Model compounds for (6-4) photolyases: A comparative flavin induced cleavage study of oxetanes and thietanes

Article abstract of DOI:10.1039/b503205a

Thietanes were used in the past as mimics for an unstable oxetane intermediate formed during the repair of mutagenic (6-4) lesions. The thietane derivatives were found to be not repaired, raising the question of how well thietanes are cleaved by single el

Full text of DOI:10.1039/b503205a

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A R T I C L E
Model compounds for (6–4) photolyases: a comparative flavin
induced cleavage study of oxetanes and thietanes†
Marcus G. Friedel,a Michaela K. Cichonb and Thomas Carell*a
a Department of Chemistry, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13,
81377, Munich, Germany. E-mail: Thomas.Carell@cup.uni-muenchen.de
b Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Str., 35032,
Marburg, Germany
Received 4th March 2005, Accepted 24th March 2005
First published as an Advance Article on the web 18th April 2005
Thietanes were used in the past as mimics for an unstable oxetane intermediate formed during the repair of
mutagenic (6–4) lesions. The thietane derivatives were found to be not repaired, raising the question of how well
thietanes are cleaved by single electron donation compared to oxetanes. We have prepared two flavin-containing
oxetane and thietane model compounds for the (6–4) photolyase catalyzed repair process and we show that both are
efficiently cleaved by a reduced and deprotonated flavin. Thietanes are therefore excellent models. The lack of their
repair can be attributed to lack of binding.
Introduction
UV irradiation of cells leads to severe damage of the genome.1,2
The main lesions formed are cyclobutane–pyrimidine dimers
(CPD) and (6–4) photoproducts.3 The (6–4) lesion is presumably
more mutagenic than the CPD lesion.4,5 It is formed in a
Paterno´–Bu¨chi reaction between adjacent pyrimidine bases
giving first an oxetane intermediate, which is thermally not
stable and rearranges above −80 ◦C to the (6–4) photo adduct
(Fig. 1a).6 The lesion is repaired in certain organisms by a
particular enzyme called (6–4) photolyase.7 The enzyme converts
the photoproduct back into the parent pyrimidines via a light-
dependent process. For the homologous CPD photolyases,8
which are responsible for the repair of the UV-induced CPD
lesions, it is known that they contain a reduced and deprotonated
flavin (FADH−). The flavin, upon excitation by light, donates an
electron to the CPD and cleaves (cycloreverts) the lesion.9 This
mechanism is well understood through crystallographic,10–12
enzymatic13,14 and model compound studies.15–18 In contrast,
the repair process catalyzed by the (6–4) photolyase is less
well characterized and structural information is lacking.19,20
Due to the high sequence homology between CPD and (6–4)
photolyases and the fact that both enzymes contain a flavin
as the essential cofactor, Kim et al. proposed the (6–4) repair
mechanism depicted in Fig. 1a.21 They postulate that the binding
of the enzyme causes a thermal rearrangement of the (6–4) lesion
back to the oxetane intermediate. Although calculations predict
that this process may be energetically very costly to proceed
spontaneously in the active site,22 Todo et al. identified two
histidine residues which might catalyze the rearrangement by
a protonation/deprotonation process.23
Fig. 1 a) Depiction of the formation and repair of the mutagenic (6–4)
The key step of the proposed repair mechanism is the electron
transfer-induced cleavage of the oxetane intermediate by a light-
induced electron transfer. Quantum chemical calculations (in the
gas phase) predict that oxetanes cleave spontaneously after single
electron donation or after single electron abstraction.24 Laser
flash photolysis experiments by Falvey and coworkers25 and
Miranda et al.26 with model compounds showed that the
cleavage of oxetanes is indeed possible with different electron
donors and acceptors in a light-dependent reaction. With the
photoproduct via the oxetane intermediate. b) Stable thietane analogues
of the repair intermediate used in enzymatic studies.
model compound 1 (Scheme 1) we demonstrated recently that
a covalently-linked flavin is also able to split the oxetane upon
irradiation when the flavin is in the reduced and deprotonated
state providing strong support for the repair mechanism
proposed by Kim et al.21
In order to investigate the enzymatic repair reaction, Clivio
and Fourrey28–32 and Taylor and Liu33 prepared the stable
thietanes 2 and 3 (Fig. 1b), which were designed to mimic the
key oxetane intermediate of the repair reaction. Sancar and co-
workers20 tested these stable thio-analogues and found that both
are not repaired by the enzyme due to a lack of binding.
† Electronic supplementary information (ESI) available: MALDI-
TOF mass spectra of collected HPLC-peaks. See http://www.rsc.org/
suppdata/ob/b5/b503205a/
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 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 3 7 – 1 9 4 1
1 9 3 7
©

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Scheme 2 Synthesis of thietane model compound 4.
Scheme 1 Synthesis of oxetane model compound 1.
These results challenge the mechanism and raise three main
questions: 1) are thio-analogues of (6–4) lesions suitable models
for the (6–4) repair intermediate? 2) Is it possible to cleave a
thietane with flavin in an analogous reaction to that of an
oxetane? 3) Are thietanes cleaved preferentially by single electron
oxidation or reduction? In order to address these three critical
questions we synthesized the thio-analogue 4 of our initial model
compound 1 and compared both model compounds 1 and 4 in
irradiation experiments.
Results and discussion
The covalently linked flavin- and oxetane-containing model
compound 1 is able to mimic the critical putative cycloreversion
step, catalyzed by (6–4) photolyase with relatively high quantum
yield (φ = 0.11).19 The splitting of the oxetane is only possible via
the reductive pathway. The cycloreversion is induced by single
electron donation to the oxetane from a flavin in the reduced
and deprotonated state. The synthesis of this model compound
is depicted in Scheme 1. Irradiation of 5 in the presence of
benzophenone furnished the stable oxetane 6. Hydrogenolytic
cleavage of the benzyl ester and condensation of the obtained
oxetane acid 7 with 10-amino-ethylflavin 818,34 gave model
compound 1 in the form of a yellow powder.27
The thietane model compound 4 was prepared in a sim-
ilar way (Scheme 2). Irradiation of 9 in the presence of
thiobenzophenone, which was synthesized from benzophenone
with the Lawesson-reagent,35 yielded the stable thietane 10.
The irradiation was repeated five times because of the low
reaction yield. Deprotection of the tert-butyl ester of 10 with
trifluoroacetic acid and coupling of the thietane acid 11 with
10-amino-ethylflavin 12 furnished the model compound 4 in the
form of a yellow solid.
To allow a better separation by HPLC of the model com-
pounds 1 and 4 and of the putative cleavage products 13 and 14
(Fig. 2b), the flavins in 1 and 13 were prepared with a pentyl side
chain at N3. In contrast, the flavins in 4 and 14 possess a methyl
group at N3.
Fig. 2 a) Chromatograms (HPLC) obtained after irradiation of a
mixture of the model compounds 1 and 4. Elution times 14: 10.7 min,
13: 15.1 min, 4: 20.5 min, 1: 27.1 min. Conditions: Nucleosil RP-C18
(5 lm) column (4 × 250 mm), detection at 450 nm, gradient: A = water,
B = acetonitrile, 40–60% B over 25 min, flow rate: 0.7 mL min−1. b)
Cleavage reaction of 1 and 4 to give 13 and 14, respectively.
Irradiation experiments were performed with solutions of
a mixture of both model compounds (1 + 4) in ethylene
glycol (10−4 M), under anaerobic conditions, in a 500 lL
fluorescence cuvette stoppered with a rubber septum to allow
direct comparison of both compounds. The flavins were reduced
and deprotonated by addition of sodium dithionite solution
(100 lL, 0.06 M) and triethylamine (60 lL). Irradiation was
carried out in a fluorimeter with monochromatic light (366
5 nm). During the experiments, samples were taken from the
assay solution after defined time intervals with a microsyringe.
These samples were subsequently reoxidized through shaking
with exposure to air for at least 1 h. The samples were
analyzed by reversed phase HPLC. The result of a typical
irradiation experiment is depicted in Fig. 2a. The chromatogram
1 9 3 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 3 7 – 1 9 4 1

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before irradiation (0 min) shows the mixture of the two model
compounds 1 and 4. Due to the different alkylchains at N3 of the
flavins, they elute with very different retention times at 27.1 min
(oxetane model compound 1) and 20.5 min (thietane model
compound 4). After 5 min of irradiation two new peaks elute at
15.1 min and 10.7 min. These peaks increase in intensity during
irradiation, while the amount of the model compounds 1 and 4
decrease. The two new peaks are the reaction products (13 + 14)
of the splitting reaction of compound 1 and 4. The peak at
15.1 min corresponds to the cleavage product 13, arising from the
oxetane 1, and the peak at 10.7 min corresponds to the cleavage
product 14 from the thietane 4. This assignment was confirmed
by co-injection of both independently synthesized cleavage
products (13 + 14) and by mass spectrometry. For the MS-
analysis all four peaks from the HPLC runs were collected and
measured by MALDI-TOF (mass spectra in the supplementary
material†). To exclude intermolecular processes the irradiation
experiments were repeated with solutions containing the single
model compounds. They gave the same results for the cleavage
efficiency as in the comparative irradiations.
To investigate if the thietane cleavage required a reduced and
deprotonated flavin further irradiation studies were performed.
We observed no detectable cleavage with the flavin in the oxidized
state or under reductive but acidic conditions, which gives the
reduced but neutral flavin FlH2. The irradiation of thietane 10,
without a flavin, in the presence of sodium dithionite and base
gave also no cleavage of the four-membered ring. These control
experiments demonstrate that the flavin is strictly needed in
its reduced and deprotonated state (FlH−) to efficiently cleave
model compound 4.
Our experiments show that the reductive splitting reaction
of the four-membered ring does not depend on the nature of
the heteroatom in the ring. This leads to the conclusion that
the thio-analogue compounds, used in the enzyme assay of
Sancar et al.,20 should in principle be cleavable with the reduced
flavin cofactor present in the (6–4) photolyase. These results
confirm the observation from the enzymatic study that the thio-
analogue compounds 2 and 3 were not repaired because they
were not bound by the (6–4) photolyase. This allows us to finally
assume that the enzyme can recognize only the (6–4) lesion and
not the oxetane intermediate directly. The lesion then requires
rearrangement. It is interesting to note that in our irradiation
experiments the thietane is cleaved, by a factor of two, more
efficiently.
were referenced to DMSO (d = 2.50 ppm) in DMSO-d6 and
CH3OH (d = 3.31 ppm) in CD3OD. 13C-NMR spectra were
recorded on Bruker ARX 200 (50 MHz), AMX 300 (75 MHz),
AMX 500 (125 MHz), AMX 600 (150 MHz) and Varian XL400
(100 MHz) spectrometers. The chemical shifts were referenced
to DMSO (d = 39.43 ppm) in DMSO-d6 and CH3OH (d =
49.05 ppm) in CD3OD. Standard pulse sequences were employed
for H 2D NOESY, H,1H and H,13C correlation studies. IR
spectra were recorded in KBr and measured with a Bruker IFS
25 Fourier transform infrared spectrophotometer and Perkin-
Elmer FT-IR spectrum 100. Mass spectra and high-resolution
mass spectra were measured on Finnigan MAT TSQ 700,
Finnigan MAT 95, Finnigan MAT 90 and Bruker Autoflex II
(MALDI-TOF) mass spectrometers. HPLC was performed with
a Merck-Hitachi system equipped with L-7400 UV and L7480
fluorescence detectors. Analytical separations were performed
with a Macherey-Nagel Nucleosil 100–5 C18 (250 × 4 mm)
column.
1
1
1
All solvents were of the quality puriss. p.a., or purum. Purum
solvents were distilled prior to use. The commercially available
reagents were used as received without further purification.
Irradiation experiments
A stirred solution of 1 (10−4 M) and 4 (10−4 M) in ethylene glycol
in a fluorescence cuvette, stoppered with a rubber septum, was
degassed by bubbling N2 through the solution for 30 min to
ensure anaerobic conditions. For reduction and deprotonation
of the flavin, sodium dithionite (100 lL, 0.06 M in water) and
triethylamine (60 lL) were added and the solution stirred for
further 30 min. Both procedures were carried out under the
exclusion of light. The irradiation was carried out within a
fluorescence spectrometer (JASCO-FP-750) at 366 nm ( 5 nm)
with constant flow of nitrogen through the solution. During
irradiation samples (20 lL) were taken from the assay solution
after defined time intervals via a microsyringe and diluted with
water (20 lL). Subsequent reoxidation of the flavin was achieved
through shaking and exposure to air for at least 1 h under
exclusion of light. The samples were analyzed by reversed phase
HPLC under the following conditions: Nucleosil RP-C18 (5 lm)
column (4 × 250 mm); A = water, B = acetonitrile; 40–60% B
over 25 min; detection at 450 nm, flow rate: 0.7 mL min−1.
(6-Methyl-3,5-dioxo-8,8-diphenyl-7-oxa-2,4-diaza-bicyclo-
[4.2.0]oct-2-yl)-acetic acid benzyl ester 6. Thymine acetic acid
benzyl ester 5 (2.03 g, 7.40 mmol) was dissolved in acetonitrile
(200 mL) under sonication. Benzophenone (2.70 g, 14.8 mmol)
was added and the solution was degassed by bubbling N2
through the solution for 0.5 h. The solution was subsequently
irradiated for 6 h with a 150 W (TQ-150) medium-pressure
mercury lamp in a quartz irradiation apparatus. Acetonitrile
was removed in vacuo and the product was isolated by flash
chromatography (ethyl acetate–n-hexane 2 : 8 to 1 : 1).
Recrystallisation from ethyl acetate–n-hexane (2 : 8) gave 6
Conclusions
Two model compounds 1 and 4 were successfully synthesized,
containing a stable oxetane and a stable thietane respectively,
both covalently linked to flavin moieties. The model compounds
mimic the critical intermediate postulated during the repair of
UV photo lesions catalyzed by (6–4) photolyase. With these
model compounds we show that upon irradiation only the
reduced and deprotonated flavin is able to cleave the oxetane
and the thietane ring. The result suggests that, in agreement with
the proposed mechanism from Kim and co-workers,21 electron
transfer proceeds from the flavin to the oxetane as the key step
of the repair reaction. In addition, the cleavage of the thietane
model compound under the same conditions shows that the thio-
analogues of the (6–4) photoproduct previously used by Sancar
et al.20 are suitable model systems. The fact that the repair was
not observed is due to a lack of binding and not because thietanes
are uncleavable by single electron donation.
(645 mg, 19%) as a white solid. Mp: 158 ◦C; mmax(KBr)/cm−1
=
3445w, 3193w, 3067w, 2931w, 2876w, 1747m, 1726s, 1692s,
1479m, 1448w, 1409w, 1391w, 1370w, 1360w, 1287w, 1253w,
1208m, 1192m, 974w, 833w, 782w, 748w, 727w, 696m, 645w,
596w, 512w; 1H-NMR (300 MHz, (CD3)2SO): d = 1.50 (s,
3H; CH3), 4.09 (d, J = 17.6 Hz, 1H; N–CHaH), 4.38 (d, J =
17.3 Hz, 1H; N–CHHb), 4.97 (s, 1H; CH), 5.11 (d, J = 12.6 Hz,
1H; O–CHaH), 5.17 (d, J = 12.3 Hz, 1H; O–CHHb), 7.19–7.42
(m, 15H; aryl-H), 10.48 (s, 1H; NH); 13C-NMR (125 MHz,
(CD3)2SO): d = 24.43, 66.30, 67.69, 69.39, 77.39, 92.22, 126.37
(2C), 126.72 (2C), 128.87 (2C), 129.36 (2C), 129.47 (2C), 129.69
(2C), 129.73 (3C), 136.92, 140.87, 145.65, 152.57, 169.77,
171.36; m/z (ESI+): 479 (100%, M + Na+); HRMS (ESI+): calc.
for C27H24N2O5Na (M + Na+): 479.1583, found: 479.1594.
Experimental
General
Melting points are uncorrected. 1H-NMR spectra were recorded
on Bruker ARX 200 (200 MHz), AMX 300 (300 MHz) and
Varian XL400 (400 MHz) spectrometers. The chemical shifts
(6-Methyl-3,5-dioxo-8,8-diphenyl-7-oxa-2,4-diaza-bicyclo-
[4.2.0]oct-2-yl)-acetic acid 7. To a stirred solution of 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 3 7 – 1 9 4 1
1 9 3 9

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(833 mg, 1.83 mmol) in glacial acetic acid (10 mL) was added
a suspension of 10% Pd/C (20 mg, 0.02 mmol) in glacial acetic
acid (2 mL). The solution was stirred under a H2 atmosphere
for 2 h at atmospheric pressure. The reaction mixture was
filtered through celite and the filter cake was washed with
hot acetic acid (80 mL). The solvent was removed in vacuo.
Recrystallisation from ethyl acetate–n-hexane (2 : 3) afforded 7
unreacted starting material, was separated by filtration and
the starting material was reisolated by recrystallisation from
chloroform–pentane. The filtrate was evaporated to dryness in
vacuo. The irradiation was repeated four times. Overall 1.50 g
(6.20 mmol) thymine-acetic acid tert-butyl ester and 1.25 g
(6.20 mmol) of thiobenzophenone were irradiated. The collected
soluble fractions were purified by flash chromatography using
a gradient of methanol in chloroform (0 to 9%) to give 10
(total amount: 108 mg, 4%) as a reddish–brown foam. Mp:
93–95 ◦C; mmax(KBr)/cm−1 = 3420m, 3254w, 3060w, 2978w,
2931w, 1740s, 1708s, 1467m, 1446m, 1367m, 1274m, 1230m,
1155s, 1086w, 1034w, 984w, 943w, 912w, 842w, 750m, 707m,
666w, 621w, 572w, 547w, 500w, 473w; 1H-NMR (400 MHz,
CD3OD): d = 1.48 (s, 9 H; C(CH3)3), 1.80 (s, 3 H; CH3), 3.96
(d, J = 17.6 Hz, 1 H; N–CHaH), 4.27 (d, J = 17.6 Hz, 1 H;
N–CHHb), 4.79 (s, 1 H; CH(6)), 7.15–7.45 (m, 8 H; aryl-H),
7.78–7.81 (m, 2 H; aryl-H); 13C-NMR (100 MHz, CD3OD):
d = 24.43, 28.34, 46.87, 56.80, 63.27, 66.92, 83.65, 128.15 (2C),
128.36 (2C), 128.85 (2C), 128.99, 129.01, 129.89 (2C), 143.35,
143.96, 152.37, 169.39, 171.93; m/z (FAB+): 57 (20%), 77 (18),
89 (16), 107 (15), 121 (23), 136 (47), 154 (54), 165 (21), 185 (29),
199 (100, S(Ph)2 + H+), 221 (11), 241 (14, M − S(Ph)2 + H+),
289 (7), 307 (6), 383 (7), 439 (5, M + H+), 461 (8, M + Na+);
HRMS (FAB+): calc. for C24H27N2O4S (M + H+): 439.1691,
found: 439.1680.
(384 mg, 57%) as a white solid. Mp: 210 ◦C; mmax(KBr)/cm−1
=
3451w, 3188w, 3061m, 1725s, 1666m, 1492m, 1448w, 1405w,
1391w, 1356w, 1287w, 1256w, 1228w, 1130w, 1082w, 1030w,
996w, 974w, 956w, 939w, 921w, 891w, 807w, 783w, 748w, 704w,
1
674w, 663w, 640w, 597w, 534w, 513w, 497w, 451w; H-NMR
(300 MHz, (CD3)2SO): d = 1.63 (s, 3H; CH3), 3.91 (d, J =
17.6 Hz, 1H; N–CHaH), 4.30 (d, J = 17.6 Hz, 1H; N–CHHb),
4.94 (s, 1H; CH), 7.28–7.47 (m, 10H; aryl-H), 10.49 (s, 1H;
NH), 12.93 (bs, 1H; CO2H); 13C-NMR (150 MHz, (CD3)2SO):
d = 23.25, 47.90, 65.16, 76.08, 90.84, 124.96 (2C), 125.43 (2C),
127.50 (2C), 128.07 (2C), 128.39 (2C), 139.61 (2C), 144.48,
151.17, 170.04; m/z (ESI+): 389.7 (70%, M + Na+), HRMS
(ESI+): calc. for C20H18N2O5Na (M + Na+): 389.1113, found:
389.1127.
N-[2-(7,8-Dimethyl-2,4-dioxo-3-pentyl-3,4-dihydro-2H-benzo-
[g]pteridin-10-yl)-ethyl]-2-(6-methyl-3,5-dioxo-8,8-diphenyl-7-
oxa-2,4-diaza-bicyclo[4.2.0]oct-2-yl)-acetamide 1. Oxetane 7
(100 mg, 0.27 mmol), 1-hydroxybenzotriazole (HOBt) (44 mg,
0.32 mmol) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-
uronium tetrafluoroborate (TBTU) (104 mg, 0.32 mmol)
were dissolved in anhydrous DMF (3.6 mL) and stirred for
10 min at room temperature. A solution of flavin 8 (141 mg,
0.03 mmol) in anhydrous DMF (3 mL) and triethylamine (2 mL,
14.3 mmol) were added. The solution was stirred for 30 min at
room temperature. The reaction was diluted with chloroform
(100 mL), washed with water (3 × 100 mL), dried (magnesium
sulfate), filtered and the solvent was removed in vacuo. Diethyl
ether was added to the orange–red oil, the precipitate was
filtered and dried under a reduced pressure. The crude product
was purified by flash chromatography (CHCl3–MeOH 20 : 1)
and afforded 1 (174 mg, 92%) as an orange solid. Mp: 170 ◦C;
mmax(KBr)/cm−1 = 3413w, 3061w, 2954w, 2859w, 1709m, 1657m,
1585m, 1548s, 1463m, 1448m, 1407w, 1383w, 1339w, 1268m,
1230m, 1206m, 1080w, 1017w, 974w, 914w, 807w, 750w, 703w,
668w, 596w, 503w, 435w; 1H-NMR (300 MHz, (CD3)2SO): d =
0.65 (t, 3H; CH3 Pentyl), 1.10 (m, 4H; CH2 pentyl), 1.36 (m,
5H; CH2 pentyl, CH3 oxetane), 2.26 (s, 3H; Ar–CH3), 2.31
(s, 3H; Ar–CH3), 3.33 (m, 3H; N–CH2, N–CHaH–CO), 3.65
(t, J = 7.3 Hz, 2H; CH2 pentyl), 3.94 (d, J = 16.6 Hz 1H;
N–CHHb–CO), 4.41 (s, 1H; CH oxetan), 4.44–4.59 (m, 2H;
N–CH2), 7.10–7.20 (m, 10H; aryl-H), 7.75 (s, 1H; aryl-H), 7.76
(s, 1H; aryl-H), 8.13 (s, 1H; NH), 10.27 (s, 1H; NH); 13C-NMR
(100 MHz, (CD3)2SO): d = 13.82, 18.65, 20.72, 21.83, 22.81,
26.95, 28.56, 35.70, 38.21, 43.01, 48.89, 65.63, 76.27, 90.73,
115.98, 124.84 (2C), 125.43 (2C), 127.54, 128.11 (2C), 128.47
(2C), 131.07 (2C), 134.13 (2C), 135.83, 136.18, 139.61, 144.48,
146.58, 149.01, 151.18, 154.74, 159.33, 168.35, 169.67; m/z
(ESI−): 378 (18%), 432 (17), 459 (10), 493 (3), 569 (19), 600 (4),
702 (100, M − H+), 738 (64), 765 (52), 792 (12), 837 (8); HRMS
(ESI+): calc. for C39H41N7O6Na (M + Na+): 726.3016, found:
726.3022.
(6-Methyl-3,5-dioxo-8,8-diphenyl-7-thia-2,4-diaza-bicyclo-
[4.2.0]oct-2-yl)-acetic acid 11. Thietane 10 (10 mg, 22.0 nmol)
was dissolved in TFA (100 lL) and stirred for 2 h. The acid
was removed in vacuo and the crude product was purified by
flash chromatography (CHCl3–MeOH 10 : 1) to give 11 (8 mg,
95%) as a white solid. Mp : >230 ◦C; mmax(KBr)/cm−1 = 3430s,
3060w, 2926w, 2854w, 1697s, 1616m, 1479m, 1445m, 1396w,
1380w, 1360w, 1311w, 1274m, 1209w, 1188w, 1145w, 1087w,
1
1032w, 911w, 747w, 707w, 578w, 544w, 502w, 468w; H-NMR
(400 MHz, (CD3)2SO): d = 1.65 (s, 3 H; CH3), 3.58 (d, J(NCH2a,
NCH2b) = 16.8 Hz, 1 H; NCH2a), 4.15 (d, J(NCH2b, NCH2a) =
16.8 Hz, 1 H; NCH2b), 4.92 (s, 1 H; CH(6)), 7.14–7.44 (m,
8 H; CHar), 7.66–7.69 (m, 2 H; CHar), 10.34 (s, 1 H; NH);
13C-NMR (100 MHz, (CD3)2SO): d = 23.70, 46.15, 54.96, 61.13,
64.56, 126.73 (2C), 126.77 (2C), 127.43 (2C), 127.54, 127.84,
128.24 (2C), 142.10, 142.37, 149.77, 170.01; m/z (FAB−): 91
(35%), 112 (32), 182 (100), 183 (8, M − S(Ph)2 − H+), 197 (29,
S(Ph)2–H+), 381 (54, M − H+); HR-MS (MALDI+): calc. for
C20H18N2O4SNa (M + Na+): 405.0885, found: 405.0881.
2-(6-Methyl-3,5-dioxo-8,8-diphenyl-7-thia-2,4-diaza-bicyclo-
[4.2.0]oct-2-yl)-N-[2-(3,7,8-trimethyl-2,4-dioxo-3,4-dihydro-2H-
benzo[g]pteridin-10-yl)-ethyl]-acetamide 4. Thietane 11 (8.0 mg,
21.0 lmol), HOBt (4.0 mg, 32.0 lmol) and TBTU (10.0 mg,
32.0 lmol) were dissolved in anhydrous DMF (0.5 mL) and
stirred for 30 min at room temperature. A solution of flavin
12 (9.0 mg, 21.0 lmol) in anhydrous DMF (0.5 mL) and
triethylamine (0.15 mL, 1.11 mmol) were added. The solution
was stirred for 60 min at room temperature. The reaction was
diluted with chloroform (10 mL), washed with water (3 ×
15 mL), dried (magnesium sulfate), filtered and the solvent
removed in vacuo. Diethyl ether was added to the orange–red oil,
the precipitate was filtered and dried under a reduced pressure.
The crude product was purified by flash chromatography
(CHCl3–MeOH 10 : 1), which afforded 4 (10 mg, 71%) as a
yellow solid. Mp: 181–183 ◦C; mmax(KBr)/cm−1 = 3435br, 2924w,
1707m, 1654m, 1584m, 1548s, 1462w, 1354w, 1275w, 1231w,
1202w, 1152w, 1036w, 806w, 748w, 708w, 576w; 1H-NMR
(400 MHz, (CD3)2SO): d = 1.43 (s, 3 H; C(5)CH3), 2.41 (s,
3 H; CarCH3), 2.55 (s, 3 H; CarCH3), 3.28 (s, 3 H; NCH3),
3.44–3.49 (m, 2 H; NCH2aCO, NHCH2aCH2N), 3.60–3.66 (m,
1 H; NHCH2bCH2N), 3.73 (s, 1 H; CH(6)), 4.14 (d, J(NCH2bCO,
NCH2aCO) = 17.2 Hz, 1 H; NCH2bCO), 4.61–4.67 (m, 1 H;
NHCH2CH2aN), 4.75–4.83 (m, 1 H; NHCH2bCH2bN), 7.13–
7.41 (m, 8 H; CHar), 7.60–7.62 (m, 2 H; CHar), 7.79 (s, 1 H;
(6-Methyl-3,5-dioxo-8,8-diphenyl-7-thia-2,4-diaza-bicyclo-
[4.2.0]oct-2-yl)-acetic acid tert-butyl ester 10. Thymine-acetic
acid tert-butyl ester 9 (0.30 g, 1.24 mmol) and thiobenzophenone
(0.25 g, 1.24 mmol) were dissolved in an ultrasonic bath in
acetonitrile (150 mL). The solution was degassed by bubbling
Ar through it for 0.5 h. The solution was subsequently irradiated
at 20 ◦C with a 150 W (TQ-150) medium-pressure mercury
lamp in a Pyrex irradiation apparatus with a cut-off at 300 nm
until the deep blue colour disappeared (45 h). The solvent
was removed in vacuo and the crude mixture was treated with
i-hexane–ethyl acetate (4 : 1). The insoluble part, containing
1 9 4 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 3 7 – 1 9 4 1

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CHar), 7.94 (s, 1 H; CHar), 8.07 (t, J(NH, NCH2) = 6.0 Hz, 1
H; NH), 10.40 (s, 1 H; NH); 13C-NMR (150 MHz, (CD3)2SO):
d = 18.73, 20.81, 22.90, 27.92, 35.47, 42.97, 45.71, 54.08, 61.34,
64.82, 115.82, 126.80, 126.95, 127.08 (2C), 127.75 (2C), 127.87
(2C), 128.02 (2C), 131.17, 131.21, 134.02, 135.87, 136.04,
141.48, 142.46, 146.50, 148.79, 149.66, 154.89, 159.51, 167.72,
169.41; m/z (FAB+): 107 (16%), 137 (66), 154 (100), 289 (14),
307 (32), 460 (3), 664 (4, M + H+); HR-MS (MALDI+): calc.
for C35H34N7O5S (M + H+): 664.2342, found: 664.2320.
11 T. Tamada, K. Kitadokoro, Y. Higuchi, K. Inaka, A. Yasui, P. E. de
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Acknowledgements
We thank the Deutsche Forschungsgemeinschaft, the Fonds
of the German Chemical Industry and the EU Marie Curie
Network programme ClustoxDNA for financial support.
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