 0.05 and 0.1 mM within an experimental 1, ranging from 0.092 in water/THF (80:20) to 0.050 in
error of ±5%. This ruled out the possibility of intermo- water/THF (5:95). This further demonstrates that efficient
lecular photosensitization between one molecule of the splitting occurs in solvents of high polarity.
model compound in the excited state and another molecule
compound in the ground state, that is, the excited trypto- Table 2. Splitting efficiencies for compounds 1 and 2 in water/THF
phan unit of one model molecule being responsible for the binary solvent.
cleavage of the dimer unit of another model molecule.
Solvent mixture
THF:H O, v/v)
Compound 1
Compound 2
spl
(
2
Φ
Q
φ
Φ
1
9
9
8
6
00:0
5:5
0:10
0:20
0:40
0.016
0.050
0.059
0.069
0.077
0.091
0.091
0.092
0.093
0.36
0.54
0.65
0.70
0.73
0.75
0.78
0.80
0.81
0.045
0.092
0.091
0.098
0.105
0.121
0.117
0.115
0.115
0.021
0.054
0.057
0.062
0.070
0.076
0.078
0.078
0.079
Quantum Yields of Splitting of 1 and 2
To measure the quantum yields for the splitting of the
model compounds Φ [Φ = (rate of dimer splitting)/(rate of
photons absorbed)], all sample solutions (Ϸ 5×10–5 m, 50:50
4
2
0
0:60
0:80
:100
3
mL) were prepared in the corresponding solvents and
placed in quartz cuvettes (10×10 mm) with a Teflon stop-
per and then irradiated with 295 nm light from a fluores-
cence spectrometer. By measuring the increase in the ab-
sorbance at 273 nm (∆A273) after certain intervals of time,
The quantum yields of compound 2 are lower than those
a rate for the dimer splitting was obtained from the straight of compound 1 in polar solvents, and similar in low-polar
line resulting from a plot of ∆A273 against irradiation time. solvents. Hydrophobic indoles can often form π-stacked ag-
The intensity of the excitation light beam was measured by gregates particularly in polar solvents. Hence, the lower
ferrioxalate actinometry.[ From the intensity of the light quantum yields for compound 2 may result from intramol-
beam and the absorbance of the sample at 295 nm before ecular tryptophan–tryptophan stacking which inhibits the
irradiation, the rate of photon absorption was calculated. electron-transfer reaction in polar solvents. Small differ-
Based on these results, the quantum yields for the dimer ences in the UV absorption spectra of compounds 1 and 2
splitting of 1 and 2 were calculated and the values are listed were observed in water, but not in THF, which supports
21]
in Table 1.
this explanation.
The data show that the quantum yields for the splitting
of 1 and 2 are strongly solvent-dependent. The values of Φ
decrease with solvents in the order water Ͼ alcohols Ͼ di-
Fluorescence Emission of 1 and 2
polar aprotics Ͼ nonpolar, ranging from 0.093 in H O to
2
0
0
.004 in THF/hexane (5:95) for 1, and 0.079 in H O to
.003 in THF/hexane (5:95) for 2. The order of the solvents (symbolized as TrpH–D) as well as the fluorescence emis-
Figure 3A shows the fluorescence emissions of 1 and 2
2
reflects their ability to solvate and stabilize charge. Hence, sion of the free tryptophan methyl ester 5 (TrpH, H denotes
the efficiency of dimer splitting is enhanced in solvents of the N1 proton) in water at room temperature. The corre-
high polarity.
sponding results for 1, 2 and 5 in THF are shown in Fig-
Furthermore, the solvent dependence of Φ was measured ure 3B. It can be seen that the fluorescence emission of the
in solutions of water/THF in various ratios (Table 2). A tryptophan unit in 1 and 2 are quenched by the covalently
gradual decrease in the quantum yield was observed upon linked thymine dimer and that the extent of fluorescence
addition of increasing amounts of THF and the lowest quenching [Equation (1), where F is the fluorescence inten-
value of Φ was obtained in pure THF. The systematic re- sity] is highly sensitive to the polarity of the solvent.
Table 1. Dependence of splitting efficiency on solvent for compounds 1 and 2.
Solvents[a]
Compound 1
Compound 2
Φ[b]
Q
φ
spl
Φ[b]
Q
φ
spl
Water
0.093
0.041
0.072
0.070
0.066
0.063
0.016
0.014
0.013
0.004
0.81
0.48
0.65
0.61
0.53
0.48
0.36
0.49
0.41
0.27
0.115
0.085
0.111
0.115
0.125
0.131
0.044
0.029
0.032
0.015
0.079
0.036
0.057
0.062
0.055
0.041
0.021
0.012
0.016
0.003
0.76
0.46
0.50
0.52
0.46
0.45
0.32
0.39
0.50
0.34
0.104
0.078
0.114
0.119
0.120
0.091
0.066
0.031
0.032
0.009
Acetonitrile
Methanol
Ethanol
Propanol
2-Propanol
THF
Ethyl acetate
1,4-Dioxane
THF/hexane (5:95)
[a] Arranged in order of decreasing dielectric constant. [b] Average of two determinations; 10 nm bandwidth.
1100 © 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org Eur. J. Org. Chem. 2005, 1097–1106

Photosensitized Splitting of Thymine Dimer by a Covalently Linked Tryptophan
FULL PAPER
Effect of pH on the Splitting Reaction
The electron-transfer reaction from the excited trypto-
phan residue to the attached dimer gives a zwitterionic in-
·
+
·–
termediate TrpH –D , which has a high driving force for
charge recombination and therefore is a very short-lived in-
termediate. Since the pK for the deprotonation at the N1
a
·+
site of the indole in the tryptophan radical cation (TrpH )
is about 4, it will deprotonate in neutral aqueous solution
to give a neutral radical (Trp ). If the deprotonation of the
tryptophan radical cation within TrpH –D occurs prior
to the cleavage of the dimer radical anion, a non-zwitter-
ionic intermediate Trp –D would form [Equation (2)] that
should have a longer lifetime, consequently yielding a more
efficient splitting.
[
22]
·
·
+
·–
·
·–
pKa
TrpH –D w Trp –D + H+
·
+
·–
·
·–
(2)
·+
·–
To examine whether the deprotonation of TrpH –D
does occur prior to the cleavage, we investigated the effect
of pH on the splitting reaction. The quantum yields for the
photosplitting of 1 and 2 on irradiation with 295 nm light
in solvent mixtures of water/THF (80:20) buffered over the
pH range of 1 to 13 is shown in Figure 4. The pH-depend-
Figure 3. Relative fluorescence emission intensities of tryptophan
methyl ester 5 and the model compounds 1 and 2 in water (A) and ent measurements show small changes from pH 3 to 10. No
in THF (B) upon excitation at 290 nm.
notable change in Φ was observed between pH 3 and 5. At
pH Ͻ 3, both the dimer and the tryptophan are possibly
protonated, which results in a slight decrease in the values
of Φ. From the UV spectra of the model compounds in
solutions at various pH, a new absorption peak at about
Q = 1 – (FTrpH–D/FTrpH
)
(1)
240 nm appears at pH 10–11 (inset of Figure 4), which may
be indicative of deprotonation of the dimer since the pKa
of the deprotonation of the cyclobutane uracil dimer is
The values of Q increase with increasing polarity of the
solvents (Table 1). The fluorescence quenching of the tryp-
tophan unit in 1 or 2 is not a result of significant absorption
of exciting light by the dimer in 1 or 2. As the molar extinc-
tion coefficient of the thymine dimer 7 is much less than
that of the tryptophan methyl ester 5 at 290 nm, for exam-
[
23]
10.7.
Thus, electron transfer to a dimer anion would be
inhibited when pH Ͼ pKa.
–
1
–1
3
–1
–1
ple, in water ε290 = 40 m cm for 7 and 5.4×10 m cm
for 5, an internal filter effect should not be significant.
Fluorescence quenching is probably a consequence of in-
tramolecular electron transfer from the excited tryptophan
to the linked dimer. Since there is almost no overlap be-
tween the emission spectra of the tryptophan methyl ester 5
and the thymine dimer 7 (not shown), singlet–singlet energy
transfer is an improbable mode of fluorescence quenching
in the model compounds. Hence, electron transfer should
be the predominant mode of fluorescence quenching in 1
and 2. The data in Table 1 show that electron transfer from
excited tryptophan to the linked dimer becomes more ef-
ficient in more polar solvents. This implies that the forma-
Figure 4. Investigation of effect of pH on the splitting efficiency of
1
and 2 in water/THF (80:20) solutions with 0.1 m buffer. Inset: UV
–5
tion of the charge-separated species would reasonably be absorption spectra of 3×10 m1 in water/THF (80:20) solutions at
expected to be accelerated in strongly polar media, which pH1–13.
would allow decay processes caused by electron transfer to
predominate, and fluorescence and nonradiative processes
to be suppressed.
Because breaking a N–D bond requires more energy
than a N–H bond, the deprotonation (Ka = [H ][Trp ]/
+
·
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Q.-H. Song, W.-J. Tang, X.-M. Hei, H.-B. Wang, Q.-X. Guo, S.-Q. Yu
FULL PAPER
·
+
[
TrpH ]) of the tryptophanal radical cation in D O would quantum yields for dimer splitting and in the fluorescence
2
be inhibited, that is, the equilibrium in D O would be quenching (Q) with solvent polarity allow the effect of sol-
2
shifted towards the protonated state to give the neutral vent on the two pairs of competitions to be evaluated.
tryptophan radical Trp· in a lower concentration than in
H O.
2
The efficiency of electron transfer can be discussed in
[
16]
terms of the following reasonable assumption.
The rate
If the deprotonation occurs prior to the cleavage of dimer constants for fluorescence and the nonradiative relaxation
·+
·–
·
·–
radical anion in TrpH –D , the concentration of Trp –D
pathways are unaltered by attachment of the dimer to the
in D O will be lower than that in H O. Thus, the value of tryptophan [i.e., kЈ (5) = k (1 or 2) and kЈ (5) = k (1 or
2
2
f
f
nr
nr
Φ would be lower in D O than in H O, that is, a deuterium 2)] and electron transfer (k ) can occur in the excited model
2
2
et
isotope effect on the splitting of the dimer would be ob- species. On this basis and from Equation (1), Equation (3),
served. Hence, an experiment to measure the deuterium iso- Equation (4)and Equation (4), it was deduced that the effi-
tope effect was performed to examine whether the N1– ciency of electron transfer is equal to the degree of fluores-
H(D) bond of indole breaks prior to cleavage. Under the cence quenching, that is, φet = Q.
same irradiation conditions, however, there were no differ-
ences in the splitting rates of compound 1 in H O and D O
2
2
F
TrpH–D ϰ k
f
/(k
f
+ knr + ket
)
(3)
(4)
(5)
within experimental error, that is, the bond breaking of the
N1–H(D) did not occur before cleavage of the dimer.
Therefore, the above results show that deprotonation of
f f
FTrpH ϰ kЈ /(kЈ + kЈnr)
·+
·–
the tryptophan radical cation within TrpH –D is slower
than both the splitting of the dimer radical anion and the
f
φet=ket/(k + knr + ket)
·+
·–
charge recombination of TrpH –D .
Thus, values of φspl were calculated from φspl = Φ/Q and
Discussion
are listed in Table 1 and Table 2. It is evident that the split-
ting efficiencies of the dimer radical anion are remarkably
dependent on the solvent polarity. On the basis of the val-
ues of Q and φspl in Table 1 and Table 2, it might reasonably
be expected that electron transfer ket and splitting kspl are
accelerated in solvents of high polarity, or that fluorescence
and back electron-transfer are suppressed.
The photophysical and photochemical processes of the
model compounds are illuminated by the simple mechanism
shown in Scheme 4. Upon irradiation with light of λ Ͼ
290 nm, a tryptophan unit in the model compound absorbs
a photon to produce an excited state of the tryptophan. The
excited state has the following relaxation pathways: fluores-
This solvent dependence contrasts with the results of ear-
cence (k ), internal conversion and intersystem crossing (to-
f
[10,15,16]
lier work
in which the solvent dependence of the
gether represented by k ), and electron transfer to the re-
lated dimer (k ). The sum of the efficiencies of these pro-
cesses is 1, that is, φ + φ + φet = 1. The charge-separated
nr
splitting reaction was investigated in detail by Rose and co-
workers. In their work, the splitting efficiency of the model
compounds was found to be rather strongly solvent-de-
pendent and increased with decreasing solvent polarity, for
et
f
·–
nr
·
+
species (TrpH –D ) formed by electron transfer can un-
dergo two processes, splitting (φspl) or back electron-trans-
fer (φbet), which results in an unproductive reversal, and
their efficiencies add up to 1. There are two pairs of compe-
titions in these processes: 1) the photophysical process (kf
and k ) and electron transfer (k ) and 2) the splitting (kspl)
[10]
[15]
[16]
[16]
example, in 1,4-dioxane Φ = 0.2,
0.4
and 0.24
and
[10]
[15]
in water Φ = 0.06,
0.02
and 0.05.
The results can
be explained in terms of the competition between the split-
ting and back electron-transfer within the charge-separated
intermediate. Since low-polar media strongly destabilize the
intermediate, charge recombination by back electron-trans-
fer becomes so exothermic as to lie in the Marcus inverted
nr
et
and back electron-transfer (kbet). In the two pairs of compe-
titions, ket and kspl contribute to the observed quantum
yield of dimer splitting. The relative quantum efficiencies of
these processes determine the observed quantum yield of
[
24]
region,
which would significantly reduce the rate of the
transfer reaction. In the methoxybenzene-containing model
dimer splitting, Φ = φetφspl.
[16]
compounds (12 ), the quantum yields for the dimer split-
ting are higher and the degree of fluorescence quenching is
lower (i.e. φ is lower) in solvents with lower polarity. Thus,
et
the quantum yields calculated for the dimer radical anion
[
16]
φspl are higher in low-polar media. This implies that sol-
vents of low polarity are unfavorable for electron transfer,
but favor the cleavage of the dimer radical anion by sup-
pressing back electron-transfer.
However, the solvent dependence observed in our work
is in accord with conclusions drawn from studies of flavin-
Scheme 4.
The observed quantum yield for dimer splitting reflects dimer model compounds.[11,18] In contrast to the strong sol-
the efficiency of the overall photosensitized cycloreversion vent dependence, the splitting reaction in flavin-dimer sys-
of the dimer by the linked tryptophan. The variations in the tems was found to have a rather low total solvent depen-
1102
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 1097–1106

Photosensitized Splitting of Thymine Dimer by a Covalently Linked Tryptophan
FULL PAPER
dence. This weak solvent dependence was demonstrated by k is Boltzmann’s constant] and leads to an efficient split-
B
·
[10,15,16]
the formation of a non-zwitterionic intermediate, FlH – ting.
·
–
–
Dimer , which was formed by a shift in charge in the FlH –
[
11,18]
Dimer species.
k
bet = AЈexp[– ∆G° + λ
s
)2/4λ
s
k
B
T]
(8)
This difference in solvent behavior may be due to differ-
ences in the distances (spacers) between the electron ac-
ceptor (dimer) and donor (chromophore) in the model com-
In contrast, the solvent reorganization energy for our
pounds, for example, the 1,3-dimethyluracil dimers model compounds with a relatively long spacer (a large
2, 13[10] and 14
[
16]
[15]
have relatively short spacers and the
1
RDA) is higher, such that λ Ͼ –∆G°. As the polarity of the
s
thymine dimers 1 and 2 have long spacers. The solvent reor- solvent decreases, the back-electron-transfer reaction
ganization energy (λ ) involved in moving an electron from wouldn’t enter the Marcus inverted region. In the Marcus
s
the donor to the acceptor without rearranging the solvation normal region (λ Ͼ –∆G°), the zwitterionic intermediate
s
shell can be determined from Equation (6),
can be stabilized by high-polar media and suppress the un-
productive charge recombination process, kbet, to result in
efficient splitting.
2
)–1 + (2r
)–1 – (RDA) ](εop – ε
–1
–1
–1)
λ
s
= e [(2r
D
A
s
(6)
In another indole-dimer system, 15, which has a longer
spacer and a dimer derived from thymine and orotic
[
25]
where εop and ε are the high frequency and static dielectric acid, a more efficient splitting reaction occurred in water
s
constants of the solvent, respectively, rD and rA are donor (Φ = 0.081) than in ethanol (Φ = 0.068). The corresponding
[
10]
and acceptor radii and RDA is the center-to-center distance values of Φ were 0.06 and 0.24 for 13 and 0.02 and 0.12
[
15]
between the ions. Because of the very short spacers (a small for 14
in the two solvents, respectively. Therefore, the
RDA) for 12, 13 and 14, the solvent reorganization energy length of spacer may play an important role in both the
·+
·–
for charge recombination for the chromophore –dimer
species is small. Although –∆G°, which can be determined of the chromophore-dimer model compounds.
splitting efficiency and the solvent dependence of splitting
from Equation (7),
The X-ray crystal structures of E. coli photolyase re-
vealed a hole, the putative substrate-binding site, which has
the right dimensions to fit the pyrimidine dimer, and the
residues lining its sides are hydrophobic on one side and
2
–∆G° = ED
·+/D
– EA/A·– – e /εr
(7)
[
7]
polar on the other. This asymmetry was suggested to fit
and EA/A·– are potentials for one-electron oxi- well with the asymmetric polarity of a pyrimidine dimer in
where ED
·+/D
dation of the donor and reduction of the acceptor, respec- which the cyclobutane ring is hydrophobic and the opposite
tively, and ε and r are the static dielectric constant of the edges of the dimer have oxygen and nitrogen atoms capable
[
7]
solvent and the RDA in Equation (6), respectively, would of forming hydrogen bonds. This specific hole may con-
also decrease, this decrease is less than λs. tribute to the accelerating cleavage of the dimer radical
As the polarity of the solvent decreases, such that anion and to the suppression of the unproductive back elec-
λ Ͻ –∆G°, the back-electron-transfer reaction enters the tron-transfer.
s
Marcus inverted region[ in which the rate constant for the
24]
The splitting efficiencies in solutions of various pH and
·+
·–
back electron-transfer (kbet) is reduced [Equation (8), where deuterium isotope experiments showed that TrpH –D
Eur. J. Org. Chem. 2005, 1097–1106
www.eurjoc.org © 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1103

Q.-H. Song, W.-J. Tang, X.-M. Hei, H.-B. Wang, Q.-X. Guo, S.-Q. Yu
FULL PAPER
does not deprotonate prior to the cleavage of the dimer rad- taining pocket observed in the X-ray crystal structure of E.
ical anion (Scheme 4), that is, kde Ͻ kspl. The tryptophan coli. photolyase may be required to increase the efficiency
radical cation would deprotonate at N1 to form a neutral of catalytic repair.
radical in neutral solution. The process is completed within
about 1 µs, and in Ͼ100 µs in acidic solutions at pH 3–4,
The pH-dependent measurements show that deproton-
ation of the tryptophan radical cation after photoinduced
[
26]
and in 300 ns for the radical cation of the tryptophan resi- electron transfer is slow and doesn’t occur prior to cleavage.
3
06
·+
[27]
due ( TrpH ) in photolyase. The first two figures show As the splitting and back electron-transfer after the splitting
[
12]
that the deprotonation rate is slower than the rate for the are expected to be complete within 0.5–2 ns, the splitting
splitting of the dimer radical anion (200 ns
56 ns ). In alkaline solution, the tryptophan radical cat- 5×10 s .
ion was short-lived, thus it is possible that the deproton- repairs the substrate with high efficiency (Φ = 0.56) upon
[
13]
and process in the enzyme is much faster than that in solution,
[
14]
6 –1 [14]
5
Hence, when the tryptophan residue directly
[26]
[
9]277
·+
ation process was more favourable than the splitting of the irradiation with light of λ = 280 nm,
TrpH would not
dimer radical anion. However, the dimer will also depro- be expected to deprotonate before splitting of the dimer
tonate at N3 in alkaline solution at pH Ͼ 10 which would radical anion. This fast splitting of the dimer radical anion
inhibit electron transfer to the dimer anion. Because the in the enzyme, which competes with the unproductive back
values of φspl are much less than 1 (Table 1 and Table 2), electron-transfer, should be a very important factor for high
φspl + φbet = 1, the back-electron-transfer reaction should enzymatic repair efficiency.
·+
·–
dominate over dimer splitting in the TrpH –D species,
that is, kspl Ͻ kbet. Hence, the rate constants would increase
Experimental Section
in the order kde Ͻ kspl Ͻ kbet
.
General Remarks: All materials were obtained from commercial
suppliers and used without further purification. Solvents of techni-
cal quality were distilled prior to use. DMF was dried overnight
Conclusions
4 4
with MgSO and was distilled after removal of anhydrous MgSO .
In summary, by using synthesized model compounds 1 Ethanol, propanol, 2-propanol and ethyl acetate were dried over-
2 3 2 3
night with K CO and were distilled after removal of the K CO .
and 2 to mimic the enzymatic repair reaction, the photosen-
sitized splitting reaction of the cis-syn thymine dimer by
covalently linked tryptophan to give photoproducts 3 and
Water was deionized and then double distilled. Acetonitrile and
methanol were spectroscopic grade from commercial suppliers and
were used without further purification. Tetrahydrofuran and 1,4-
4
was confirmed to be a clean conversion upon irradiation
1
13
dioxane were distilled from sodium metal. H and C NMR spec-
tra were measured with a Bruker AV 300 spectrometer operating
at 300 and 75 MHz, respectively. FTIR spectroscopy was measured
with a Bruker Vector22 Intrared Spectrometer. Mass spectra were
with light of λ Ͼ 290 nm.
The splitting reaction is strongly solvent-dependent and
the splitting efficiencies increase in increasingly polar sol-
vents. Fluorescence quenching of the tryptophan unit by obtained with a Micromass GCF TOF mass spectrometer. Elemen-
the attached dimer shows that efficient electron transfer tal analyses were measured with an Elemental Vario EL III spec-
from the excited tryptophan unit to the dimer occurs in trometer.
polar solvents. This result is in agreement with the observa-
Measurement of the Quantum Yield for Splitting: To measure the
[
16]
tion from non-indole-dimer model compounds (12 ). quantum yield for the splitting of the model compounds, samples
Furthermore, the quantum yields of the dimer radical anion (3 mL) were irradiated with 295 nm light from a 970 CRT fluores-
exhibit the same solvent dependence. Thus, back electron- cence spectrometer (Shanghai Analysis Instrument) containing a
transfer, which competes with the cleavage of the dimer rad-
ical anion, is suppressed. Therefore, solvents of high po-
larity enhance the efficiency of the forward electron transfer
from the excited tryptophan moiety to the dimer and sup-
press the unproductive back electron-transfer (charge re-
combination) thereby allowing the model compounds to
undergo highly efficient splitting reactions.
125-W Xe lamp and a monochromator with a 10-nm slit. To mea-
sure the extent of dimer splitting the increase in the absorbance at
approximately 273 nm due to the regeneration of the 5,6-double
bonds of the pyrimidine units was monitored. After certain inter-
vals of time, the absorbance of the irradiated solutions was re-
corded with a Lambda 45 UV/Vis spectrometer (Perkin-Elmer In-
struments). Before each UV spectrum could be recorded a short
period of time (about 2 min) after irradiation was required in order
The solvent dependence of the splitting efficiencies ob- to obtain a homogeneous irradiated mixture. Potassium ferrioxal-
served in this work contrasts with earlier solvent-dependent ate actinometry[ was performed in triplicate before and after
21]
[
10,15,16]
studies.
These differences in solvent dependence may sample irradiation to measure the intensity of irradiation (295 nm).
Within an experimental error of ±5%, the quantum yields for split-
be a result of the different lengths of spacers between the
ting were constant irrespective of whether or not N
through solutions prior to irradiation. The values of Φ for 1 in
three representative aerated and N -saturated solvents (in parenthe-
ses) are as follows: H O, 0.093 (0.091); propanol, 0.066 (0.065); and
2
was passed
donor and acceptor in the model compounds. Compared
with model systems[
10,15,16]
with short spacers, the solvent
2
reorganization energy for the back electron-transfer within
charge-separated species with longer spacers is higher and
does not lie in the Marcus inverted region.
2
THF, 0.016 (0.017). Hence, aerated solutions were employed in all
quantum yield measurements unless otherwise noted.
The solvent dependence in our model systems, which
The absorbances at 273 (A273) and 295 nm (A295) were measured
drawn from studies of flavin-di- at certain intervals of time after irradiation. The change in A273
supports conclusions[
11,18]
mer model compounds, implies that the polar flavin-con- (∆A273) of the solution depends on the extent of splitting of the
104 www.eurjoc.org
1
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2005, 1097–1106

Photosensitized Splitting of Thymine Dimer by a Covalently Linked Tryptophan
FULL PAPER
model compounds. As the model compounds fully split to form
products 3 and 4, the changes in the molar extinction coefficients
M.p. 153–154 °C. IR (KBr): ν˜ = 3039 (m), 2994 (m), 1690 (s), 1464
(m), 1201 (m), 1013 (m), 797 (m) cm . H NMR (300 MHz, [D ]
6
–
1 1
(
∆ε273) were obtained from the UV absorption spectra of 1 and 2
DMSO): δ = 1.25 (t, J = 7.0 Hz, 3 H, CH
2.77 (d, J = 5.7 Hz, 2 H, CH ), 3.97 (d, J = 5.7 Hz, 2 H, NCH
4.15 (q, J = 7.0 Hz, 2 H, CH CH
(s, 1 H, NH). C NMR (75 MHz, [D
14.0 (CH CH ), 32.9 (CH ), 43.8 (CH ), 60.2 (CH
2
CH
3
), 1.91 (s, 3 H, CH
3
),
),
and the splitting products 3 and 4; the value of ∆ε273 employed was
1
compound was obtained from ∆A273/∆ε273
The percentage of splitting of the model compounds was kept
within 5% in all measurements of the quantum yields. A plot of
2
2
4
–1
–1
.92×10 m cm . The splitting concentration (cspl) of the model
2
3
), 7.19 (s, 1 H, CH), 8.76 ppm
]DMSO): δ = 11.9 (CH ),
CH ), 108.2 (C),
1
3
.
6
3
2
3
2
2
2
3
141.8 (CH), 150.8, 164.3, 170.7 ppm. TOF-MS (CI): C10
H
14
N
2
O
4
+
c
spl against the irradiation time (t/min) fits a straight line well (all
[M + 1] : 227.1032; found 227.1025.
correlation coefficients, R, Ͼ 0.996). The slope B of the straight line
gives the rate of splitting of the model compounds. The intensity of
Ethyl cis-[4a]-cisoid-[4a,4b]-cis-[4b]-Dodecahydro-4a,4b-dimethyl-
2,4,5,7-tetraoxocyclobuta[1,2-d:4,3-d]dipyrimidine-1,8-dipropionate
(7): The ethyl ester 6 (2.00 g, 8.8 mmol) was dissolved in acetone/
MeCN (200 mL, 1:4) and the solution was degassed for 15 min in
an ultrasonic bath. The solution was irradiated for 20 h with a 300-
W high-pressure Hg lamp in a Pyrex photochemical reactor. Dur-
ing the irradiation the solution was purged with nitrogen. The reac-
tion mixture was evaporated to dryness in vacuo. The residual oil
was dissolved in EtOAc and subjected to column chromatography
–
1
the incident light I
0
(unit: Einstein min ) was measured by using
ferrioxalate actinometry.[ The intensity of the light absorbed (I
21]
a
)
).
–
A295
by a solution was calculated from Beer’s law, I
From these values the quantum yields could be calculated: Φ =
BV /I , where V is the volume of the irradiated solution,
×10–3 L.
a
= I
0
(1 – 10
0
a
0
3
Measurement of Steady-State Fluorescence Emission: Fluorescence
emission spectra were measured at room temperature with a Per-
kin-Elmer Instruments LS55 Luminescence Spectrometer. To deter-
mine the degree of fluorescence quenching of 1 and 2, the fluores-
cence intensities of 1 and 2 were compared with the fluorescence
intensity of tryptophan 5, that is, Q = 1 – (FTrpH–D/FTrpH). Equal
concentrations of tryptophan residue of the tryptophan-dimer and
the free tryptophan 5 were used in the same solvent. An excitation
wavelength of 290 nm was used, which corresponds to an absorp-
tion maximum of the model compounds resulting from an nǞπ*
transition of the indole unit.
(
silica gel-H, petroleum ether/EtOAc/MeOH, 3:2:0.1) to yield the
isomers 7. Recrystallization from petroleum ether/EtOAc yielded
the cis-syn isomer 7 as colorless needles (720 mg, 36%). M.p. 157–
1
58 °C. IR (KBr): ν˜ = 3221 (m), 2985 (m), 1722 (s), 1679 (s), 1486
–1
1
(
m), 1211 (m), 1019 (m), 790 (m) cm . H NMR (300 MHz, [D
DMSO): δ = 1.18 (m, 6 H, CH CH ), 1.30 (s, 6 H, CH ), 2.59 (m,
), 3.76 (m, 2 H, NCH ), 4.05 (m,
+ CH), 10.31 ppm (s, 2 H, NH). 13C NMR (75 MHz,
]DMSO): δ = 14.0 (CH CH ), 18.0 (CH ), 31.6 (CH ), 41.8
), 46.3 (C), 58.6 (CH), 60.1 (CH CH ), 151.7, 170.3,
(452.46): calcd. C 53.09, H 6.24, N 12.38;
found C 53.24, H 6.01, N 12.17. TOF-MS (CI): C20 [M +
6
]
2
3
3
4
6
H, CH
2
), 3.01 (m, 2 H, NCH
2
2
H, OCH
2
[
D
6
2
3
3
2
(
CH
2
2
3
28 4 8
171.1 ppm. C20H N O
pH-Dependent Measurement of the Quantum Yield for Splitting: To
explore the effect of pH on the splitting reaction, Φ values were
determined in solutions at various pH; water/tetrahydrofuran
28 4 8
H N O
+
1] : 453.1985; found 453.1987.
(
80:20) solvent mixtures were used with 0.1 m buffer, over a pH
cis-[4a]-cisoid-[4a,4b]-cis-[4b]-Dodecahydro-4a,4b-dimethyl-2,4,5,7-
range of 1 to 13. HCl/KCl buffers were used for pH 1–2, 0.1 m HCl/
PAP (PAP = potassium hydrogen phthalate) buffers for pH 3, 0.1 m
NaOH/PAP buffers for pH 4–5, potassium phosphate buffers were
used for pH 6–11 and NaOH/KCl buffers were used for pH 12–13.
tetraoxocyclobuta[1,2-d:4,3-d]dipyrimidine-1,8-dipropionic Diacid
11): The diester 7 (540 mg, 1.19 mmol) was dissolved in 5 m hydro-
(
chloride (12 mL). The reaction mixture was stirred refluxed for 1 h
and then the reaction solution was concentrated in vacuo. The pro-
Tryptophan Methyl Ester (5):[28] Thionyl chloride (12 g, 100 mmol)
was added dropwise to methanol (50 mL) at –5 °C whilst stirring.
The mixture was stirred for a further 0.5 h at –5 °C. Tryptophan
duct was washed several times with Et O and dried in vacuo to
yield a white powder (465 mg, 98%). M.p. 246–248 °C. IR (KBr):
2
ν˜ = 3214 (m), 3098 (m), 2920 (m), 1716 (s), 1485 (m), 1290 (m),
–
1 1
1
213 (m) cm . H NMR (300 MHz, [D
CH ), 2.53 (m, 4 H, CH ), 2.99 (m, 2 H, NCH
NCH ), 4.05 (s, 2 H, CH), 10.31 (s, 2 H, NH), 12.36 ppm (s, 2 H,
6
]DMSO): δ = 1.29 (s, 6 H,
(10.0 g, 49 mmol) was added to the reaction solution. The ice bath
3
2
2
), 3.73 (m, 2 H,
was removed and the reaction mixture was allowed to stir for 5 h.
The solution was heated to reflux and then concentrated in vacuo.
The precipitate was recrystallized from ethanol/diethyl ether. The
tryptophan methyl ester hydrochloride was dissolved in water, satu-
rated sodium hydrogen carbonate solution was added to give pH 7–
2
1
3
OH). C NMR (75 MHz, [D
6 3
]DMSO): δ = 18.1 (CH ), 31.7
(
CH ), 42.0 (CH ), 46.4 (C), 58.9 (CH), 151.8, 170.5, 172.9 .
2
2
Model Compound 1: A solution of the cis-syn-thymine dimeric di-
carboxylic acid 11 (200 mg, 0.51 mmol) and an excess of BOP
8
and the product 5 was extracted with ethyl acetate. The organic
phase was washed with water, dried with MgSO , filtered and con-
centrated in vacuo to obtain 5 as a white powder (9.8 g, 92%). M.p.
4
(
600 mg, 1.29 mmol) were dissolved in DMF (6 mL) and the mix-
ture was stirred at room temperature for 30 min. After the addition
of a solution of tryptophan methyl ester 5 (120 mg, 0.55 mmol) in
DMF (2 mL), the reaction was stirred for 2 h at room temperature.
Then pentylamine (0.12 mL, 1.04 mmol) was added to the reaction
mixture, and stirring was continued for another 5 h at room tem-
perature. The reaction mixture was diluted with water (100 mL)
9
1
2
0–91 °C. IR (KBr): ν˜ = 3359(m), 3295 (w), 1732 (s), 1572 (m),
–
1 1
227 (m), 742 (m) cm . H NMR (300 MHz, CDCl
3
): δ = 3.17 (m,
), 7.07 (s, 1
H, CH ), 3.72 (s, 3 H, CH ), 3.84 (m, 1 H, CHCH
2
3
2
1
3
H, CH), 7.37 (m, 4 H, Hindole), 8.16 ppm (s, 1 H, NH). C NMR
75 MHz, CDCl ): δ = 30.7 (CH ), 52.0 (CH ), 54.8 (CH), 110.3,
11.3, 118.5, 119.2, 121.9, 123.2, 127.3, 136.3, 175.7 ppm. TOF-
(
1
3
2
3
and extracted with CHCl
ers were separated, dried with MgSO
3
(3×200 mL). The combined organic lay-
, filtered and concentrated
+
14 2 2
MS (CI): C12H N O [M + 1] : 219.1134; found 219.1125.
4
Ethyl 1,2,3,4-Tetrahydro-5-methyl-2,4-dioxopyrimidine-1-propionate in vacuo. The remaining crude product was purified by column
(6): Thymine (10.0 g, 79.3 mmol) was refluxed with ethyl acrylate
3
chromatography (silica gel-H, CHCl /MeOH, 50:1). Compound 1
(
10.6 g, 106 mmol) in the presence of hydroquinone (100 mg, poly-
was obtained as a white powder (69 mg, 21%). M.p. 183–185 °C.
IR (KBr): ν˜ = 3348 (m), 3078 (m), 2953 (m), 2932 (m), 2864 (w),
1713 (s), 1650 (s), 1489 (m), 1289 (m), 1216 (m), 1108 (w), 745
merization inhibitor) in ethanol (70 mL) containing NaOH
276 mg) for 20 h. After evaporation of the solvent, the residual
(
–
1
1
material was washed several times with cold ethanol and recrys-
tallized from ethanol to yield 6 as colorless needles (7.6 g, 42%).
(w) cm . H NMR (300 MHz, [D
6
]DMSO): δ = 0.85 (t, 3 H,
), 1.30 (m, 6 H,
CH CH ), 1.20 (s, 3 H, CH ), 1.23 (s, 3 H, CH
2
3
3
3
Eur. J. Org. Chem. 2005, 1097–1106
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1105

Q.-H. Song, W.-J. Tang, X.-M. Hei, H.-B. Wang, Q.-X. Guo, S.-Q. Yu
FULL PAPER
(
+
CH
CHCH
H, CH), 4.48 (m, 1 H, CHCH
H, CH NH), 8.47 (d, 1 H, CHNH), 10.27 (d, 2 H, NH), 10.86 ppm (m), 1361 (m), 1218 (m) cm . H NMR (300 MHz, [D
2
)
3
CH
3
), 2.48 (m, 4 H, CH
), 3.75 (m, 2 H, NCH
), 7.24 (m, 5 H, Hindole), 7.94 (m, 1
2
), 3.05 (m, 6 H, NCH
), 3.89 (m, 2 white powder (185 mg, 68%). M.p. 183–184 °C. IR (KBr): ν˜ = 3362
(m), 2958 (m), 2929 (m), 1703 (s), 1671 (s), 1651 (s), 1547 (m), 1471
2
+ NHCH
2
of the tryptophan methyl ester 5. Compound 4 was obtained as a
2
), 3.57 (s, 3 H, OCH
3
2
2
–
1 1
2
6
]DMSO): δ
), 1.26 (m, 6 H, (CH CH ),
), 2.41 (t, J = 6.4 Hz, 2 H, CH ), 2.99 (m, 2 H,
), 3.81 (t, J = 6.4 Hz, 2 H, NCH ), 7.36 (s, 1 H, CH), 7.91
), (s, 1 H, NHCH ), 11.19 ppm (s, 1 H, NH). C NMR (75 MHz,
8.8 (CH), 59.0 (CH), 109.5, 111.5, 118.0, 118.5, 121.0, 123.6, [D ]DMSO): δ = 11.9 (CH ), 13.8 (CH CH ), 21.8 (CH ), 28.5
27.0, 136.1, 151.5, 170.0, 170.1, 170.2, 170.4, 170.5, 172.4 ppm. (CH ), 28.7 (CH ), 34.4 (CH ), 38.4 (CH ), 44.7 (CH ), 107.9 (C),
(665.74): calcd. C 59.54, H 6.51, N 14.73; found C
13
(
(
2
(
d, 1 H, NHindole). C NMR (75 MHz, [D
CH CH ), 17.9 (CH ), 18.1 (CH ), 21.8 (CH
8.6 (CH ), 28.7 (CH ), 33.0 (CH ), 33.4 (CH ), 38.5 (CH
CH ), 42.7 (CH ), 46.0 (C), 46.7 (C), 51.8 (OCH ), 53.3 (CHCH
6
]DMSO): δ = 13.8
), 27.0 (CHCH ),
), 42.4
= 0.82 (t, J = 6.8 Hz, 3 H, CH
1.71 (s, 3 H, CH
NHCH
2
CH
3
2
)
3
3
2
3
3
3
2
2
3
2
2
2
2
2
2
2
2
1
3
2
2
3
2
2
5
1
C
6
3
2
3
2
2
2
2
2
2
33
H
43
N
7
O
8
141.9 (CH), 150.7, 164.3, 169.3 ppm. TOFMS (CI): C13
[M + 1] : 268.1661; found 268.1664.
H
21
N
3
O
3
+
+
5
9.59, H 5.94, N 14.45. ESI-MS (m/z): 666 [M] , 688 [M – H +
+
Na] .
Model Compound 2: A solution of the cis-syn-thymine dimer di-
carboxylic acid 11 (100 mg, 0.25 mmol) and an excess of BOP
Acknowledgments
(
300 mg, 0.65 mmol) were dissolved in DMF (4 mL) and the mix-
The authors are grateful for financial support from the National
Natural Science Foundation of China (Grant No. 20332020,
ture was stirred at room temperature for 30 min. After the addition
of a solution of tryptophan methyl ester 5 (120 mg, 0.55 mmol) in
DMF (2 mL), the reaction was stirred for 5 h at room temperature.
The reaction mixture was diluted with water (100 mL) and ex-
30000036, 20273066, and 30470444).
tracted with CHCl
were separated, dried with MgSO
vacuo. The remaining crude product was purified by column
chromatography (silica gel-H, CHCl /MeOH, 50:1Ǟ20:1). Com-
pound 2 was obtained as a white powder (51 mg, 25 %). M.p. 203–
05 °C. IR (KBr): ν˜ = 3372 (m), 3062 (m), 2954 (w), 1742 (s), 1710
3
(3×200 mL). The combined organic layers
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, filtered and concentrated in
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2
–
1
(
s), 1655 (s), 1487 (m), 1284 (m), 1204 (m), 1103 (m), 740 (m) cm .
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6] T. Carell, L. T. Burgdorf, L. M. Kundu, M. Cichon, Curr. Opin.
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1
H NMR (300 MHz, [D
H, CH ), 2.38 (m, 4 H, CH
.55 (s, 3 H, OCH ), 3.58 (s, 3 H, OCH
.88 (m, 2 H, CH), 4.49 (m, 2 H, CHCH
.47 (m, 2 H, CHNH), 10.24 (s, 1 H, NH), 10.25 (s, 1 H, NH),
6
]DMSO): δ = 1.12 (s, 3 H, CH
), 3.04 (m, 6 H, NCH + CHCH
), 3.72 (m, 2 H, NCH
3
), 1.19 (s,
3
3
3
8
1
3
2
2
2
),
),
[
7] H. W. Park, S.-T. Kim, A. Sancar, J. Deisenhofer, Science 1995,
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89, 900–904.
13
0.81 (s, 1 H, NHindole), 10.84 ppm (s, 1 H, NHindole). C NMR
]DMSO): δ = 17.9 (CH ), 27.0 (CHCH ), 32.9 (CH ),
), 45–46 (C), 51.9 (OCH ), 53.3 (CHCH ), 58.7 (CH),
09.4, 111.5, 118.0, 118.5, 121.1, 123.6, 127.0, 136.1, 151.5, 170.2,
70.5, 172.5 ppm. C40 10 (796.84): C 60.29, H 5.57, N 14.06;
[
[
[
[
[
[
[
[
10] S.-T. Kim, R. F. Hartman, S. D. Rose, Photochem. Photobiol.
990, 52, 789–794.
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(75 MHz, [D
6
3
2
2
1
4
1
1
2.4 (CH
2
3
2
1
44 8
H N O
12] S.-T. Kim, M. Volk, G. Rousseau, P. F. Heelis, A. Sancar, M.-
E. Michel-Beyerle, J. Am. Chem. Soc. 1994, 116, 3115–3116.
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Radiat. Biol. 1974, 25, 521–523.
+
found C 59.21, H 5.34, N 13.55. ESI-MS (m/z): 797 [M] , 819 [M –
H + Na] .
+
1-(Carboxyethyl)thymine Tryptophan Amide (3): The ethyl ester 6
14] S. R. Yeh, D. E. Falvey, J. Am. Chem. Soc. 1991, 113, 8557–
(230 mg, 1.02 mmol) was dissolved in 5 m hydrochloride (10 mL)
8558.
and the reaction mixture was refluxed for 1 h. The reaction solution
was then concentrated in vacuo. The residue and an excess of BOP
15] S.-T. Kim, S. D. Rose, J. Photochem. Photobiol. B: Biol. 1992,
2, 179–191.
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54.
1
(600 mg, 1.29 mmol) were dissolved in DMF (6 mL) and stirred at
room temperature for 30 min. After the addition of a solution of
tryptophan methyl ester 5 (240 mg, 1.10 mmol) in DMF (2 mL),
the reaction was stirred for 5 h at room temperature. The reaction
mixture was purified by column chromatography on aluminium ox-
ide (100–200 mesh, EtOAc/MeOH, 20:1Ǟ2:1). Compound 3 was
obtained as a white powder (155 mg, 38%). M.p. 110–112 °C. IR
8
17] A. P. M. Eker, J. K. C. Hessels, R. H. Dekker, Photochem. Pho-
tobiol. 1986, 44, 197–205.
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Perkin Trans. 2 1997, 1217–20.
[
[
20] B. Castro, G. Evin, C. Selve, R. Seyer, Synthesis 1977, 413–469.
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J. Phys. Chem. 1991, 95, 3636–3643.
(
(
(
2
=
+
KBr): ν˜ = 3399 (m), 3057 (m), 2954 (m), 1738 (s), 1671 (s), 1460
–1
1
m), 1220 (m), 1127 (m), 1010 (m), 746 (m) cm . H NMR
300 MHz, [D ]DMSO): δ = 1.69 (s, 3 H, CH ), 2.46 (t, J = 6.4 Hz,
), 3.06 (m, 2 H, CHCH ), 3.55 (s, 3 H, OCH ), 3.77 (t, J
6.6 Hz, 2 H, NCH ), 4.49 (m, 1 H, CHCH ), 7.23 (m, 6 H, CH
indole), 8.47 (d, 1 H, CHNH), 10.83 (s, 1 H, NHindole),
6
3
[
[
H, CH
2
2
3
2
2
23] M. S. Jorns, J. Am. Chem. Soc. 1992, 114, 3133–3136.
H
[24] G. L. Closs, J. R. Miller, Science 1988, 240, 440–447 and refer-
13
1
(
(
1.19 ppm (s, 1 H, NH). C NMR (75 MHz, [D
CH ), 27.1 (CHCH ), 34.0 (CH ), 44.3 (CH ), 51.8 (OCH
CHCH
36.0, 141.9 (CH), 150.7, 164.3, 169.8, 172.3 ppm. TOF-MS (CI):
6
]DMSO): δ = 11.9
ences cited therein.
), 53.2 [25] T. Young, S.-T. Kim, J. R. V. Camp, R. F. Hartman, S. D. Rose,
3
2
2
2
3
Photochem. Photobiol. 1988, 8, 635–641.
2
), 108.0 (C), 109.4, 111.5, 118.0, 118.5, 121.0, 123.6, 127.0,
[
[
26] D. V. Bent, E. Hayon, J. Am. Chem. Soc. 1975, 97, 2612–2619.
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Brettel, P. Mathis, Biochem. Biophys. Acta 2004, 1655, 64–70.
1
+
C
20
H
22
N
4
O
5
: [M + 1] : 399.1668; found 399.1667.
-(Carboxyethyl)thymine n-Pentyl Amide (4): Compound 4 was syn-
thesized similarly to compound 3 but with n-benzylamine instead
1
[28] M. Brenner, W. Huber, Helv. Chim. Acta 1953, 36, 1109–1115.
Received: September 6, 2004
1106
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Eur. J. Org. Chem. 2005, 1097–1106