Synthesis and stereochemical assignment of DNA spore photoproduct analogues

Article abstract of DOI:10.1002/chem.200600169

Investigation of the DNA repair process performed by the spore photoproduct (SP) lyase repair enzyme is strongly hampered by the lack of defined substrates needed for detailed enzymatic studies. The problem is particularly severe because the repair enzyme belongs to the class of strongly oxygen-sensitive radical (S)-adenosyl-methionine (SAM) enzymes, which are notoriously difficult to handle. We report the synthesis of the spore photoproduct analogues 1a and 1b, which have open backbones and are diastereoisomers. In order to solve the problem of stereochemical assignment, two further derivatives 2a and 2b with closed backbones were prepared. The key step of the synthesis of 2a/b is a metathesis-based macrocyclization that strongly increases the conformational rigidity of the synthetic spore photoproduct derivatives. NOESY experiments of the cyclic isomers furnished a clear cross-peak pattern that allowed the unequivocal assignment of the stereochemistry. The results were transfer red to the data for isomers 1a and 1b, which were subsequently used for enzymatic-repair studies. These studies were performed with the novel spore photoproduct lyase repair enzyme from Geobacillus stearothermophilus. The studies showed an accordance with a recent investigation performed by us with the spore photoproduct lyase from Bacillus subtilis,^[1] in that only the 5 isomer la is recognized and repaired. The ability to prepare a defined functioning substrate now paves the way for detailed enzymatic studies of the SP-lyase lesion recognition and repair process.

Full text of DOI:10.1002/chem.200600169

FULL PAPER
DOI: 10.1002/chem.200600169
Synthesis and Stereochemical Assignment of DNA Spore Photoproduct
Analogues
Marcus G. Friedel,[a] J. Carsten Pieck,[a] Jochen Klages,[b] Christina Dauth,[a]
Horst Kessler,[b] and Thomas Carell*[a]
Abstract: Investigation of the DNA
repair process performed by the spore
photoproduct (SP) lyase repair enzyme
is strongly hampered by the lackof de-
fined substrates needed for detailed en-
zymatic studies. The problem is partic-
ularly severe because the repair
enzyme belongs to the class of strongly
oxygen-sensitive radical (S)-adenosyl-
methionine (SAM) enzymes, which are
notoriously difficult to handle. We
report the synthesis of the spore photo-
product analogues 1a and 1b, which
have open backbones and are diaster-
eoisomers. In order to solve the prob-
lem of stereochemical assignment, two
further derivatives 2a and 2b with
closed backbones were prepared. The
key step of the synthesis of 2a/b is a
metathesis-based macrocyclization that
strongly increases the conformational
rigidity of the synthetic spore photo-
product derivatives. NOESY experi-
ments of the cyclic isomers furnished a
clear cross-peakpattern that allowed
the unequivocal assignment of the ster-
eochemistry. The results were transfer-
red to the data for isomers 1a and 1b,
which were subsequently used for en-
zymatic-repair studies. These studies
were performed with the novel spore
photoproduct lyase repair enzyme from
Geobacillus stearothermophilus. The
studies showed an accordance with a
recent investigation performed by us
with the spore photoproduct lyase from
Bacillus subtilis,[1] in that only the S
isomer 1a is recognized and repaired.
The ability to prepare a defined func-
tioning substrate now paves the way
for detailed enzymatic studies of the
SP-lyase lesion recognition and repair
process.
Keywords: cyclization
·
DNA
damage · enzymes · spore photo-
product · structure elucidation
Introduction
DNA packing, however, adjacent thymidines in the spore
DNA react under exposure to UV light to give a unique
DNA lesion, the spore photoproduct (SP) (Scheme 1).[6,7]
This DNA lesion, which would inhibit spore germination, is
efficiently repaired by the (S)-adenosylmethionine (SAM)-
dependent iron sulfur DNA repair enzyme SP lyase in an
early phase of spore germination.[8–14]
To date, the exact repair mechanism is unknown. Only a
model study was carried out that suggests a plausible mech-
anism.[15] In addition, our knowledge about the exact struc-
Bacteria of the Bacillus and Clostridium species form meta-
bolically dormant endospores in response to nutrient deple-
tion.[2] Spores are resistant to toxic chemicals, heat, and des-
iccation, and can survive over extreme periods of time.[3]
Among other factors, the unusual toroid packing of the ge-
netic material inside the spore[4] is one factor believed to be
responsible for the unusual 50-fold increased resistance of
the spore DNA to 254 nm UV light.[5] Due to the unusual
[a] Dipl.-Chem. M. G. Friedel, Dipl.Ing. J. C. Pieck,
Dipl.-Chem. C. Dauth, Prof. Dr. T. Carell
Department of Chemistry and Biochemistry
Ludwig-Maximilians-University Munich
Butenandtstrasse 5–13, Haus F, 81377 München (Germany)
Fax : (+49)89-218-077756
E-mail: thomas.carell@cup.uni-muenchen.de
[b] Dipl.-Chem. J. Klages, Prof. Dr. H. Kessler
Lehrstuhl für Organische Chemie II
Department für Chemie, Technische Universität München
Lichtenbergstrasse 4, 85747 Garching (Germany)
Scheme 1. Formation and repair (SP lyase) of the spore photoproduct
DNA lesion in UV-irradiated spores. TpT=thymidylyl–(3’–5’)–thymi-
dine.
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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ture of the spore lesion itself and the reasons behind the for-
mation of this photoproduct are only rudimentary. This has
mainly to do with the fact that the lesion is currently not
synthetically available due to the fact that the known syn-
thesis of the dinucleotide spore photoproduct[16] has not de-
livered sufficient yields and therefore strongly limits enzy-
matic studies.[17] In principle, the SP lesion can be formed as
the 5S or 5R stereoisomer in either an intra- or intermolecu-
lar fashion. It was revealed from a sophisticated high-per-
formance liquid chromatography (HPLC)-MS/MS study[18]
that the spore photoproduct is predominantly formed as an
intrastrand lesion (99%) and only to a minor extent as an
interstrand cross-link. Regarding the stereochemistry,
Begley et al.[16] postulated that the natural lesion might be
formed as the 5R isomer. This postulate was based on the
assumption that the DNA in spores is in a more A-like con-
formation. Within an A-type duplex, the structure would
favor formation of the R isomer due to sterical constrains. A
recent cryoelectronic investigation,[4] however, revealed that
the spore DNA exists almost in the B conformation, ques-
tioning the sterical argument.
Results and Discussion
Synthesis of 1a/b and 2a/b: The synthesis of the substrates
1a/b with open backbones was performed as depicted in
Scheme 3.[1] The first steps were the hydrogenation of thymi-
We recently described the use of SP-lesion analogues,
which lackthe central phosphodiester linkage 1a/b, as de-
fined substrates in a novel SP-lyase assay by using the SP-
lyase enzyme from Bacillus subtilis. The enzymatic study
showed clearly that only the S isomer 1a is recognized and
repaired by the repair enzyme (Scheme 2) indicating that
Scheme 3. Synthesis of the substrates with open backbones (1a/b) starting
with thymidine 3. Reagents and conditions: a) Rh/Al2O3, H2, MeOH/
H2O (1:1), RT; b) TESCl, imidazole, DMF, RT, 88%; c) SEMCl, iPr2NEt,
CH2Cl2, RT, 84%; d) TBDMSCl, imidazole, DMF, RT, 95%; e) SEMCl,
iPr2NEt, CH2Cl2, RT, 72%; f) NBS, DBPO, CCl4, 708C, 60%; g) LDA
(1.5 equiv), 5, THF, ꢀ78!08C; h) TBAF, THF, RT; rp-HPLC separation,
8a: 15%, 8b: 11%; i) SnCl4, DCM, 08C, 55% (for 1b: 75%).
dine 3 under atmospheric pressure using Rh/Al2O3 as a cata-
lyst, followed by the protection of the OH groups using
TESCl (TES=triethylsilyl) to give compound 4, and further
protection of the NH group using SEMCl (SEM=trimethyl-
silylethoxymethyl) to give the desired dihydrothymidine
building block 5. A second batch of thymidine 3 was treated
with TBDMSCl (TBDM=tert-butyldimethylsilyl) and
SEMCl to give the TBDMS- and SEM-protected compound
6. The following bromination furnished the allylbromide 7
as the second building blockneeded for the critical coupling
reaction. For the coupling of 5 with 7, we first deprotonated
compound 5 with LDA and allowed the Li enolate to react
with the allylbromide 7. Subsequent deprotection of the OH
groups using tetrabutylammonium fluoride (TBAF) gave
the SEM-protected diastereomers 8a and 8b as a 1:1 mix-
ture. Compounds 8a and 8b were finally separated by re-
versed-phase (rp) HPLC (120 Š, 3 mm, C8). Cleavage of the
SEM protecting groups was possible using SnCl4, which fur-
Scheme 2. Structure of compounds 1a and 1b that were tested in a repair
assay with the spore photoproduct lyase.[1]
the natural lesion might be 5S and not 5R configured.[1]
Herein we report the synthesis of lesion analogues 1a and
1b together with the derivatives with closed backbones, 2a/
b, which allowed the unequivocal assignment of the stereo-
chemistry of both diastereomers by using 2D NMR spectro-
scopy (NOESY). Additionally, we report initial enzymatic-
repair studies with the novel spore photoproduct lyase from
the thermophilic organism Geobacillus stearothermophilus.
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DNA Spore Photoproduct Analogues
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nished the SP-lesion monomers 1a and 1b in an overall
yield of 0.7% for each diastereomer.
5’-protecting group was selectively cleaved with TFA/H2O
to furnish the 3’-TBDMS-thymidine 13. Following protec-
tion with TESCl and SEMCl we obtained the completely
protected thymidine 14 in an overall yield of 41%. Subse-
quent bromination with NBS furnished the allylbromide 12.
Deprotonation of the dihydrothymidine compound 10 with
LDA and coupling of the enolate with the allylbromide 12
afforded the methylene-linked bis-thymidine compound
again in the form of two diastereoisomers (15a+15b). De-
protection with HF·pyridine gave the desired protected SP
analogues 9a/b. The diastereomers 9a und 9b were separat-
ed by using normal-phase (np) HPLC (100 Š, 5 mm) with an
n-heptane/ethyl acetate gradient.
Quantitative NOESY experiments of 1a and 1b to assign
the stereoconfiguration at C5 of the dihydrothymidine unit
gave no significant results due to the high flexibility of the
molecules. Particularly problematic is the ability of the mol-
ecules to freely rotate around the glycosidic bond.
In order to increase the rigidity of the SP isomers we de-
veloped a strategy to perform a macrocyclization (ring-clos-
ing metathesis, RCM) with the TBDMS-protected isomers
9a/b, which were synthesized as shown in Scheme 4. The di-
For the cyclization of the separated isomers 9a and 9b
(Scheme 5), both compounds were first reacted with 4-pen-
tenoylchloride to give the diesters 16a and 16b. The macro-
Scheme 4. Synthesis of the spore photoproduct analogues 9a/b starting
with thymidine 3. Reagents and conditions: a) Rh/Al2O3, H2, MeOH/
H2O (1:1), RT; b) TBDMSCl, imidazole, DMF, RT, 82%; c) TESCl, imi-
dazole, DMF, RT, 89% d) SEMCl, iPr2NEt, CH2Cl2, RT, 86%;
e) TBDMSCl, imidazole, DMF, RT, 95%; f) TFA/H2O (10:1), CH2Cl2,
08C, 81%; g) TESCl, imidazole, DMF, RT, 91%; h) SEMCl, iPr2NEt,
CH2Cl2, RT, 64%; i) NBS, DBPO, CCl4, 708C, 20%; j) LDA (1.5 equiv),
10, THF, ꢀ78!08C; k) HF·pyr (4%), MeCN, ꢀ308C, 34%.
Scheme 5. Synthesis of the cyclic SP isomer 2a (the synthesis of 2b was
performed under the same conditions). Reagents and conditions: a) 4-
pentenoylchloride, iPr2NEt, CH2Cl2, RT, 94% (for 16b: 80%);
b) Grubbs II catalyst, CH2Cl2, 408C, 71% (for 2b: 82%). The HPLC-pu-
rified cyclic isomers 2a and 2b were analyzed by 2D NMR (NOESY) to
assign the stereochemistry at C5 of the dihydrothymidine unit.
cyclization was finally achieved through an RCM reaction[19]
with the Grubbs II catalyst.[20,21] This reaction furnished the
24-membered rings 2a and 2b as E/Z mixtures (2a, 95:5;
2b, 85:15; Scheme 5).[22] The E/Z isomers were separated by
using np-HPLC (100 Š, 5 mm) with an n-hexane/ethyl ace-
tate gradient. Only the major isomers were used for further
investigation. The exact geometry of the double bond of the
major isomers 2a and 2b could not be determined due to
the complex coupling pattern and the lackof resolution of
the double-bond proton signals in the NMR spectra (see the
Supporting Information for the 1H NMR spectra).
hydrothymidine building block 10 was synthesized in four
steps analogously to building block 5. Thymidine 3 was first
hydrogenated and subsequently protected in the 5’-position
with TBDMSCl to give the 5’-TBDMS-dihydrothymidine
11. Protection of the OH(3’) group was performed with
TESCl. Final protection of the ring imide with SEMCl gave
the dihydrothymidine building block 10.
The allylbromide building block 12 was synthesized in
five steps also starting from thymidine 3. First, both OH
groups were protected with TBDMS. In a second step, the
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T. Carell et al.
NMR investigations of 2a and
2b: For the 2D NMR analyses,
1
all H and 13C NMR signals of
2a and 2b were assigned by
using 1H,1H COSY, 1H,13C
COSY (HMQC), and HMBC
experiments. NOESY experi-
ments were performed for the
stereochemical assignment of
the stereo center at C5 of the
dihydrothymidine unit.
In the case of isomer 2a, the
proton in the 6-position of the
pyrimidine ring (B) shows NOE
contacts to H(3’), H(5’), and
H(2’) above the sugar plane.
Therefore, H(6B) is positioned
above the sugar ring and the
base is in the anti conformation
(Figure 1D). The CH2 group in
the 6-position of the dihydro-
thymidine unit (A) shows NOE
contacts to the methyl group
that are almost identical for
both protons (Figure 1A). This
shows that the methyl group is
positioned between the two H
atoms (Ha and Hb). The proton
of this CH2 group with a signal
at d=3.33 ppm (Ha) features a
strong cross peakto the proton
in the 2’-position (d=2.15 ppm
(Hc), Figure 1B) above the
sugar ring (Figure 2A). In addi-
tion, proton Ha shows a strong
NOE contact to the proton,
with a signal at d=2.68 ppm
(He), of the CH2 bridge togeth-
er with a weaker contact to the
proton with
2.28 ppm (Hf, Figure 1C). The
proton He features strong
a signal at d=
a
cross peakto the methyl group
(Figure 2B) and the proton Hf a
strong NOE contact to the
CH(6B) proton (Figure 1D).
All together, this cross-peak
pattern can only be explained
when the CH2 bridge is point-
ing to the front and the methyl
group to the back. This, howev-
er, is only possible when isomer
Figure 1. Depiction of the first set of essential NOESY cross peaks in CDCl3 (600 MHz) for the assignment of
the stereoconfiguration of 2a.
2a is S configured (Figure 3). This assignment is supported
by additional cross peaks between the CH2 group (6A) and
H(1’), which indicate that the H atoms of the CH2 group are
not located above the sugar ring (Figure 2C) excluding
other conformations that would be in agreement with the
observed cross-peakpattern. Hence, we can conclude that
isomer 2a is the S-configured compound.
For isomer 2b, the pyrimidine ring (B) is also in the anti
conformation. The H(6) proton with signal at d=7.41 ppm
possesses NOE contacts to the sugar protons above the ring
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DNA Spore Photoproduct Analogues
FULL PAPER
tacts to the proton in the 2’-po-
sition (Hc) with almost the
same intensity (Figure 4B). So
proton Hc is located in the
middle between the protons of
the CH2 group. Additional
cross peaks to other sugar pro-
tons are not present. Contacts
to the CH2 bridge between the
bases are observed only for
proton Hb (d=3.67 ppm). The
cross peakto the H atom with
a signal at d=2.75 ppm (He) is
slightly larger relative to the
cross peakto the H atom with
a signal at d=2.68 ppm (Hf,
Figure 4B). The distance from
the CH2 group (6A) to the
methyl group is almost identical
for both protons (Figure 4C). In
addition, NOE contacts be-
tween the CH2 bridge and the
methyl group were observed; a
strong one to the signal at d=
2.68 ppm (Hf) and a weaker
one to the signal at d=
2.75 ppm (He, Figure 4C). The
CH(6B) proton shows a strong
cross peakto the CH bridge
2
proton with
2.75 ppm
a
(He,
signal at d=
Figure 4A),
which is the same proton that
the CH2(6A) protons showed a
cross peakto (H b, Figure 4B),
and a weaker cross peak to the
proton with
a signal at d=
2.68 ppm (Hf), which shows a
strong signal to the methyl
group (Figure 4C).
Figure 2. Depiction of the second set of essential NOESY cross peaks in CDCl3 (600 MHz) for the assignment
of the stereoconfiguration of 2a.
In principle, the observed
NOESY signal pattern for 2b could be explained by both
the 5S and 5R stereochemistry. But, in the case of the S con-
figuration, large sterical interactions between the 5’-TBDMS
group and the backbone as well as the bases would have to
be accepted, which is unlikely (Figure 5, right). In the case
of the R configuration, sterical repulsion is negligible
(Figure 5, left). Consequently, we assign the R configuration
to isomer 2b.
In order to correlate the results of the stereochemical as-
signment of compounds 2a/b to the fully deprotected SP iso-
mers 1a/b, small amounts of 9a and 9b were deprotected by
using TBAF to generate the SEM-protected diastereomers
8a and 8b as depicted in Scheme 6. These isomers were also
isolated during the synthesis of 1a/b and separated by using
rp-HPLC (see above and ref. [1]).
Figure 3. Three-dimensional depiction of 2a in the S configuration show-
ing the observed strong NOE contacts. The SEM and TBDMS protecting
groups were exchanged for H atoms for clarity.
plane (H(5’), H(2’)/Hc, and H(3’); Figure 4A). The two pro-
tons of the CH2 group (d=3.14 (Ha) and 3.67 ppm (Hb)) at
the 6-position of the dihydrothymidine unit show NOE con-
A comparison of the HPL chromatograms from 8a and
8b derived either from 9a and 9b or from the synthesis of
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T. Carell et al.
mers 8a and 8b, samples corre-
sponding to the HPLC peaks
were collected and the mass
was determined with MALDI-
TOF analysis (see the Support-
ing Information).
Enzymatic studies: With the un-
equivocally assigned diaster-
eomers 1a and 1b as defined
substrates in hand, we per-
formed enzymatic studies with
the purified and reconstituted
spore
photoproduct
lyase
from G. stearothermophilus
(splG).[8,11,23–25] We performed
enzymatic assays under differ-
ent conditions and analyzed
them by using rp-HPLC follow-
ing
method.[1] The enzyme contains
reduced [4Fe+4S]+ cluster
a
previously described
a
that is believed to transfer an
electron to the SAM cofactor,
resulting in the formation of
methionine (Met) and the
highly reactive 5’-deoxyadeno-
sine radical (Scheme 7). This
unstable radical is highly reac-
tive and forms 5’-deoxyadeno-
sine (5’AdoH), which can be
easily detected at l=260 nm by
using HPLC.
This was tested in the first
assay solution containing only
SAM and splG. As shown in
Figure 6A, we could observe
the formation of 5’AdoH. The
production of 5’AdoH by the
holo-splG is 1.2 molecules of 5’-
deoxyadenosine per molecule
of enzyme. In a second batch
we added the R isomer 1b to
the assay solution and observed
only the peaks for 5’AdoH and
compound 1b (Figure 6B). In a
third batch we added the S
isomer 1a. Only here we ob-
served the formation of a new
peakat 12.9 min, which corre-
sponds to the desired repair
product, thymidine (Figure 6C).
The thymidine formation is
Figure 4. Depiction of the set of essential NOESY cross peaks in MeOH (600 MHz) for the assignment of the
stereoconfiguration of 2b.
Figure 5. Three-dimensional depiction of 2b in the R (left) and S (right) configuration showing the observed
strong NOE contacts. The SEM and TBDMS protecting groups were exchanged for H atoms for clarity.
1a and 1b shows that the order of appearance stays the
same (see the Supporting Information). Therefore, 1a must
be the S isomer and 1b the R isomer of the fully deprotect-
ed SP isomers. To ensure the identity of the deprotected iso-
time dependent and the calculated reaction yield for the
splG-driven repair of the S-isomer substrate is 0.2 mol prod-
uct formation per 1 mol splG per hour. In a control experi-
ment containing only compound 1a and SAM and no splG
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DNA Spore Photoproduct Analogues
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Conclusion
In summary, we were able to synthesize and separate the di-
astereomers with open backbones, 1a and 1b, which func-
tion as substrates for the DNA repair enzyme SP lyase from
spores. The stereochemical assignment of the synthetic SP
isomers 1a and 1b was possible by using the cyclic SP iso-
mers 2a and 2b. The key step of the synthesis of 2a/b was a
macrocyclization to increase the rigidity of the spore photo-
product derivatives. In NOESY experiments of the cyclic
isomers we obtained cross-peakpatterns that allowed the as-
signment of the stereochemistry for both diastereomers 2a
and 2b. We were able to correlate these results to isomers
1a and 1b to assign the stereochemistry of the lesion ana-
logues. Additionally, enzymatic DNA repair studies using 1a
and 1b as defined substrates and the SP lyase from G. stearo-
thermophilus were performed. These studies showed, as in
the case of our recently published repair study with the
spore photoproduct lyase from B. subtilis,[1] that only the S
isomer 1a is recognized and repaired by splG. These syn-
thetic stereochemically defined
Scheme 6. Deprotection of isomers 9a and 9b. Reagents and conditions:
a) TBAF, THF, RT.
and pure spore photoproduct
derivates pave the way for de-
tailed enzymatic studies of the
SP-lyase lesion recognition and
repair process.
Experimental Section
General information: All solvents
were of the quality puriss. p.a., or
purum. Purum solvents were distilled
prior to use. The commercially availa-
ble reagents were used as received
without further purification. Analytical
thin-layer chromatography (TLC) was
carried out using aluminum-based
plates (Kieselgel 60 F254) from Merck.
Plates were visualized under UV light
(l=254 nm) or by staining them with
anisaldeyhde solution. Flash chroma-
tography was carried out by using
MerckKieselgel 60 (0.040–0.063 mm)
with N2 overpressure. Samples were
applied as saturated solutions in an ap-
propriate solvent. Melting points are
Scheme 7. Postulated repair mechanism of the spore photoproduct lyase.[8,15]
1
uncorrected. H NMR spectra were re-
enzyme, we neither observed the formation of 5’AdoH nor
the formation of thymidine (Figure 6D) showing that their
formation is strictly enzyme dependent.
corded on Bruker DRX 200 (200 MHz), AMX 300 (300 MHz), ARX 300
(300 MHz), AMX 400 (400 MHz), AMX 500 (500 MHz), AMX 600
(600 MHz), Varian Oxford 200 (200 MHz), and Varian XL 400
(400 MHz) spectrometers. The chemical shifts were referenced to DMSO
(d=2.50 ppm) in [D6]DMSO, CH3OH (d=3.31 ppm) in CD3OD, and
CHCl3 (d=7.26 ppm) in CDCl3. 13C NMR spectra were recorded on
Bruker ARX 200 (50 MHz), AMX 300 (75 MHz), ARX 300 (75 MHz),
AMX 500 (125 MHz), AMX 600 (150 MHz), and Varian XL 400
(100 MHz) spectrometers. The chemical shifts were referenced to DMSO
(d=39.43 ppm) in [D6]DMSO, CH3OH (d=49.05 ppm) in CD3OD, and
CHCl3 (d=77.00 ppm) in CDCl3. Standard pulse sequences were em-
ployed for 1H,1H and 1H,13C NMR correlation studies. IR spectra of
solids were recorded in KBr, and of liquids as thin films. IR spectra were
measured with Bruker IFS 88 and Perkin–Elmer FTIR spectrum 100 in-
The formation of 5’AdoH and thymidine was confirmed
by coinjection (HPLC) and mass spectrometry. In these en-
zymatic studies we observed, as in the case of the recently
published study with the spore photoproduct lyase from Ba-
cillus subtilis,[1] that only the S isomer 1a is accepted as a
substrate and repaired by the enzyme. These investigations,
performed with the SPL enzyme from a different organism,
therefore support the idea that the spore repair enzymes
can only repair the 5S isomers of the spore photoproduct.
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T. Carell et al.
Celite and the solvent was removed
under reduced pressure. The crude
product was azeotropically dried three
times with pyridine and dissolved in
anhydrous DMF (20 mL). Imidazole
(3.00 g, 44.1 mmol)
and
TESCl
(4.16 mL, 24.8 mmol) were added and
the reaction mixture was stirred over-
night at RT. The reaction mixture was
diluted with CHCl3 (30 mL), washed
with saturated aqueous sodium bicar-
bonate (3”40 mL), dried (MgSO4),
and the solvent was removed in vacuo.
Purification by using flash chromatog-
raphy (silica gel, pentane/ethyl acetate
7:3) provided
(3.45 g, 88%).
4 as a white solid
Rf =0.35 (pentane/ethyl acetate 7:3);
1H NMR (300 MHz, [D6]DMSO): d=
0.52–0.62 (m, 12H; 2”Si
0.87–0.96 (m, 18H; 2”Si
ACHTREUNG
AHCTREUNG
1.05 (d, J=6.6 Hz, 3H; C(5)CH3),
1.73–1.82 (m, 1H; CH2(2’)), 2.11–2.22
(m, 1H; CH2(2’)), 2.55–2.64 (m, 1H;
CH(5)), 2.91–2.99 (m, 1H; CH2(6)),
3.35–3.40 (m, 1H; CH2(6)), 3.55–3.68
(m, 3H; CH(4’), CH2(5’)), 4.22–4.33
(m, 1H; CH(3’)), 6.11 (t, J=6.6 Hz,
1H; CH(1’)), 10.23 ppm (s, 1H; NH);
13C NMR (75 MHz, [D6]DMSO): d=
Figure 6. HPL chromatograms of enzymatic studies. The enzyme reaction and rp-HPLC analyses were per-
formed as described in the Experimental Section. A) HPLC analysis of the assay containing 5 nmol splG and
SAM. B) HPLC analysis of the assay containing 5 nmol splG, SAM, and the (R)-1b. C) HPLC analysis of the
assay containing 5 nmol splG, SAM, and (S)-1a. Only in this case an additional peakwas observed, which was
identified as the desired repair product thymidine. D) HPLC analysis of the negative control containing SAM
and (S)-1a but no splG. In all four chromatograms the y axis shows the intensity of the absorbance at l=
260 nm.
4.0 (Si
6.8 (2”Si
34.8 (CH(5)), 36.5 (CH2(2’)), 41.5
(CH2(6)), 62.7 (CH2(5’)), 72.1
A
N
AHCTREUNG
(CH(3’)), 83.1 (CH(1’)), 85.6 (CH(4’)),
153.2 (CO), 173.3 ppm (CO); MS
struments. Mass spectra and high-resolution mass spectra were measured
(FAB+): m/z (%): 495 (20) [M+Na]+, 443 (13), 413 (6), 311 (10), 213
on Finnegan TSQ 7000, Finnegan MAT 95S, Finnegan MAT 95Q, Finne-
gan MAT 90 (FAB), Finnegan LTQ-FT, PE Sciex Q-Star Pulsar i, and
Bruker Autoflex II (MALDI-TOF) instruments. Analytical HPLC was
performed with Merck–Hitachi systems equipped either with L-7400 UV
and L-7480 fluorescence detectors or with L7420 UV/Vis and L-7455
DAD detectors. Preparative HPLC was performed with a Merck–Hitachi
system equipped with a L-7480 UV detector. Analytical separations were
performed with Macherey–Nagel Nucleosil 120-3 C8, Nucleosil 100-5,
and Nucleosil 120-3 columns. Preparative separations were performed
with Macherey–Nagel Nucleosil 120-3 C8 and Nucleodur 100-5 columns.
(18), 145 (40), 115 (93), 87 (100), 59 (36).
N3-Trimethylsilylethoxymethyl-3’,5’-O-di(triethylsilyl)-5,6-dihydrothymi-
dine (5):[16]
A solution containing 4 (3.64 g, 7.70 mmol), iPr2NEt
(5.27 mL, 30.8 mmol), and SEMCl (2.04 mL, 11.6 mmol) in anhydrous
CH2Cl2 (20 mL) was stirred for 4 d. After 24 h additional iPr2NEt
(5.27 mL, 30.8 mmol) and SEMCl (2.04 mL, 11.6 mmol) were added. The
reaction mixture was diluted with CHCl3 (30 mL), washed with saturated
aqueous sodium bicarbonate (3”50 mL), dried (MgSO4) and the solvent
was removed in vacuo. Flash chromatography (silica gel, pentane/ethyl
acetate 10:1) gave 5 as a colorless oil (3.89 g, 84%).
NMR measurements: All NMR spectra for the stereochemical analysis
Rf =0.43 (pentane/ethyl acetate 9:1); 1H NMR (300 MHz, [D6]DMSO):
were recorded at 298 K on
a 600 MHz Bruker DMX spectrometer
d=ꢀ0.04 (s, 9H; Si
C
ACHTREUNG
(Bruker, Karlsruhe, Germany) equipped with a quadrupole resonance
probe head with actively shielded x, y, and z gradients. The experiments
were carried out on a 18 mm sample of 2a (9 mg dissolved in 500 mL
CDCl3) and a 16 mm sample of 2b (8 mg dissolved in 500 mL CD3OD).
The spectra were processed by using XWINNMR (Bruker) and analyzed
with either XWINNMR or SPARKY software.[26] Resonance assignments
were obtained from standard 1H TOCSY,[27,28] 1H COSY,[29,30] 13C,1H
HSQC,[31–33] and 13C,1H HMBC[34] spectra. Stereospecific assignments of
the prochiral methylene groups were derived from NOESY spectra,
which were acquired with a mixing time of 150 ms and suppression of
zero quantum artifacts.[35, 36] The spectra were recorded with 32 scans per
increment. The spectral width was 6009 Hz in both the direct and the in-
direct dimension, sampled with 2048 and 512 complex points, respective-
ly. All dimensions were apodized with a p/2-shifted square sine-bell func-
tion zero filling to provide a processed spectrum of 4096”1024 complex
points.
AHCTREUNG
(d, J=7.0 Hz, 3H; C(5)CH3), 1.81–1.88 (m, 1H; CH2a(2’)), 2.15–2.24 (m,
1H; CH2b(2’)), 2.72–2.79 (m, 1H; CH(5)), 2.99 (dd, J=12.3, J=10.3 Hz,
1H; CH2a(6)), 3.41 (dd, J=12.9, J=5.6 Hz, 1H; CH2b(6)), 3.50 (t, J=
8.0 Hz, 2H; SiCH2CH2O), 3.60–3.62 (m, 2H; CH2(5’)), 3.66–3.68 (m, 1H;
CH(4’)), 4.30–4.32 (m, 1H; CH(3’)), 5.05 (s, 2H; OCH2N), 6.17 ppm (t,
J=7.3 Hz, 1H; CH(1’)); 13C NMR (75 MHz, [D6]DMSO): d=ꢀ1.2 (Si-
A
(CH2CH3)3), 4.3 (Si
A
G
13.1 (C(5)CH3), 17.6 (OCH2CH2Si), 35.1 (CH(5)), 36.7 (CH2(2’)), 40.4
(CH2(6)), 62.6 (CH2(5’)), 65.8 (OCH2CH2Si), 69.3 (NCH2O), 72.0
(CH(3’)), 84.0 (CH(1’)), 85.8 (CH(4’)), 152.9 (CO), 172.6 ppm (CO); MS
(FAB+): m/z (%): 625 (8) [M+Na+], 603 (3) [M+H+], 573 (5), 427 (5),
213 (13), 145 (48), 116 (100), 87 (71), 73 (53), 59 (23), 44 (11).
3’,5’-O-Di(tert-butyldimethylsilyl)thymidine (17):[38,39] Thymidine
3
(2.00 g, 8.26 mmol) was dissolved in anhydrous DMF. Imidazole (3.38 g,
49.6 mmol) and TBDMSCl (3.74 g, 24.8 mmol) were added and the solu-
tion was stirred overnight at RT. The reaction mixture was diluted with
CHCl3 (200 mL), washed with water (3”200 mL), dried (MgSO4), and
the solvent was removed in vacuo. Purification by using flash chromatog-
3’,5’-O-Di(triethylsilyl)-5,6-dihydrothymidine (4):[16,37] Thymidine
3
(2.00 g, 8.26 mmol) was dissolved in MeOH/water (50 mL, 1:1) and Rh/
Al2O3 (100 mg, 5% Rh) was added. The suspension was stirred under an
H2 atmosphere at RT for 3 d. The reaction mixture was filtered through
6088
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 6081 – 6094

DNA Spore Photoproduct Analogues
FULL PAPER
raphy (silica gel, pentane/ethyl acetate 1:1) provided 17 as a white foam
(3.70 g, 95%).
ous phase was extracted with CHCl3 (3”20 mL). The collected extracts
were dried (MgSO4) and the solvent was removed in vacuo.
1
Rf =0.30 (CHCl3/MeOH 20:1); H NMR (300 MHz, [D6]DMSO): d=0.06
For deprotection of the OH groups, a TBAF solution was prepared by
dissolving TBAF (2.33 g, 7.40 mmol) in anhydrous THF (10 mL) and
adding molecular sieves (4 Š). The solution was stirred at RT for 1.5 h to
remove traces of water. The crude mixture of the coupling was dissolved
in anhydrous THF (20 mL) and added to the TBAF solution. The reac-
tion mixture was stirred at RT for 1.5 h until all starting material had re-
acted. The mixture was filtered, diluted with CHCl3 (100 mL), washed
with aqueous sodium bicarbonate (150 mL), and the aqueous phase was
extracted with CHCl3 (3”100 mL). The collected organic phases were
dried (MgSO4) and the solvent was removed in vacuo. An initial purifica-
tion was achieved by using flash chromatography (silica gel, CHCl3/
MeOH 10:1) to give 8a/b as a mixture of diastereomers (328 mg, 60%).
(s, 6H; Si
(CH3)2), 0.07 (s, 6H; Si
9H; C(CH3)3), 1.76 (s, 3H; C(5)CH3), 2.00–2.09 (m, 1H; CH2a(2’)), 2.12–
G
N
ACHTREUNG
2.22 (m, 1H; CH2b(2’)), 3.67–3.74 (m, 2H; CH2(5’)), 3.75–3.79 (m, 1H;
CH(4’)), 4.32–4.38 (m, 1H; CH(3’)), 6.14 (t, J=6.3 Hz, 1H; CH(1’)), 7.41
(s, 1H; CH(6)), 11.33 ppm (s, 1H; NH); 13C NMR (75 MHz,
[D6]DMSO): d=ꢀ5.3 (SiCH3), ꢀ5.3 (SiCH3), ꢀ4.8 (SiCH3), ꢀ4.6
(SiCH3), 12.4 (C(5)CH3), 17.9 (C
A
G
N
25.9 (C(CH3)3), 39.3 (CH2(2’)), 62.9 (CH2(5’)), 72.2 (CH(3’)), 83.8
R
(CH(1’)), 86.8 (CH(4’)), 109.7 (C(5)), 135.7 (CH(6)), 150.6 (CO),
163.8 ppm (CO); MS (FAB+): m/z (%): 493 (35) [M+Na+], 471 (17)
[M+H+], 281 (10), 213 (13), 145 (46), 127 (15), 115 (16), 89 (40), 73
(100).
N3-Trimethylsilylethoxymethyl-3’,5’-O-di(tert-butydimethylsilyl)thymidine
(6):[16] SEM protection was carried out as described above for 5. Product
6 was isolated as a colorless oil (3.34 g, 72%).
The diastereomers were separated by using rp-HPLC using a 250/10 Nu-
cleosil 120-3 C8-column (Macherey–Nagel) with a water/acetonitrile gra-
dient (0!6% acetonitrile in 20 min, 6!9% acetonitrile in 30 min, 9!
100% acetonitrile in 5 min; flow rate: 3 mLminꢀ1; detection wavelength:
250 nm). The mixture was dissolved in water/acetonitrile (30 mL, 1:1)
and for each separation 1 mL of the solution was injected through a
Rheodyne valve on the column. After HPLC purification, the S isomer
8a (82.0 mg, 15%) and the R isomer 8b (58.0 mg, 11%) were isolated as
colorless oils.
Rf =0.33 (pentane/ethyl acetate 9:1); 1H NMR (500 MHz, [D6]DMSO):
d=ꢀ0.04 (s, 9H; Si
(CH3)2), 0.81–0.85 (m, 2H; SiCH2CH2O), 0.88 (s, 9H; C
9H; C(CH3)3), 1.84 (s, 3H; C(5)CH3), 2.12 (ddd, J=13.3, J=6.3, J=
(CH3)3), 0.08 (s, 6H; Si
ACHTREUNG
A
ACHTREUNG
ACHTREUNG
3.4 Hz, 1H; CH2a(2’)), 2.17–2.22 (m, 1H; CH2b(2’)), 3.57 (t, J=8.0 Hz,
2H; SiCH2CH2O), 3.72 (dd, J=11.4, J=3.8 Hz, 1H; CH2a(5’)), 3.78 (dd,
J=11.5, J=4.2 Hz, 1H; CH2b(5’)), 3.81–3.85 (m, 1H; CH(4’)), 4.35–4.40
(m, 1H; CH(3’)), 5.22 (s, 2H; OCH2N), 6.18 (t, J=6.5 Hz, 1H; CH(1’)),
7.51 ppm (s, 1H; CH(6)); 13C NMR (125 MHz, [D6]DMSO): d=ꢀ5.4 (2”
Isomer (S)-8a: Rf =0.30 (CHCl3/MeOH 10:1); 1H NMR (500 MHz,
[D6]DMSO): d=ꢀ0.06 (s, 9H; Si
0.84 (m, 4H; 2”SiCH2CH2O), 1.03 (s, 3H; C(5A)CH3), 1.81 (ddd, J=
13.1, J=6.0, J=2.5 Hz, 1H; CH2a(2’A)), 1.96–2.08 (m, 2H; CH2b(2’A),
CH2a(2’B)), 2.15 (ddd, J=13.1, J=6.0, J=3.3 Hz, 1H; CH2b(2’B)), 2.32
A
G
A
ACHTREUNG
SiCH3), ꢀ4.8 (SiCH3), ꢀ4.7 (SiCH3), ꢀ1.3 (Si
17.6 (SiCH2CH2O), 17.8 (C(CH3)3), 18.1 (C
(C(CH3)3), 39.6 (CH2(2’)), 62.7 (CH2(5’)), 66.5 (SiCH2CH2O), 69.8
ACHTREUNG
A
ACHTREUNG
A
N
ACHTREUNG
(d, J=13.9 Hz, 1H; C(5B)CH2a), 2.78 (d, J=13.7 Hz, 1H; C(5B)CH2b),
3.14 (d, J=13.3 Hz, 1H; CH2a(6A)), 3.19 (d, J=13.3 Hz, 1H; CH2b(6A)),
ACHTREUNG
(OCH2N), 72.1 (CH(3’)), 85.1 (CH(1’)), 87.1 (CH(4’)), 108.9 (C(5)), 135.1
(CH(6)), 150.6 (CO), 162.8 ppm (CO); MS (FAB+): m/z (%): 601 (17)
[M+H+], 543 (15), 229 (34), 213 (30), 199 (47), 145 (100), 115 (21).
3.38–3.41 (m, 3H; CH2
3.50–3.55 (m, 2H; SiCH2CH2O(B)), 3.56–3.59 (m, 2H; CH2
3.64 (m, 1H; CH(4’A)), 3.79–3.83 (m, 1H; CH(4’B)), 4.11–4.15 (m, 1H;
CH(3’A)), 4.23–4.27 (m, 1H; CH(3’B)), 4.96 (d, J=9.6 Hz, 1H;
OCH2aN(A)), 5.04 (d, J=9.9 Hz, 1H; OCH2bN(A)), 5.15 (d, J=9.9 Hz,
1H; OCH2aN(B)), 5.17 (d, J=9.9 Hz, 1H; OCH2bN(B)), 6.15–6.22 (m,
2H; 2”CH(1’)), 7.84 ppm (s, 1H; CH(6B)); 13C NMR (125 MHz,
ACHTREUNG
AHCTREUNG
G
ACHTREUNG
N3-Trimethylsilylethoxymethyl-3’,5’-O-di(tert-butydimethylsilyl)-5-bromo-
E
ACHTREUNG
methyl-2’-deoxyuridine (7):[40] The protected thymidine
6
(1.00 g,
1.66 mmol), NBS (621 mg, 3.49 mmol), and benzoyl peroxide (12.0 mg,
0.05 mmol) were dissolved in carbon tetrachloride (20 mL). The reaction
was heated at 708C for 1 h. The reaction was allowed to cool and was fil-
tered through a sintered funnel. The solvent was removed in vacuo to
yield a crude yellow oil. Carrying out quickflash chromatography (silica
gel, pentane/ethyl acetate 9:1) gave 7 as a colorless oil (683 mg, 60%),
which decomposed at room temperature but was stable at ꢀ208C.
[D6]DMSO): d=ꢀ1.2 (2”Si
(SiCH2CH2O), 20.0 (C(5A)CH3), 31.7 (C(5B)CH2), 36.2 (CH2
(CH2(2’B)), 41.4 (C(5A)), 44.4 (CH2(6A)), 61.4 (CH2(5’B)), 62.0 (CH2-
(5’A)), 65.7 (SiCH2CH2O(A)), 66.5 (SiCH2CH2O(B)), 69.7 (OCH2N(A)),
70.0 (OCH2N(B)), 70.6 (CH(3’B)), 70.9 (CH(3’A)), 84.0 (CH(1’A)), 85.3
(CH(1’B)), 86.3 (CH(4’A)), 87.8 (CH(4’B)), 107.6 (C(5B)), 138.6
ACHTREUNG
AHCTREUNG
A
ACHTREUNG
AHCTREUNG
A
N
ACHTREUNG
Rf =0.33 (pentane/ethyl acetate 9:1); 1H NMR (300 MHz, CDCl3): d=
A
N
ACHTREUNG
(CH(6B)), 150.4 (CO(2B)), 152.4 (CO(2A)), 163.0 (CO(4B)), 173.5 ppm
(CO(4A)); IR (film): n˜max =3400s, 2952s, 1710s, 1666s, 1465s, 1394w,
1365w, 1338w, 1279m, 1247m, 1199m, 1091s, 998s, 917w, 860s, 837s,
766m, 694w, 619w cmꢀ1; MS (MALDI+): m/z (%): 783 (17) [M+K+],
767 (86) [M+Na+], 306 (27), 284 (100), 215 (17), 175 (12); HRMS
(ESI+): m/z calcd for C32H56N4NaO12Si2: 767.3331; found: 767.3296
[M+Na+].
0.00 (s, 9H; Si
A
G
U
(s, 9H; C(CH3)3), 0.91 (s, 2H; SiCH2CH2O), 0.94 (s, 9H; C
A
ACHTREUNG
2.04 (m, 1H; CH2a(2’)), 2.34 (ddd, J=8.6, J=5.6, J=2.7 Hz, 1H;
CH2b(2’)), 3.64–3.72 (m, 2H; SiCH2CH2O), 3.77 (dd, J=11.3, J=2.6 Hz,
1H; CH2a(5’)), 3.89 (dd, J=11.6, J=2.6 Hz, 1H; CH2b(5’)), 4.27 (d, J=
10.3 Hz, 1H; CH2aBr), 4.30 (d, J=10.3 Hz, 1H; CH2bBr), 4.35–4.41 (m,
1H; CH(3’)), 5.41 (s, 2H; OCH2N), 6.30 (dd, J=7.6, J=5.6 Hz, 1H;
CH(1’)), 7.88 ppm (s, 1H; CH(6)); 13C NMR (75 MHz, CDCl3): d=ꢀ5.4
Isomer (R)-8b: Rf =0.30 (CHCl3/MeOH 10:1); 1H NMR (500 MHz,
(2”SiCH3), ꢀ4.9 (SiCH3), ꢀ4.7 (SiCH3), ꢀ1.5 (Si
A
[D6]DMSO): d=ꢀ0.06 (s, 9H; Si
0.85 (m, 4H; 2”SiCH2CH2O), 1.02 (s, 3H; C(5A)CH3), 1.77–1.84 (m,
1H; CH2a(2’A)), 1.96–2.04 (m, 1H; CH2b(2’A)), 2.07–2.14 (m, 2H; CH2-
(2’B)), 2.19 (d, J=14.2 Hz, 1H; C(5B)CH2a), 2.83 (d, J=13.9 Hz, 1H;
C(5B)CH2b), 3.11 (d, J=13.2 Hz, 1H; CH2a(6A)), 3.31 (d, J=13.1 Hz,
1H; CH2b(6A)), 3.41–3.48 (m, 4H; CH2(5’A), SiCH2CH2O(A)), 3.49–3.61
(m, 4H; CH2(5’B), SiCH2CH2O(B)), 3.63–3.68 (m, 1H; CH(4’A)), 3.77–
3.82 (m, 1H; CH(4’B)), 4.11–4.16 (m, 1H; CH(3’A)), 4.22–4.27 (m, 1H;
CH(3’B)), 4.75 (t, J=5.4 Hz, 1H; OH(5’A)), 4.92 (d, J=9.8 Hz, 1H;
OCH2aN(A)), 4.98 (t, J=5.0 Hz, 1H; OH(5’B)), 5.07 (d, J=9.9 Hz, 1H;
OCH2bN(A)), 5.10–5.19 (m, 3H; OH(3’A), OCH2N(B)), 5.26 (d, J=
A
G
18.1 (SiCH2CH2O), 18.4 (C(CH3)3), 25.7 (C(CH3)3), 26.0 (C
G
ACHTREUNG
(CH2Br), 41.9 (CH2(2’)), 63.0 (CH2(5’)), 67.7 (SiCH2CH2O), 70.2
(OCH2N), 72.2 (CH(3’)), 86.2 (CH(1’)), 88.2 (CH(4’)), 110.9 (C(5)), 137.8
(CH(6)), 150.4 (CO), 161.3 ppm (CO); MS (FAB+): m/z (%): 751 (25),
679 (8) [M+H+], 653 (13), 623 (18), 599 (13), 543 (6), 287 (10), 213 (23),
145 (100).
A
ACHTREUNG
AHCTREUNG
AHCTREUNG
A
ACHTREUNG
N3
(A/B)-Di(trimethylsilylethoxymethyl)-5-(a-thymidyl)-5,6-dihydrothy-
A
A
ACHTREUNG
A
ACHTREUNG
midine (8a/b): Dihydrothymidine 5 (446 mg, 0.74 mmol), azeotropically
dried with anhydrous toluene, was dissolved in anhydrous THF (4 mL)
and cooled to ꢀ788C. A freshly prepared LDA solution (diisopropyl-
amine (156 mL, 1.11 mmol), BuLi (0.70 mL, 1.60m in hexane) in anhy-
drous THF (2 mL) left at 08C for 1 h) was slowly added and the reaction
mixture was stirred at ꢀ788C for 2 h before the addition of 7 (500 mg,
0.74 mmol) dissolved in anhydrous THF (6 mL). The reaction mixture
was stirred at ꢀ788C for 1.5 h and at 08C for 1.5 h. The reaction was
quenched by adding aqueous sodium bicarbonate (12 mL) and the aque-
AHCTREUNG
AHCTREUNG
4.1 Hz, 1H; OH(3’B)), 6.17–6.20 (m, 2H; 2”CH(1’)), 7.73 ppm (s, 1H;
N
CH(6B)); 13C NMR (125 MHz, [D6]DMSO): d=ꢀ1.2 (2”Si
C
36.1 (CH
(CH2(5’A)), 65.6 (SiCH2CH2O(A)), 66.5 (SiCH2CH2O(B)), 69.6
(OCH2N(A)), 70.0 (OCH2N(B)), 70.6 (CH(3’B)), 71.0 (CH(3’A)), 83.9
A
N
AHCTREUNG
A
ACHTREUNG
Chem. Eur. J. 2006, 12, 6081 – 6094
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6089

T. Carell et al.
(CH
A
(1’B)), 86.4 (CH
U
(4’B)), 107.9
Al2O3 (100 mg, 5% Rh) was added. The suspension was stirred under an
H2 atmosphere at RT for 2 d. The reaction mixture was filtered through
Celite and the solvent was removed under reduced pressure. The crude
product was azeotropically dried three times with pyridine and dissolved
in anhydrous DMF (11 mL). Imidazole (1.54 g, 22.7 mmol) and
TBDMSCl (1.71 g, 11.3 mmol) were added and the reaction mixture was
stirred for 1 h at RT. The reaction mixture was diluted with CHCl3
(50 mL), washed with saturated aqueous sodium bicarbonate (7”
100 mL), dried (MgSO4), and the solvent was removed in vacuo. Purifica-
tion by using flash chromatography (silica gel, CHCl3/MeOH 20:1) pro-
vided 11 as a white foam (3.01 g, 82%).
(C(5B)), 138.5 (CH(6B)), 150.5 (CO(2B)), 152.1 (CO(2A)), 162.9
(CO(4B)), 173.3 ppm (CO(4A)); IR (film): n˜max =3442s, 2952s, 1653s,
1465m, 1364w, 1278w, 1247w, 1200w, 1091m, 916w, 860m, 836m, 760m,
694w, 667w cmꢀ1; MS (MALDI+): m/z (%): 783 (3) [M+K+], 767 (29)
[M+Na+], 306 (30), 253 (6), 242 (100), 197 (5); HRMS (ESI+): m/z calcd
for C32H56N4NaO12Si2: 767.3331; found: 767.3343 [M+Na]+.
(5R)-(a-Thymidyl)-5,6-dihydrothymidine (1b): Isomer (R)-8b (59.0 mg,
0.08 mmol) was dissolved in anhydrous CH2Cl2 (7 mL) and the solution
was cooled to 08C. A solution of SnCl4 (0.80 mL, 1m in CH2Cl2) was
added dropwise. The reaction mixture was stirred at 08C for 1 h. The re-
action was quenched by adding methanolic sodium hydroxide (3 mL, 4%
NaOH). The solvent was removed under reduced pressure. The crude
product was suspended in methanol (4 mL), and centrifuged and deca-
nted to remove the tin salts. This procedure was repeated twice. The sol-
vent was removed from the collected solutions in vacuo. Purification by
using flash chromatography (silica gel, CHCl3/MeOH 4:1) provided 1b as
a white solid (27.0 mg, 75%).
1
Rf =0.20 (CHCl3/MeOH 20:1); H NMR (400 MHz, [D6]DMSO): d=0.04
(s, 3H; Si
A
G
N
J=7.2 Hz; 3H, CH3), 1.79–1.85 (m, 1H; CH2a(2’)), 2.03–2.10 (m, 1H;
CH2b(2’)), 2.53–2.64 (m, 1H; CH(5)), 2.99 (dd, J=12.4, J=10.0 Hz, 1H;
CH2a(6)), 3.38 (dd, J=12.8, J=5.6 Hz, 1H; CH2b(6)), 3.60–3.67 (m, 3H;
CH(4’), CH2(5’)), 4.10–4.11 (m, 1H; CH(3’)), 5.13–5.14 (m, 1H; OH),
6.11–6.14 (m, 1H; CH(1’)), 10.19 ppm (s, 1H; NH); 13C NMR (100 MHz,
Rf =0.10 (CHCl3/MeOH 4:1); m.p. 155–1588C; 1H NMR (500 MHz,
[D6]DMSO): d=0.98 (s, 3H; C(5A)CH3), 1.77 (ddd, J=12.8, J=6.2, J=
[D6]DMSO): d=ꢀ5.5 (SiCH3), ꢀ5.5 (SiCH3), 12.6 (CH3), 17.9 (C
C
AHCTREUNG
2.5 Hz, 1H; CH2a
2H; CH2(2’B)), 2.25 (d, J=14.2 Hz, 1H; C(5B)CH2a), 2.73 (d, J=
13.3 Hz, 1H; C(5B)CH2b), 3.05 (d, J=13.1 Hz, 1H; CH2a(6A)), 3.23 (d,
J=13.1 Hz, 1H; CH2b(6A)), 3.41 (t, J=5.3 Hz, 2H; CH2(5’A)), 3.51–3.57
(m, 2H; CH2(5’B)), 3.59–3.63 (m, 1H; CH(4’A)), 3.74–3.79 (m, 1H; CH-
(4’B)), 4.07–4.12 (m, 1H; CH(3’A)), 4.21–4.27 (m, 1H; CH(3’B)), 4.81 (t,
J=5.5 Hz, 1H; OH(5’A)), 5.02 (t, J=5.3 Hz, 1H; OH(5’B)), 5.30 (d, J=
4.1 Hz, 1H; OH(3’A)), 5.30 (d, J=4.1 Hz, 1H; OH(3’B)), 6.08–6.17 (m,
A
N
(CH2(5’)), 70.3 (CH(3’)), 82.7 (CH(1’)), 85.3 (CH(4’)), 152.8 (CO),
173.0 ppm (CO); MS (FAB+): m/z: 740 [2M+H+], 381 [M+Na+], 359
[M+H+]; HRMS (FAB+): m/z calcd for C16H31N2O5Si: 359.2002; found:
359.2002 [M+H]+.
ACHTREUNG
G
3’-O-Triethylsilyl-5’-O-tert-butyldimethylsilyl-5,6-dihydrothymidine (18):
The TBDMS-protected dihydrothymidine 11 (2.97 g, 8.29 mmol), imida-
zole (1.69 g, 24.9 mmol), and TESCl (2.09 mL, 12.4 mmol) were dissolved
in anhydrous DMF (13 mL). The reaction mixture was stirred overnight
at RT. The solution was diluted with CHCl3 (60 mL), washed with satu-
rated aqueous sodium bicarbonate (7”120 mL), dried (MgSO4), and the
solvent was removed in vacuo. Carrying out flash chromatography (silica
gel, i-hexane/ethyl acetate 7:3) gave 18 as a white foam (3.50 g, 89%).
U
R
2H; 2”CH(1’)), 7.65 (s, 1H; CH(6B)), 10.05 (s, 1H; NH(A)), 11.22 ppm
(s, 1H; NH(B)); 13C NMR (125 MHz, [D6]DMSO): d=19.5 (C(5A)CH3),
31.1 (C(5B)CH2), 36.0 (CH2
(CH2(5’B)), 62.2 (CH2(5’A)), 70.8 (CH
(1’A)), 84.3 (CH(1’B)), 86.2 (CH(4’A)), 87.6 (CH
ACHTREUNG
A
G
E
ACHTREUNG
G
G
G
ACHTREUNG
Rf =0.31 (iHex/ethyl acetate 7:3); 1H NMR (400 MHz, [D6]DMSO): d=
139.1 (CH(6B)), 150.4 (CO(2B)), 152.4 (CO(2A)), 163.7 (CO(4B)),
174.2 ppm (CO(4A)); IR (KBr): n˜max =3419s, 2924m, 2854w, 1697s,
0.05 (s, 3H; Si
Si(CH2CH3)3), 0.87 (s, 9H;
(CH2CH3)3)), 1.06 (d, J=7.2 Hz, 3H; CH3), 1.78–1.83 (m, 1H; CH2a(2’)),
A
N
1476m, 1389w, 1278w, 1205m, 1091m, 1050m, 766w cmꢀ1
; MS
A
C
ACHTREUNG
(MALDI+): m/z (%): 507 (100) [M+Na+], 444 (5), 412 (5), 390 (6), 314
AHCTREUNG
2.14–2.21 (m, 1H; CH2b(2’)), 2.56–2.66 (m, 1H; CH(5)), 2.97 (dd, J=
12.4, J=10.4 Hz, 1H; CH2a(6)), 3.37 (dd, J=12.8, J=5.6 Hz, 1H;
CH2b(6)), 3.60–3.66 (m, 3H; CH(4’), CH2(5’)), 4.27–4.29 (m, 1H;
CH(3’)), 6.10–6.14 (dd, J=6.4, J=6.4 Hz, 1H; CH(1’)), 10.22 ppm (s,
1H; NH); 13C NMR (100 MHz, [D6]DMSO): d=ꢀ5.6 (SiCH3), ꢀ5.6
(10), 306 (46), 288 (29), 284 (35), 268 (15), 254 (12), 165 (13); HRMS
(ESI+): m/z calcd for C20H28N4NaO10
:
507.1703; found: 507.1694
: 523.1443; found: 523.1434
[M+Na]+; m/z calcd for C20H28KN4O10
[M+K]+.
(5S)-(a-Thymidyl)-5,6-dihydrothymidine (1a): SEM deprotection was
carried out as described above for 1b. Isomer (S)-1a was isolated as a
white solid (27.0 mg, 55%).
Rf =0.10 (CHCl3/MeOH 4:1); m.p. 155–1588C; 1H NMR (500 MHz,
(SiCH3), 4.2 (Si
G
U
A
ACHTREUNG
(CH2(5’)), 71.7 (CH(3’)), 82.8 (CH(1’)), 85.3 (CH(4’)), 152.9 (CO),
173.0 ppm (CO); MS (FAB+): m/z: 969 [2M+Na+], 496 [M+Na+], 474
[M+H+]; HRMS (MALDI+): m/z calcd for C22H44N2NaO5Si2: 495.2686;
found: 495.2637 [M+Na]+.
N3-Trimethylsilylethoxymethyl-3’-O-triethylsilyl-5’-O-tert-butyldimethyl-
silyl-5,6-dihydrothymidine (10):[16] SEM protection was carried out as de-
scribed above for 5. Product 10 (3.80 g, 86%) was isolated as a colorless
oil.
[D6]DMSO): d=0.99 (s, 3H; C(5A)CH3), 1.77 (ddd, J=13.2, J=6.2, J=
2.8 Hz, 1H; CH2a
A
G
N
(ddd, J=13.2, J=5.9, J=3.1 Hz, 1H; CH2b
AHCTREUNG
1H; C(5B)CH2a), 2.72 (d, J=14.0 Hz, 1H; C(5B)CH2b), 3.05 (d, J=
12.9 Hz, 1H; CH2a(6A)), 3.17 (d, J=13.0 Hz, 1H; CH2b(6A)), 3.36 (s,
13H; CH2
(m, 1H; CH
(3’B)), 4.77 (t, J=5.5 Hz, 1H; OH
(5’B)), 5.19 (d, J=4.2 Hz, 1H; OH
(3’B)), 6.09 (dd, J=7.9, J=6.2 Hz, 1H; CH
CH(1’B)), 7.72 (s, 1H; CH(6B)), 10.11 (s, 1H; NH(A)), 11.23 ppm (s,
1H; NH(B)); 13C NMR (125 MHz, [D6]DMSO): d=20.2 (C(5A)CH3),
30.8 (C(5B)CH2), 35.9 (CH2(2’A)), 41.1 (C(5A)), 45.5 (CH2(6A)), 61.50
(CH2(5’B)), 62.0 (CH2(5’A)), 70.7 (CH(3’B)), 70.8 (CH(3’A)), 83.0 (CH-
(1’A)), 84.2 (CH(1’B)), 86.0 (CH(4’A)), 87.6 (CH(4’B)), 108.7 (C(5B)),
A
(5’B), CH
G
A
ACHTREUNG
A
N
Rf =0.19 (iHex/ethyl acetate 9:1); 1H NMR (400 MHz, [D6]DMSO): d=
A
N
ꢀ0.04 (s, 9H; Si
0.59 (q, J=8.0 Hz, 6H; Si
0.87 (s, 9H; C(CH3)3), 0.93 (t, J=8.0 Hz, 9H; Si
A
G
U
A
ACHTREUNG
AHCTREUNG
ACHTREUNG
G
ACHTREUNG
7.2 Hz, 3H; CH3), 1.82–1.88 (m, 1H; CH2a(2’)), 2.15–2.22 (m, 1H;
CH2b(2’)), 2.73–2.78 (m, 1H; CH(5)), 3.00 (dd, J=12.8, J=10.0 Hz, 1H;
CH2a(6)), 3.38 (dd, J=12.8, J=5.6 Hz, 1H; CH2b(6)), 3.50 (t, J=8.0 Hz,
2H; SiCH2CH2O), 3.62–3.68 (m, 3H; CH(4’), CH2(5’)), 4.28–4.31 (m,
1H; CH(3’)), 5.05 (s, 2H; OCH2N), 6.15–6.18 ppm (dd, J=6.4, J=6.4 Hz,
1H; CH(1’)); 13C NMR (100 MHz, [D6]DMSO): d=ꢀ5.7 (SiCH3), ꢀ5.6
AHCTREUNG
A
G
A
ACHTREUNG
A
G
A
ACHTREUNG
139.1 (CH(6B)), 150.4 (CO(2B)), 152.5 (CO(2A)), 163.8 (CO(4B)),
174.4 ppm (CO(4A)); IR (KBr): n˜max =3393s, 2918m, 2253w, 1693s,
1474w, 1389w, 1278w, 1232w, 1093m, 1051m, 1026m, 1002m, 827w,
766w, 574w cmꢀ1; MS (ESI+): m/z (%): 523 (57) [M+K+], 407 (16), 336
(12), 303 (100), 257 (17), 122 (45); HRMS (ESI+): m/z calcd for
C20H28KN4O10: 523.1443; found: 523.1450 [M+K]+.
(SiCH3), ꢀ1.4 (Si
C
G
U
G
ACHTREUNG
36.5 (CH2(2’)), 40.2 (CH2(6)), 62.6 (CH2(5’)), 65.5 (OCH2CH2Si), 69.0
(NCH2O), 71.5 (CH(3’)), 83.7(CH(1’)), 85.4 (CH(4’)), 152.6 (CO),
172.3ppm (CO); MS (FAB+): m/z: 604 [M+H+]; HRMS (MALDI+):
m/z calcd for C28H58N2NaO6Si3: 625.3500; found: 625.3496 [M+Na]+.
5’-O-tert-Butyldimethylsilyl-5,6-dihydrothymidine (11):[37–39] Thymidine 3
(2.50 g, 10.3 mmol) was dissolved in MeOH/water (50 mL, 1:1) and Rh/
6090
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 6081 – 6094

DNA Spore Photoproduct Analogues
FULL PAPER
3’-O-tert-Butyldimethylsilylthymidine (13):[41] The bis-TBDMS-protected
thymidine 17 (3.53 g, 7.50 mmol) was dissolved in CH2Cl2 (41 mL) and
cooled to 08C. A TFA/water mixture (4.1 mL, 10:1, cooled to 08C) was
added dropwise to the solution. The reaction mixture was stirred for 4 h
at 08C. The solution was diluted with cooled CH2Cl2 (9 mL), washed
with ice-cold water (50 mL) and with saturated aqueous sodium chloride
(25 mL), dried (MgSO4), and the solvent was removed in vacuo. Carrying
out flash chromatography (silica gel, CHCl3/MeOH 20:1) gave 13 as a
white foam (2.17 g, 81%).
13C NMR and mass spectral data were unobtainable due to the rapid de-
composition of 12.
N3
(A/B)-Di(trimethylsilylethoxymethyl)-5’(A),3’(B)-O-di(tert-butyldime-
G
thylsilyl)-5-(a-thymidyl)-5,6-dihydrothymidine (15a/b): Dihydrothymi-
dine 10 (87 mg, 0.14 mmol), azeotropically dried with anhydrous toluene,
was dissolved in anhydrous THF (0.8 mL) and cooled to ꢀ788C. A fresh-
ly prepared LDA solution (diisopropylamine (31 mL, 0.22 mmol), BuLi
(0.14 mL, 1.60m in hexane) in anhydrous THF (0.4 mL) left at 08C for
1 h) was slowly added and the reaction mixture was stirred at ꢀ788C for
2 h before the addition of 12 (100 mg, 0.15 mmol), dissolved in anhydrous
THF (1.2 mL). The reaction mixture was stirred at ꢀ788C for 1.5 h and
at 08C for 1.5 h. The reaction was quenched by adding aqueous sodium
bicarbonate (5 mL) and the aqueous phase was extracted with CHCl3
(3”5 mL). The collected extracts were dried (MgSO4) and the solvent
was removed in vacuo.
1
Rf =0.18 (CHCl3/MeOH 20:1); H NMR (400 MHz, [D6]DMSO): d=0.08
(s, 6H; Si
N
N
1H; CH2a(2’)), 2.15–2.21 (m, 1H; CH2b(2’)), 3.51–3.61 (m, 2H; CH2(5’)),
3.74–3.77 (m, 1H; CH(4’)), 4.39–4.42 (m, 1H; CH(3’)), 5.06–5.08 (m, 1H;
OH), 6.13–6.16 (m, 1H; CH(1’)), 7.66 (s, 1H; CH(6)), 11.28 ppm (s, 1H;
NH); 13C NMR (100 MHz, [D6]DMSO): d=ꢀ4.9 (SiCH3), ꢀ4.8 (SiCH3),
12.2 (CH3), 17.7 (C
N
N
The crude mixture of the coupling reaction was dissolved in anhydrous
acetonitrile (1.29 mL) and cooled to ꢀ308C. A HF·pyridine solution (4%
in acetonitrile, 2.92 mL, 937 mmol) was added and the reaction mixture
was stirred at ꢀ308C for 40 min. The reaction was quenched by adding
methoxytrimethylsilane, stirred at ꢀ308C for 1 h, and the solvent was re-
moved in vacuo. An initial purification was achieved by using flash chro-
matography (silica gel, i-hexane/ethyl acetate 4:1!3:2) to give 9a/b as
colorless oil (48.0 mg, 34%).
(CH2(5’)), 72.1 (CH(3’)), 83.7 (CH(1’)), 87.3 (CH(4’)), 109.4 (C(5)), 136.0
(CH(6)), 150.4 (CO), 163.6 ppm (CO); MS (FAB+): m/z: 714 [2M+H+],
379 [M+Na+], 357 [M+H+], 117 [TBS+H+].
3’-O-tert-Butyldimethylsilyl-5’-O-triethylsilylthymidine (19): TES protec-
tion was carried out as described above for 18. Product 19 was isolated as
a colorless oil (2.53 g, 91%).
Rf =0.36 (iHex/ethyl acetate 7:3); 1H NMR (400 MHz, [D6]DMSO): d=
0.08 (s, 6H; Si
A
G
The diastereomers were separated by using np-HPLC with a 250/10 Nu-
cleodur 100-5 column (Macherey–Nagel) and an n-heptane/ethyl acetate
gradient (0!70% ethyl acetate in 70 min, 70!100% ethyl acetate in
5 min; flow rate: 3 mLminꢀ1; detection wavelength: 260 nm). The mix-
ture was dissolved in n-heptane/ethyl acetate (9 mL, 1:1) and for each
separation 1 mL of the solution was injected through a Rheodyne valve
on the column. After HPLC purification, the isomers (S)-9a (13.0 mg,
9%) and (R)-9b (18.0 mg, 13%) were isolated as colorless oils.
9H; C(CH3)3), 0.93 (t, J=8.0 Hz, 9H; Si
G
ACHTREUNG
2.03–2.09 (m, 1H; CH2a(2’)), 2.17–2.24 (m, 1H; CH2b(2’)), 3.67–3.75 (m,
2H; CH2(5’)), 3.76–3.80 (m, 1H; CH(4’)), 4.36–4.39 (m, 1H; CH(3’)),
6.14–6.17 (dd, J=6.4, J=6.4 Hz, 1H; CH(1’)), 7.47 (s, 1H; CH(6)),
11.32 ppm (s, 1H; NH); 13C NMR (100 MHz, [D6]DMSO): d=ꢀ5.0
(SiCH3), ꢀ4.9 (SiCH3), 3.8 (Si
A
(CH3), 17.6 (C(CH3)3), 25.6 (C
U
ACHTREUNG
(CH(1’)), 86.7 (CH(4’)), 109.4 (C(5)), 135.6 (CH(6)), 150.3 (CO),
163.6 ppm (CO); MS (FAB+): m/z: 494 [M+Na+], 472 [M+H+], 117
[TES+H+] and [TBS+H+]; HRMS (MALDI+): m/z calcd for
C22H42N2NaO5Si2: 493.2530; found: 493.2541 [M+Na]+.
N3-Trimethylsilylethoxymethyl-3’-O-tert-butyldimethylsilyl-5’-O-triethylsi-
lylthymidine (14): SEM protection was carried out as described above for
5. Product 14 was isolated as a colorless oil (2.04 g, 64%).
Isomer (S)-9a: Rf =0.45 (iHex/ethyl acetate 3:2); 1H NMR (400 MHz,
CDCl3): d=ꢀ0.02 (s, 9H; Si
3H; Si(CH3)), 0.07 (s, 3H; Si(CH3)), 0.11 (s, 6H; 2”Si
(m, 22H; 2”SiCH2CH2O, 2 ” C(CH3)3), 1.22 (s, 3H; CH3), 2.00–2.18 (m,
3H; CH2(2’A), CH2a(2’B)), 2.36 (ddd, J=13.2, J=6.0, J=2.8 Hz, 1H;
CH2b(2’B)), 2.57 (d, J=14.0 Hz, 1H; C(5B)CH2a), 2.82 (d, J=14.0 Hz,
1H; C(5B)CH2b), 3.13 (d, J=12.8 Hz, 1H; CH2a(6A)), 3.23 (d, J=
12.8 Hz, 1H; CH2b(6A)), 3.57–3.69 (m, 5H; 2”SiCH2CH2O, CH2a(5’A)),
3.71 (dd, J=12.0, J=2.4 Hz, 1H; CH2a(5’B)), 3.76–3.79 (m, 1H; CH-
(4’A)), 3.83 (dd, J=10.0, J=4.0 Hz, 1H; CH2b(5’A)), 3.97 (dd, J=12.0,
J=2.4 Hz, 1H; CH2b(5’B)), 4.01–4.03 (m, 1H; CH(4’B)), 4.34–4.38 (m,
1H; CH(3’A)), 4.44–4.46 (m, 1H; CH(3’B)), 5.13 (d, J=9.6 Hz, 1H;
A
N
A
N
ACHTREUNG
AHCTREUNG
A
ACHTREUNG
AHCTREUNG
Rf =0.24 (iHex/ethyl acetate 9:1); 1H NMR (400 MHz, [D6]DMSO): d=
ACHTREUNG
ꢀ0.05 (s, 9H; Si
Si(CH2CH3)3), 0.83 (t, J=8.0 Hz, 2H; SiCH2CH2O), 0.87 (s, 9H; C-
(CH3)3), 0.92 (t, 3J=8.0 Hz, 9H; Si
(CH2CH3)3), 1.83 (s, 3H; CH3), 2.08–
A
N
AHCTREUNG
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
2.14 (m, 1H; CH2a(2’)), 2.18–2.25 (m, 1H; CH2b(2’)), 3.56 (t, J=8.0 Hz,
2H; SiCH2CH2O), 3.71 (dd, J=11.2, J=3.6 Hz, 1H; CH2a(5’)), 3.77 (dd,
J=11.2, J=4.0 Hz, 1H; CH2b(5’)), 3.80–3.83 (m, 1H; CH(4’)), 4.36–4.39
(m, 1H; CH(3’)), 5.21 (s, 2H; OCH2N), 6.15–6.19 (m, 1H; CH(1’)),
7.56 ppm (s, 1H; CH(6)); 13C NMR (100 MHz, [D6]DMSO): d=ꢀ5.0
A
ACHTREUNG
OCH2aN(A)), 5.17 (d, J=9.6 Hz, 1H; OCH2bN(A)), 5.34 (d, J=9.6 Hz,
1H; OCH2aN(B)), 5.37 (d, J=9.6 Hz, 1H; OCH2bN(B)), 6.14–6.18 (m,
1H; CH
C
N
(SiCH3), ꢀ4.9 (SiCH3), ꢀ1.5 (Si
(CH2CH3)3), 12.7 (CH3), 17.4 (SiCH2CH2O), 17.6 (C
(CH3)3), 39.4 (CH2(2’)), 62.1 (CH2(5’)), 66.3 (SiCH2CH2O), 69.6
A
ACHTREUNG
(SiCH3), ꢀ4.5 (SiCH3), ꢀ1.29 (Si
U
G
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
(OCH2N), 71.8 (CH(3’)), 84.9 (CH(1’)), 86.9 (CH(4’)), 108.6 (C(5)), 134.9
(CH(6)), 150.4 (CO), 162.6 ppm (CO); MS (FAB+): m/z: 1225 [2M+H+
], 624 [M+Na+], 602 [M+H+], 117 [TES+H+] and [TBS+H+]; HRMS
(MALDI+): m/z calcd for C28H56N2NaO6Si3: 623.3344; found: 623.3319
[M+Na]+.
A
G
ACHTREUNG
AHCTREUNG
E
ACHTREUNG
G
G
G
ACHTREUNG
(C(5B)), 139.9 (CH(6B)), 150.7 (CO(2B)), 152.4 (CO(2A)), 164.0
(CO(4B)), 174.5 ppm (CO(4A)); MS (MALDI+): m/z: 1012 [M+K+],
996 [M+Na+]; HRMS (MALDI+): m/z calcd for C44H83N4NaO12Si4:
994.4982; found: 994.4942 [M+Na]+.
N3-Trimethylsilylethoxymethyl-3’-O-tert-butyldimethylsilyl-5’-O-triethyl-
silyl-5-bromomethyl-2’-deoxyuridine (12):[40] The bromination was carried
out as described above for 7. Product 12 was isolated as a colorless oil
(112 mg, 20%), which decomposed at room temperature but was stable
at ꢀ208C.
Isomer (R)-9b: Rf =0.45 (iHex/ethyl acetate 3:2); 1H NMR (400 MHz,
CDCl3): d=ꢀ0.03 (s, 9H; Si
3H; SiCH3), 0.08 (s, 3H; SiCH3), 0.10 (s, 3H; SiCH3), 0.11 (s, 3H;
SiCH3), 0.88–0.91 (m, 22H; 2”SiCH2CH2O, 2 ” C(CH3)3), 1.22 (s, 3H;
CH3), 2.08–2.18 (m, 3H; CH2(2’A), CH2a(2’B)), 2.26 (ddd, J=13.2, J=
6.0, J=3.6 Hz, 1H; CH2b(2’B)), 2.62 (d, J=14.4 Hz, 1H; C(5B)CH2a),
(CH3)3), ꢀ0.02 (s, 9H; Si
(CH3)3), 0.06 (s,
Rf =0.30 (iHex/ethyl acetate 9:1); 1H NMR (200 MHz, CDCl3): d=0.00
AHCTREUNG
(s, 9H; Si
(CH2CH3)3), 0.89–0.93 (m, 11H; SiCH2CH2O, C
9H; Si(CH2CH3)3), 1.94–2.09 (m, 1H; CH2a(2’)), 2.29–2.40 (m, 1H;
A
ACHTREUNG
G
ACHTREUNG
A
ACHTREUNG
AHCTREUNG
ACHTREUNG
2.82 (d, J=14.4 Hz, 1H; C(5B)CH2b), 3.06 (d, J=13.2 Hz, 1H;
CH2a(6A)), 3.29 (d, J=13.2 Hz, 1H; CH2b(6A)), 3.57 (dd, J=10.0, J=
CH2b(2’)), 3.64–3.72 (m, 2H; SiCH2CH2O), 3.79–3.98 (m, 3H; CH(4’),
CH2(5’)), 4.23 (d, J=10.6 Hz, 1H; CH2aBr), 4.32 (d, J=10.6 Hz, 1H;
CH2bBr), 4.39–4.42 (m, 1H; CH(3’)), 5.41 (s, 2H; OCH2N), 6.28–6.34 (m,
1H; CH(1’)), 7.97 ppm (s, 1H; CH(6)).
2.8 Hz, 1H; CH2a
ACHTREUNG
12.4, J=2.4 Hz, 1H; CH2a
N
G
ACHTREUNG
Chem. Eur. J. 2006, 12, 6081 – 6094
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6091

T. Carell et al.
3.87–3.89 (m, 1H; CH
A
-
0.10 (s, 6H; Si
(CH3)3), 0.90 (s, 9H; C
1H; CH2a(2’A)), 2.13–2.22 (m, 2H; CH2b
6H; CH2b(2’B), 2”CH2=CHCH2CH2, C(5B)CH2a), 2.41–2.46 (m, 4H; 2”
ACHTREUNG
G
G
R
C
C
ACHTREUNG
G
A
ACHTREUNG
J=10.0 Hz, 1H; OCH2aN(A)), 5.21 (d, J=9.6 Hz, 1H; OCH2bN(A)),
5.34 (d, J=9.6 Hz, 1H; OCH2aN(B)), 5.37 (d, J=9.6 Hz, 1H;
AHCTREUNG
CH2=CHCH2CH2), 2.87 (d, J=14.0 Hz, 1H; C(5B)CH2b), 3.20 (d, J=
13.2 Hz, 1H; CH2a(6A)), 3.46 (d, J=12.8 Hz, 1H; CH2b(6A)), 3.49–3.55
(m, 2H; SiCH2CH2O(A)), 3.57–3.62 (m, 2H; SiCH2CH2O(B)), 3.79 (dd,
OCH2bN(B)), 6.29–6.33 (m, 1H; CH
C
U
(SiCH3), ꢀ5.2 (SiCH3), ꢀ4.7 (SiCH3), ꢀ4.5 (SiCH3), ꢀ1.3 (Si
G
J=11.2, J=3.2 Hz, 1H; CH2a
CH2b(5’A)), 3.94–3.96 (m, 1H; CH
4.20 (dd, J=12.0, J=4.0 Hz, 1H; CH2a
6.0 Hz, 1H; CH2b(5’B)), 4.38–4.42 (m, 1H; CH
2”CH2=CH, OCH2aN(A)), 5.14–5.21 (m, 2H; OCH2bN(A), CH
ACHTREUNG
ꢀ1.3 (Si
18.5 (C
(C(5B)CH2), 37.1 (CH2
(CH2(6A)), 62.0 (CH2(5’B)), 64.4 (CH2
67.6 (SiCH2CH2O(B)), 70.3 (OCH2N(A)), 70.5 (OCH2N(B)), 72.0 (CH-
(3’B)), 73.8 (CH(3’A)), 84.6 (CH(1’A)), 85.0 (CH(4’A)), 86.1 (CH(1’B)),
88.2 (CH
A
ACHTREUNG
A
G
ACHTREUNG
R
N
ACHTREUNG
AHCTREUNG
U
ACHTREUNG
H
ACHTREUNG
R
ACHTREUNG
ACHTREUNG
5.26 (d, J=10.0 Hz, 1H; OCH2aN(B)), 5.30 (d, J=10.0 Hz, 1H;
OCH2bN(B)), 5.74–5.88 (m, 2H; 2”CH2=CH), 6.16 (t, J=6.8 Hz, 1H;
A
G
A
A
ACHTREUNG
A
(CO(2A)), 163.8 (CO(4B)), 174.1 ppm (CO(4A)); MS (MALDI+): m/z:
1012 [M+K+], 996 [M+Na+]; HRMS (MALDI+): m/z calcd for
C44H83N4NaO12Si4: 994.4982; found: 994.4942 [M+Na]+.
CH
C
G
(SiCH3), ꢀ4.7 (SiCH3), ꢀ4.5 (SiCH3), ꢀ1.2 (2”Si
U
ACHTREUNG
18.9 (SiCH2CH2O), 19.1 (SiCH2CH2O), 19.3 (C
(C(5A)CH3), 26.3 (C(CH3)3), 26.6 (C
Diester 16a: The protected SP model 9a (10.0 mg, 10.3 mmol), iPr2NEt
(4.30 mL, 25.8 mmol), and 4-pentenoylchloride (2.40 mL, 22.7 mmol) were
dissolved in anhydrous CH2Cl2 (1 mL) and stirred at RT for 6 h. After
2 h and 4 h stirring, additional iPr2NEt (4.30 mL, 25.8 mmol) and 4-pente-
noylchloride (2.40 mL, 22.7 mmol) were added, respectively. The reaction
mixture was diluted with CHCl3 (4 mL), washed with aqueous sodium bi-
carbonate (5 mL), and the aqueous phase was extracted with CHCl3 (2”
5 mL). The collected organic phases were dried (MgSO4) and the solvent
was removed in vacuo. Purification by using flash chromatography (silica
gel, i-hexane/ethyl acetate 9:1!1:1) provided 16a as a colorless oil
(11.0 g, 94%).
N
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
A
G
A
ACHTREUNG
(C(5B)), 116.2 (2”CH2=CH), 137.9 (CH2=CH), 138.0 (CH2=CH), 139.7
(CH(6B)), 152.1 (CO(2B)), 154.2 (CO(2A)), 164.8 (CO(4B)), 174.1
(ester-CO), 174.2 (ester-CO), 175.1 ppm (CO(4A)); IR (KBr): n˜max
=
3080w, 2953s, 2928s, 2856m, 1741s, 1719s, 1670s, 1461s, 1361m, 1276w,
1249s, 1170w, 1092s, 1031w, 992w, 917w, 860m, 835s, 778m, 695w,
669w cmꢀ1; MS (FAB+): m/z (%): 1159 (2) [M+Na+], 213 (7), 145 (10),
136 (17), 89 (27), 81 (31), 73 (100), 55 (17); HRMS (ESI+): m/z calcd for
C54H96N4NaO14Si4: 1159.5898; found: 1159.5909 [M+Na]+.
Rf =0.73 (iHex/ethyl acetate 7:3); 1H NMR (400 MHz, [D4]MeOH): d=
ꢀ0.01 (s, 9H; Si
0.16 (s, 6H; Si(CH3)2), 0.85–0.91 (m, 4H; 2”SiCH2CH2O), 0.94 (s, 9H;
(CH3)3), 0.96 (s, 9H; C(CH3)3), 1.23 (s, 3H; C(5A)CH3), 2.03 (ddd, J=
13.6, J=5.2, J=1.2 Hz, 1H; CH2a(2’A)), 2.16–2.24 (m, 2H; CH2b(2’A),
CH2a(2’B)), 2.30 (ddd, J=13.6, J=6.4, J=4.0 Hz, 1H; CH2b(2’B)), 2.35–
A
G
G
ACHTREUNG
C
A
ACHTREUNG
Lactone 2a: For the ring-closing metathesis, 16a (13.0 mg, 11.4 mmol)
was dissolved in anhydrous CH2Cl2 (5 mL) and heated to 408C. The
Grubbs-II catalyst[20,21] (0.50 mg, 0.57 mmol), dissolved in anhydrous
CH2Cl2 (1 mL), was added and the reaction mixture was stirred at 408C
for 3 h. The solvent was removed in vacuo. Carrying out flash chromatog-
raphy (silica gel, i-hexane/ethyl acetate 4:1) gave 2a as a colorless oil
(9.00 mg, 71%).
T
ACHTREUNG
H
ACHTREUNG
2.42 (m, 4H; 2”CH2=CHCH2CH2), 2.45–2.50 (m, 2H; CH2=
CHCH2CH2), 2.54–2.57 (m, 2H; CH2=CHCH2CH2), 2.69 (d, J=14.0 Hz,
1H; C(5B)CH2a), 2.75 (d, J=14.0 Hz, 1H; C(5B)CH2b), 3.25 (d, J=
13.2 Hz, 1H; CH2a(6A)), 3.30–3.35 (m, 9H; CH2b(6A), MeOH), 3.58 (t,
J=8.0 Hz,
SiCH2CH2O(B)), 3.80 (dd, J=10.8, J=3.6 Hz, 1H; CH2a
J=11.2, J=3.6 Hz, 1H; CH2b(5’A)), 3.93–3.96 (m, 1H; CH
4.08 (m, 1H; CH(4’B)), 4.27 (dd, J=12.0, J=4.0 Hz, 1H; CH2a
4.37 (dd, J=12.0, J=5.2 Hz, 1H; CH2b(5’B)), 4.48–4.52 (m, 1H; CH-
(3’B)), 4.97–5.02 (m, 2H; CH2 =CH), 5.04–5.06 (m, 1H; CH2=CH), 5.08–
5.10 (m, 1H; CH2=CH), 5.17 (s, 2H; OCH2N(A)), 5.22–5.25 (m, 1H;
2H;
SiCH2CH2O(A)),
3.66
(t,
2H;
1
Rf =0.25 (iHex/ethyl acetate 4:1); H NMR (600 MHz, CDCl3): d=ꢀ0.01
AHCTREUNG
(s, 9H; Si
N
N
G
ACHTREUNG
3H; SiCH3), 0.10 (s, 3H; SiCH3), 0.11 (s, 3H; SiCH3), 0.86–0.93 (m, 20H;
2”C(CH3)3, SiCH2CH2O), 0.96–0.99 (m, 2H; SiCH2CH2O), 1.24 (s, 3H;
C(5A)CH3), 1.85–1.90 (m, 2H; CH2a(2’A), CH2a(2’B)), 2.12–2.18 (m, 1H;
CH2b(2’A)), 2.19–2.24 (m, 1H; 1”CH=CHCH2CH2), 2.27 (d, J=13.8 Hz,
1H; C(5B)CH2a), 2.35–2.49 (m, 8H; 2”CH=CHCH2CH2, CH2b(2’B)),
A
ACHTREUNG
AHCTREUNG
AHCTREUNG
A
ACHTREUNG
ACHTREUNG
AHCTREUNG
ACHTREUNG
CH(3’A)), 5.34 (s, 2H; OCH2N(B)), 5.80–5.91 (m, 2H; 2”CH2=CH),
A
2.68 (d, J=13.8 Hz, 1H; C(5B)CH2b), 3.24 (d, J=13.8 Hz, 1H;
CH2a(6A)), 3.33 (d, J=13.8 Hz, 1H; CH2b(6A)), 3.57 (t, J=8.4 Hz, 2H;
SiCH2CH2O(A)), 3.67 (t, J=8.4 Hz, 2H; SiCH2CH2O(B)), 3.74 (dd, J=
6.25–6.30 (m, 2H; 2”CH(1’)), 7.62 ppm (s, 1H; CH(6B)); 13C NMR
(100 MHz, [D4]MeOH): d=ꢀ5.1 (SiCH3), ꢀ5.0 (SiCH3), ꢀ4.6 (SiCH3),
ꢀ4.4 (SiCH3), ꢀ1.2 (2”Si
A
A
11.4, J=2.4 Hz, 1H; CH2a
(5’A)), 4.03–4.06 (m, 1H; CH
4.21 (m, 2H; CH2a(5’B), CH
CH2b(5’B)), 5.14 (d, J=9.6 Hz, 1H; OCH2aN(A)), 5.18–5.21 (m, 2H;
OCH2bN(A), CH(3’A)), 5.36 (d, J=9.6 Hz, 1H; OCH2aN(B)), 5.41 (d,
J=9.6 Hz, 1H; OCH2bN(B)), 5.44–5.50 (m, 1H; CH=CH), 5.52–5.57 (m,
1H; CH=CH), 6.07 (t, J=6.0 Hz, 1H; CH(1’B)), 6.37 (dd, J=10.2, J=
4.8 Hz, 1H; CH
(1’A)), 7.09 ppm (s, 1H; CH(6B)); 13C NMR (150 MHz,
[D4]MeOH): d=ꢀ5.2 (SiCH3), ꢀ5.1 (SiCH3), ꢀ4.8 (SiCH3), ꢀ4.6
(SiCH3), ꢀ1.3 (2”Si(CH3)3), 18.8 (C(CH3)3), 18.9 (SiCH2CH2O), 19.0
(SiCH2CH2O), 19.3 (C(CH3)3), 21.0 (C(5A)CH3), 26.3 (C(CH3)3), 26.6
(C(CH3)3), 29.1 (CH=CHCH2CH2), 29.7 (CH=CHCH2CH2), 33.6
(C(5B)CH2), 34.4 (CH=CHCH2CH2), 34.7 (CH=CHCH2CH2), 36.3 (CH2-
(2’A)), 42.6 (CH2(2’B)), 44.9 (C(5A)), 47.1 (CH2(6A)), 64.3 (CH2(5’B)),
64.9 (CH2(5’A)), 67.9 (SiCH2CH2O(A)), 68.5 (SiCH2CH2O(B)), 71.1
(OCH2N(A)), 71.4 (OCH2N(B)), 72.9 (CH(3’B)), 76.7 (CH(3’A)), 83.8
(CH(4’A)), 85.5 (CH(1’A)), 87.0 (CH(4’B)), 87.5 (CH(1’B)), 109.8
A
-
19.1 (SiCH2CH2O(B)), 19.4 (C
26.7 (C(CH3)3), 30.0 (2”CH2=CHCH2CH2), 33.5 (C(5B)CH2), 34.4
(CH2=CHCH2CH2), 34.5 (CH2=CHCH2CH2), 35.0 (CH2(2’A)), 41.4
(CH2(2’B)), 44.2 (C(5A)), 45.8 (CH2(6A)), 64.5 (CH2(5’B)), 64.7 (CH2-
(5’A)), 67.8 (SiCH2CH2O(A)), 68.6 (SiCH2CH2O(B)), 71.2 (OCH2N(A)),
71.5 (OCH2N(B)), 73.3 (CH(3’B)), 75.9 (CH(3’A)), 84.9 (CH(4’A)), 85.7
(CH(1’A)), 86.2 (CH(4’B)), 87.3 (CH(1’B)), 109.9 (C(5B)), 116.2 (2”
(CH3)3), 22.4 (C(5A)CH3), 26.3 (C
U
A
N
ACHTREUNG
ACHTREUNG
A
ACHTREUNG
AHCTREUNG
AHCTREUNG
A
ACHTREUNG
AHCTREUNG
ACHTREUNG
G
U
ACHTREUNG
AHCTREUNG
A
N
ACHTREUNG
AHCTREUNG
CH2=CH), 137.9 (CH2=CH), 138.0 (CH2=CH), 140.7 (CH(6B)), 152.1
(CO(2B)), 154.3 (CO(2A)), 165.0 (CO(4B)), 174.1 (ester-CO), 174.3
(ester-CO), 175.3 ppm (CO(4A)); IR (KBr): n˜max =3080w, 2954s, 2929s,
2897w, 2858m, 1741s, 1719s, 1681s, 1668s, 1463s, 1360m, 1276w, 1249s,
1169w, 1093s, 1030w, 991w, 916m, 860m, 836s, 779m, 694w, 668w, 613w,
521w cmꢀ1; MS (FAB+): m/z (%): 1159 (7) [M+Na+], 213 (12), 145 (13),
136 (17), 89 (29), 81 (33), 73 (100), 55 (16); HRMS (MALDI+): m/z
calcd for C54H96N4NaO14Si4: 1159.5898; found: 1159.5887 [M+Na]+.
A
ACHTREUNG
A
ACHTREUNG
AHCTREUNG
A
N
ACHTREUNG
AHCTREUNG
A
ACHTREUNG
Diester 16b: The reaction was carried out as described above for 16a.
Isomer (R)-16b was isolated as a colorless oil (14.0 mg, 80%).
Rf =0.73 (iHex/ethyl acetate 7:3); 1H NMR (400 MHz, [D4]MeOH): d=
A
G
A
ACHTREUNG
(C(5B)), 130.8 (CH=CH), 131.0 (CH=CH), 138.4 (CH(6B)), 151.9
(CO(2B)), 154.4 (CO(2A)), 164.9 (CO(4B)), 174.1 (ester-CO), 174.4
(ester-CO), 174.6 ppm (CO(4A)); IR (film): n˜max =2927s, 2854m, 1737s,
ꢀ0.06 (s, 9H; Si
A
G
G
6092
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 6081 – 6094

DNA Spore Photoproduct Analogues
FULL PAPER
1667s, 1461s, 1360w, 1249m, 1089s, 860w, 835s, 778m cmꢀ1; MS (FAB+):
m/z (%): 1131 (4) [M+Na+], 154 (19), 136 (27), 89 (26), 81 (29), 73
(100); HRMS (ESI+): m/z calcd for C52H92N4NaO14Si4: 1131.5585; found:
1131.5577 [M+Na]+.
were further analyzed by Fourier transform ion cyclotron resonance
(FTICR) mass spectrometry (Thermo Finnigan LTQ FT) measurements.
Lactone 2b: The ring-closing reaction was carried out as described above
for 2a. Isomer (R)-2b (8.00 mg, 82%) was isolated as a colorless oil.
Rf =0.25 (iHex/ethyl acetate 4:1); 1H NMR (600 MHz, [D4]MeOH): d=
Acknowledgements
We thankthe Deutsche Forschungsgemeinschaft, the Volkswagen foun-
dation, the EU Marie Curie training and mobility program (CLUS-
TOXDNA), and the Fonds of the chemical industry for financial support.
0.02 (s, 9H; Si
(s, 3H; SiCH3), 0.17 (s, 6H; Si
SiCH2CH2O), 0.95 (s, 18H; 2”C(CH3)3), 1.34 (s, 3H; C(5A)CH3), 2.09–
2.14 (m, 2H; CH2a(2’A), CH2a(2’B)), 2.18–2.23 (m, 1H; CH2b(2’A)), 2.28–
2.41 (m, 5H; CH2b(2’B), 2”CH=CHCH2CH2), 2.43–2.50 (m, 4H; 2”
N
G
AHCTREUNG
AHCTREUNG
A
G
ACHTREUNG
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169.
CH=CHCH2CH2), 2.72 (d, J=13.8 Hz, 1H; C(5B)CH2a), 2.78 (d, J=
13.8 Hz, 1H; C(5B)CH2b), 3.19 (d, J=12.6 Hz, 1H; CH2a(6A)), 3.56–3.60
(m,
SiCH2CH2O(B)), 3.93–3.98 (m, 2H; CH2
(4’A)), 4.04–4.07 (m, 1H; CH(4’B)), 4.12 (dd, J=12.0, J=6.0 Hz, 1H;
CH2a(5’B)), 4.28 (dd, J=12.0, J=5.4 Hz, 1H; CH2b(5’B)), 4.40–4.42 (m,
1H; CH(3’B)), 5.03 (d, J=9.6 Hz, 1H; OCH2aN(A)), 5.15 (d, J=9.0 Hz,
1H; OCH2bN(A)), 5.19–5.21 (m, 1H; CH(3’A)), 5.36 (s, 2H;
OCH2N(B)), 5.50–5.60 (m, 2H; CH=CH), 6.25 (dd, J=7.8, J=6.0 Hz,
2H;
SiCH2CH2O(A)),
3.64–3.73
(m,
3H;
CH2b(6A),
AHCTREUNG
A
ACHTREUNG
A
ACHTREUNG
ACHTREUNG
AHCTREUNG
1H; CH
U
A
(SiCH3), ꢀ4.6 (SiCH3), ꢀ4.4 (SiCH3), ꢀ1.2 (Si
A
G
18.9 (C
(CH3)3), 22.3 (C(5A)CH3), 26.3 (C
CHCH2CH2), 28.7 (CH=CHCH2CH2), 34.3 (CH=CHCH2CH2), 34.4
(CH2(2’A)), 34.7 (C(5B)CH2), 34.8 (CH=CHCH2CH2), 41.2 (CH2(2’B)),
43.3 (C(5A)), 46.8 (CH2(6A)), 64.7 (CH2(5’B)), 65.2 (CH2(5’A)), 67.9
(SiCH2CH2O(A)), 68.6 (SiCH2CH2O(B)), 71.2 (OCH2N(A)), 71.4
(OCH2N(B)), 74.0 (CH(3’B)), 77.5 (CH(3’A)), 86.2 (CH(1’A)), 86.4 (2”
CH(4’)), 87.3 (CH(1’B)), 111.1 (C(5B)), 130.7 (CH=CH), 131.3 (CH=
ACHTREUNG
A
N
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
A
N
ACHTREUNG
ACHTREUNG
CH), 138.7 (CH(6B)), 152.2 (CO(2B)), 154.4 (CO(2A)), 164.6 (CO(4B)),
174.2 (ester-CO), 174.4 (ester-CO), 174.6 ppm (CO(4A)); IR (film):
n˜max =2953s, 2929s, 2857m, 1732s, 1682s, 1461m, 1361w, 1248s, 1080s,
936w, 860w, 835s, 778m cmꢀ1; MS (FAB+): m/z (%): 1131 (5) [M+Na+],
154 (19), 136 (28), 89 (30), 81 (30), 73 (100); HRMS (ESI+): m/z calcd
for C52H92N4NaO14Si4: 1131.5585; found: 1131.5568 [M+Na]+.
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2246.
Enzymatic-repair assay: SplG was overexpressed and purified as His6-
tagged recombinant protein in Escherichia coli.[42] The reconstitution of
the {Fe-S} cluster and the repair assays were performed under anaerobic
conditions in a glove box. For enzyme activity, the amount of 5’-deoxy-
adenosine and repaired SP was determined and analyzed by using rp-
HPLC. Each reaction mixture contained Tris-HCl (pH 7.0, 100 mm), KCl
(200 mm), sodium dithionite (3 mm), DTT (5 mm), and SAM (0.45 mm).
All solutions were degassed under reflux and the chemicals were trans-
ferred into the glove box as solids. One sample only contained the com-
ponents described above. To the second sample 50 mmol holo-splG was
added. The third sample contained 50 mmol splG and the 5S-configured
spore lesion. The fourth one contained the same as the third, but instead
of the 5S-configured spore lesion the 5R-configured spore lesion was
added. All the reaction mixtures were incubated for 12 h at 48C for com-
parison. After incubation, the reaction mixture was frozen in liquid nitro-
gen and stored at ꢀ808C. All the samples were thawed and centrifuged
to remove precipitated protein before being injected into the HPLC
column.
rp-HPLC analysis: For the rp-HPLC analysis, the samples were injected
directly after they had been centrifuged in order to decrease the SAM
cleavage to S-adenosylhomocysteine. Up to 50 mL of the enzyme reaction
were injected onto the rp-HPLC column (Macherey–Nagel Nucleosil
300-5 C18) equilibrated with 0.1% TFA in H2O. The products could be
separated by applying a linear gradient (0%!30% solvent B (50%
CH3CN with 0.1% TFA) in 20 min) at a flow rate of 0.7 mLminꢀ1. The
detection wavelength was 260 nm. The HPLC gradient was chosen to
allow separation and hence detection of thymidine in the assay solution,
which is the only expected product of the repair reaction. The peaks
were assigned by coinjection of thymidine and 5’-deoxyadenosine, and
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Received: February 7, 2006
Published online: June 21, 2006
6094
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 6081 – 6094