Investigation of the pathways of excess electron transfer in DNA with flavin-donor and oxetane-acceptor modified DNA hairpins

Article abstract of DOI:10.1002/chem.200600074

Oxetane is a potential intermediate that is enzymatically formed during the repair of (6-4) DNA lesions by special repair enzymes (6-4 DNA photolyases). These enzymes use a reduced and deprotonated flavin to cleave the oxetane by single electron donation.

Full text of DOI:10.1002/chem.200600074

FULL PAPER
DOI: 10.1002/chem.200600074
Investigation of the Pathways of Excess Electron Transfer in DNA with
Flavin-Donorand Oxetane-AcceptorModified DNA Hairpins
Sascha Breeger,[a, b] Martin von Meltzer,[a, b] Ulrich Hennecke,[a] and Thomas Carell*[a]
Abstract: Oxetane is a potential inter-
mediate that is enzymatically formed
during the repair of (6–4) DNA lesions
by special repair enzymes (6–4 DNA
photolyases). These enzymes use a re-
duced and deprotonated flavin to
cleave the oxetane by single electron
donation. Herein we report synthesis
of DNA hairpin model compounds
containing a flavin as the hairpin head
and two different oxetanes in the stem
structure of the hairpin. The data show
that the electron moves through the
duplex even over distances of 17 Š. At-
tempts to trap the moving electron
with N2O showed no reduction of the
cleavage efficiency showing that the
electron moves through the duplex and
not through solution. The electron
transfer is sequence dependent. The ef-
ficiency is reduced by a factor of 2 in
GC rich DNA hairpins.
Keywords: DNA · DNA repair ·
electron transfer
photolyases
·
flavin
·
Introduction
One of the main remaining questions associated with the
excess electron-transfer process is the exact electron-transfer
pathway. In principle,the electron can either hop through
the duplex or,alternatively,it can escape from the duplex
and move along the double helix or through the solvent in
form of a solvated electron as proposed by Harriman.[19]
Electron-transfer processes through the DNA duplex can
occur over large distances.[1–7] Several groups have shown
that a positive charge,generated in DNA after single-elec-
tron abstraction from the nucleoside deoxyguanosine,hops
through the duplex by using either deoxyguanosines or de-
oxyadenosines as stepping stones.[8,9] Excess electrons in
contrast hop through the duplex using the pyrimidine nu-
cleosides deoxycytidine or deoxythymidine as stepping
stones.[10,11] The sequence dependence of the long range hole
transfer process was found to be strong. Best hopping effi-
ciencies were determined for deoxyguanosine-rich sequen-
ces.[8,12,13] In contrast,the sequence dependence of the alter-
native excess electron-transfer process is less well under-
stood. Recent measurements with electron donor and ac-
ceptor modified DNA duplexes showed,however,that the
excess electron-hopping process is most efficient through de-
oxythymidine sequences and less efficient if deoxycytidines
space the electron donors and acceptors in the duplex.[14–18]
Results and Discussion
In order to shine more light on the excess electron-transfer
pathway in a DNA duplex we prepared the two series of
flavin 1 bridged DNA hairpins (2a–d and 3a–d) depicted in
Figure 1. Hairpins have in contrast to normal DNA duplexes
the advantage that they possess stable double helix struc-
tures and concentration independent melting points. The
constructed DNA hairpins contain first of all flavin 1 as the
cap. This unit injects,in its reduced and deprotonated state,
[20]
after light excitation,electrons into the duplex.
The hair-
pins contain in the stem in addition one of two different ox-
etane units 2 and 3 as electron acceptors. Oxetanes have in
general the advantage that they cleave very rapidly and irre-
versibly after single-electron reduction with rate constants
between 10ꢀ7 sꢀ1 and 10ꢀ8 sꢀ1.[21–31] To use oxetanes as elec-
tron acceptor has in addition the appeal in that these com-
pounds are believed to be intermediates in the repair of (6–
4) DNA lesions by (6–4) photolyases. Here to,repair is as-
sumed to involve single-electron donation followed by cleav-
age of the oxetane intermediate. In fact cleavage of oxe-
tanes with various electron donors to simulate and investi-
[a] Dipl.-Chem. S. Breeger,Dipl.-Chem. M. von Meltzer,
Dipl.-Chem. U. Hennecke,Prof. Dr. T. Carell
Department of Chemistry and Biochemistry
Ludwig-Maximilians-University Munich,Butenandtstrasse 5–13
Haus F,81377 Munich (Germany)
Fax : (+49)89-2180-77756
E-mail: Thomas.Carell@cup.uni-muenchen.de
[b] Dipl.-Chem. S. Breeger,Dipl.-Chem. M. von Meltzer
These authors contributed equally to the work.
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would expect faster cleavage of
the oxetane in series 3. More
importantly,in this scenario
one should be able to disrupt
the electron-transfer process
upon addition of N2O,which is
a strong scavenger for solvated
electrons.
In order to investigate again
the sequence dependence of the
electron movement along the
DNA we also changed the nu-
cleotide sequences in both hair-
pin series between the flavin
cap and the oxetane acceptor
from 3AT and 4AT (a and c) to
3GC and 4GC (b and d),re-
spectively.
Figure 1. Depiction of the two series 2a–d and 3a–d of flavin (1) capped,oxetane containing oligonucleotide
hairpins. Temperatures shown below the structures are the UV melting points. Tm measurement conditions:
Hairpin concentration=3 mm in buffer (150 mm NaCl,10 m m Tris-HCl,pH 7.4).
gate the process behind the repair reaction is of great ac-
tuality.[26,28,32] The hairpin series 2a–d contains an oxetane,
which is positioned close to the duplex. The assumed struc-
ture of the hairpins containing acceptor 2 is depicted as the
result of a modeling study in Figure 2a. It is evident that the
oxetane acceptor 2,close to the duplex distorts the helix at
the oxetane position.
Synthesis: The flavin 1 H-phosphonate was prepared as pre-
viously described.[33] For the synthesis of the oxetane accept-
or 2,we first methylated 2-thiothymine 4 (Scheme 1) with
methyl iodide and converted the resulting (2S)-methyl-3-
methylthymine 5 into 3-methylthymine 6 using HCl at
808C.[34] Irradiation of 3-methylthymine 6 in the presence of
benzophenone in acetonitrile in
a quartz apparatus furnished in
44% yield the oxetane 7 as a
pair of enantiomers. Subse-
quent glycosidation of 7 with a-
1-chloro-3,5-di-O-toluoyl-1-de-
oxyribofuranoside gave the cor-
responding nucleoside
8
in
53% as a 1:1 mixture of the
a,b-anomers. The diastereomer-
ically pure b-anomer 8 [(5S)-
configuration] was separated
and the toluoyl protecting
groups were cleaved to give
compound 9,which was finally
converted into the phosphor-
amidite 2. The crystal structure
of compound 8 (Figure 3) con-
firmed the structure of the oxe-
tane. Most interesting was the
observation that only one dia-
stereomer of the b-nucleoside 8
Figure 2. Result of modeling studies of the hairpins a) 2a and b) 3a (SYBYL 6.8 MMFF94,hairpin 2a 8 ps
MD in water,hairpin 3a energy minimized structure).
The oxetane in series 3 is placed far outside the duplex
structure as shown in Figure 2b. A semi-rigid propargyla-
mide spacer places this electron acceptor at a distance of
about 10 Š away from the nucleotide stack. In this hairpin
series the duplex structure is rigid and closed. The oxetane
acceptor points out of the major groove into the solution.
If the excess electron moves through the duplex using the
bases as stepping stones,cleavage of the oxetane acceptor in
series 2 should be fast. If however,the electron travels ex-
ternally along the DNA duplex as a solvated electron,one
was obtained. We believe that the (5S)-configured oxetane
(S)-7 reacted selectively with the a-1-chloro-3,5-di-O-toluo-
yl-1-deoxyribofuranoside to give the corresponding b-
anomer,while the (5 R)-oxetane forms selectively the a-
anomer. To our knowledge this is the first time that the ster-
eochemical information present in the nucleoside deter-
mines the configuration of the anomeric center in the glyco-
sidation reaction.
For the preparation of oxetane acceptor 3 we first alkylat-
ed thymine 11 with benzyl bromoacetate to give thymine
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Excess Electron Transfer in DNA
FULL PAPER
building-block 3 ready for incorporation into DNA hairpins
of series 3a–d using solid-phase phosphoramidite chemistry.
Synthesis of the hairpins 2a–d and 3a–d,via phosphor-
amidite chemistry,using the building blocks 2 and 3 was per-
formed on an Expedite DNA synthesizer with standard pro-
cedures. Highly base labile protected phosphoramidite
building blocks were used. The DNA hairpins were after
completion of synthesis cleaved from the solid support
under concomitant cleavage of all protecting groups with
25% aq. NH3 for the series 3 and 25% aq. NH3/EtOH (3:1)
for series 2. The DNA hairpins were subsequently purified
by reversed-phase HPLC and anion-exchange HPLC. Purity
and correct incorporation of the flavin and the oxetane ac-
ceptors was confirmed by analytical reversed phase HPLC
and MALDI-TOF mass spectrometry (see Experimental
Section).
Figure 3. X-ray crystal structure (two representations) of compound 8
confirms formation of just one diastereomer of the b-anomer (for de-
tailed information see Experimental Section).
Irradiation results and discussion: Melting-point studies of
all hairpins prepared (2a–d and 3a–d) confirmed that they
all possess melting points well above room temperature
(Figure 1). Temperature dependent CD spectra (exemplified
for hairpin 3a),as depicted in Figure 4 show that the hair-
pins adopt a B-like conformation which melts cooperatively
upon heating.[35]
All eight hairpins were finally irradiated to induce the
electron injection by the flavin. To this end they were indi-
vidually dissolved in irradiation buffer (150 mm NaCl,10 m m
Tris-HCl,pH 7.4) containing 9.8 m m sodium dithionite as
the reducing agent. The solutions were filled into small fluo-
rescence cuvettes stoppered with a rubber septum. Argon
was bubbled through the solution in order to establish anae-
robic conditions. The cuvettes were irradiated with
a
Scheme 1. Synthesis of the oxetane electron acceptor 2. a) MeI,DMF,
RT,18 h,65%; b) 3 n HCl,80 8C,8 h,93%; c) hn,benzophenone,MeCN,
3 h,44%; d) a-1’-chloro-3’,5’-di-O-toluoyl-deoxyribofuranoside,NaH,
1000 W xenon lamp equipped with a cooled cut-off l=
340 nm filter. The irradiation experiments were each per-
formed for up to 40 min. During the irradiation time small
aliquots were removed from the cuvettes and analyzed by
reversed-phase HPLC. Figure 5 shows the HPLC results ob-
tained upon irradiation of hairpin 2b as a typical example.
Clearly evident is the clean reaction of the oxetane contain-
ing hairpin to a new hairpin product. Analysis of the prod-
uct confirmed that the new hairpin is a DNA strand without
benzophenone cleaved off,which would be the expected
product of the irradiation experiment. Control experiments
established that with either no dithionite in solution or in
the absence of light no oxetane cleavage is observed. These
results prove that in these hairpins an electron transfer
occurs upon irradiation from the reduced and deprotonated
flavin to the oxetane moiety. This unit cleaves spontaneously
after single-electron reduction very similar to what is pro-
posed to happen during repair of (6–4) lesions by (6–4) pho-
tolyases and what was shown to happen in model com-
pounds.[25,26]
MeCN,RT,4 h,53%
DMTCl,pyridine,RT,4 h,84%; g)
a/b 1:1; e) K2CO3,MeOH,RT,3 h,84%; f)
b-cyanoethyltetraisopropylphosphor-
diamidite,diisopropylethylamine,THF,RT,2 h,67%.
benzyl ester 12 (Scheme 2). We then irradiated compound
12 in the presence of benzophenone in a quartz apparatus to
obtain the thymine-oxetane 13 as a racemic mixture.[25] Hy-
drogenolytic cleavage of the benzyl ester 13 over Pd/C in an
H2 atmosphere to carboxylic acid 14,activation of the acid
with TBTU/HOBt and reaction with propargylamine fur-
nished the alkyne 15. Sonogashira coupling of this oxetane–
alkyne conjugate to the unprotected 5-iododeoxyuridine
proved to be more difficult than anticipated and gave only
low yields (28%). To improve the yield,we turned to a pro-
tected nucleoside such as the 3,5-bis-tert-butyldimethylsilyl-
(TBDMS) protected 5-iododeoxyuridine. Sonogashira cou-
pling with this compound furnished the oxetane–deoxyuri-
dine conjugate 16 in a much better and much more reliable
yield (66%) as a mixture of two diastereoisomers. Cleavage
of the TBDMS groups and conversion of the unprotected
nucleoside 17 into the DMT-protected phosphoramidite
using standard procedures furnished the oxetane-acceptor
In order to determine the cleavage efficiency,we integrat-
ed the peak area of the new product at each time point and
plotted the relative amount of product formed against the ir-
radiation time. Figure 6 shows the curves obtained for hair-
pins 2a and 2b as examples.
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T. Carell et al.
d with four base pairs between
the flavin head and the oxetane
acceptor are consistently slower
compared with the hairpins
with only three base pairs. We
also observe—in agreement
with previous data—that the
cleavage efficiency in all hair-
pins with GC base pairs be-
tween the flavin donor and the
oxetane acceptor is significantly
reduced.[14] These data support
our[14] and Rokitaꢁs[15] previous
observation that GC base pairs
reduce the efficiency of the
electron-transfer process. It is
very interesting that we are
able to confirm these observa-
tions with the hairpins 3a–d in
which the acceptor unit is
placed far outside the duplex
structure.
Scheme 2. Synthesis of the oxetane acceptor 3. a) benzyl bromoacetate,K 2CO3,DMF,RT,0 8C ! RT,20 h,
68%; b) hn,benzophenone,MeCN,RT,5 h,57%; c) H 2,Pd/C,EtOAc,RT,3 h,98%; d) propargylamine,
HOBt,TBTU,NEt 3,DMF,RT,86%; e) 5-iodo-3 ’,5’-di-O-TBDMS-deoxyuridine,CuI,[PdCl 2(PPh3)2],DIEA,
DMF,RT,12 h,66%; f) HF·pyridine,pyridine,THF,RT,12 h,then MeOTMS; g) DMTCl,4-DMAP,pyridine,
The results obtained with the
oxetane acceptors 2 are not as
consistent. This is not surprising
if we consider that the oxetane
AHCTREUNG
mol sieves 4 Š,RT,54 h,41% over two steps; h)
b-cyanoethyltetraisopropylphosphordiamidite,diisopropyl-
ammonium tetrazolate,dichloromethane,RT,12 h,58%.
Figure 5. HPLC traces obtained during the irradiation experiment with
hairpin 2b. Assay solution: Hairpin concentration=20 mm in buffer
(150 mm NaCl,10 m m Tris-HCl,9.8 m m Na2S2O4,pH 7.4). Irradiation
with 1000 W xenon lamp,cut-off filter l=340 nm, T=208C. HPLC con-
ditions: Nucleosil column (120 Š,3 mm); eluting buffers (buffer A: 0.1m
NHEt3OAc in H2O; buffer B: 0.1m NHEt3OAc in MeCN); Gradient: 0–
10% B in 10 min and then up to 45% B in 45 min at a flow of
0.5 mLminꢀ1. I=relative absorbance.
Figure 4. Temperature dependent CD spectra of the hairpin 3a. Condi-
tions: Hairpin concentration=3 mm in buffer (150 mm NaCl,10 m m Tris-
HCl,pH 7.4).
The bars in Figure 7 summarize the data on the cleavage
efficiency as % reaction product formed per minute of irra-
diation. A first observation is that the oxetanes cleave clean-
ly and efficiently in all hairpins to give the expected prod-
ucts. The fast hairpins cleave with efficiencies of more than
10% product formation per minute.
Analysis of the hairpins with oxetane acceptor 3 shows
first of all the expected small distance dependence of the
electron transfer induced oxetane cleavage in accord with
data from other systems.[36] The hairpins of the series c and
close to the duplex induces a rather strong perturbation of
the DNA double-helix structure. We can,however,conclude
that the oxetane cleavage in these hairpins is always slower
compared with series 3. This is particularly obvious when we
compare hairpins 3c and 3d with 2c and 2d. In 2d and 3d
four strongly helix stabilizing GC base pairs separate the
flavin donor and the acceptor. In these series 2c,d and 3c,d
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Excess Electron Transfer in DNA
FULL PAPER
Figure 6. Time dependent formation of the product hairpins 2a and 2b
after irradiation. Assay solution: hairpin concentration=20 mm in buffer
(150 mm NaCl,10 m m Tris-HCl,9.8 m m Na2S2O4,pH 7.4). Irradiation
Figure 8. Bar graph representation of the irradiation data. Depiction of
the efficiencies of oxetane cleavage of hairpins 2a,b and 3a,b,d in percent
per minute. Assay solution,hairpin concentration: 20 mm in buffer
(150 mm NaCl,10 m m Tris-HCl,pH 7.4). &: Buffer contains 9.8 mm
Na2S2O4,Ar; &: buffer plus 80 mL 0.2m EDTA,3 mL 20 mm d-(+)-man-
nitol,Ar; &: buffer plus 80 mL 0.2m EDTA,3 mL 20 mm d-(+)-mannitol,
N2O; irradiation with 1000 W xenon lamp,cut-off filter 340 nm, T=
208C.
&
*
with 1000 W xenon lamp,cut-off filter l=340 nm, T=208C; : 2a, : 2b.
Data points were fitted exponentially using the program Microcal Origin.
ence of N2O has no effect! Based on this result we can fully
rule out that the excess electrons move along the DNA as a
solvated species. This result was confirmed in a second set
of experiments in which we replaced Na2S2O4 as the flavin
reducing agent by a photoreduction process with EDTA as
the photoreducing agent.[37] We had feared that the dithion-
ite might somehow react with the N2O. The EDTA proce-
dure provided the reduced flavin much cleaner but required
pre-irradiation. For the experiment the hairpins were dis-
solved in an EDTA containing buffer (150 mm NaCl,10 m m
Tris-HCl,30 m m EDTA,pH 7.4) and irradiated for one
minute with white light. After this,the flavin was fully re-
duced. Irradiation was then continued under the same con-
ditions as with Na2S2O4 and aliquots were removed for anal-
ysis. Comparing the data obtained with EDTA reduction in
an argon atmosphere with those obtained in the presence of
N2O revealed,however,again no difference. The involve-
ment of solvated electrons in the cleavage of the oxetane ac-
ceptor outside the duplex structure can be again ruled out.
Figure 7. Bar graph representation of the irradiation data. Depiction of
the efficiencies of oxetane cleavage of hairpins 2a–d and 3a–d in percent
per minute of irradiation. Assay solution,hairpin concentration: 20 mm in
buffer (150 mm NaCl,10 m m Tris-HCl,9.8 m m Na2S2O4,pH 7.4). Irradia-
tion with 1000 W xenon lamp,cut-off filter 340 nm, T=208C. Each graph
is an average of three independent measurements. The error bars were
calculated from the deviation.
cleavage of the oxetane outside the helix is significantly
faster. It is clear that the strong helix destruction caused by
the oxetane acceptor 2 gives rise to a reduced cleavage effi-
ciency. In contrast,placing the acceptor outside the duplex,
has only a small effect.
One exception is hairpin 2a. In this compound,cleavage
of the oxetane is particularly fast. However,this hairpin fea-
tures also the smallest melting temperature. We believe that
the three AT base pairs separating the strongly helix dis-
turbing oxetane 2 from the flavin cap are not able to en-
force a rigid double helix structure causing,for example,
slippage that brings the flavin donor closer to the oxetane
acceptor.
We next analyzed if one could disrupt the electron-trans-
fer process in series 2 and 3 with N2O as a scavenger for sol-
vated electrons. The hairpins 2a,b and 3a,b,d were selected
for this study. All hairpins were irradiated in the presence of
Na2S2O4 once under an argon and N2O atmosphere. In the
latter experiment we bubbled N2O through the solution for
about 20 min prior to the irradiation. Analysis of the oxe-
tane cleavage efficiencies showed (Figure 8) that the pres-
Conclusion
In summary we have prepared the first oligonucleotides
with oxetane electron acceptors stably and side specifically
incorporated. The oxetane could be cleaved in the presence
of a reduced and deprotonated flavin in a light dependent
process inside the DNA duplex. Similar to the repair of (6–
4) lesions by the flavin containing (6–4) photolyases[38–40] the
cleavage involves an excess electron transfer from the flavin
to the oxetane.[25,26,28,32] The presence of a reductive cleavage
mechanism is supported by the fact that the flavin was re-
quired in the reduced and deprotonated state in which it can
only donate and not accept an electron. Secondly,the ob-
served sequence and distance dependence of the cleavage
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T. Carell et al.
(5% methanol/chloroform); m.p. 1918C; 1H NMR (300 MHz,CDCl
,
process is in accord with other systems that depend on elec-
tron donation into the duplex rather than electron abstrac-
tion and hole transfer.
3
258C): d=7.41–7.23 (m,10H),6.35 (d, 3J
(H,H)=4.2 Hz,1H),2.80 (s,3H),1.77 ppm (s,3H);
ACHTREUNG
AHCTREUNG
CDCl3,25 8C): d=170.3,152.5,144.3,138.5,128.6 (2C),128.3 (2C),127.9
(2C),125.8 (2C),124.8 (2C),90.8,75.7,59.7,26.8,23.6 ppm; IR (neat):
= 3326,2978,1730,1687,1473,1448,1382,1300,1238,1128,1105,978,
Most surprising is the observation that the oxetane could
be cleaved efficiently even if it was placed 10 Š away from
the duplex. This fact together with the result that the cleav-
age in all hairpins is not diminished in the presence of N2O,
which is an efficient quencher for solvated electrons,shows
that the excess electrons move through the duplex and not
as a solvated electron through solvent or along the DNA
backbone as previously suggested![19] If the oxetane is
placed in close proximity to the center of the duplex,the
DNA double helix is significantly disturbed. We observe
that this helix destabilization has a larger effect on the effi-
ciency of the reductive oxetane cleavage than placing the
oxetane away from the duplex.
n˜
751,704 cm ꢀ1; HRMS (ESIꢀ): m/z: calcd for C19H18N2O335Cl1: 357.1006;
found 357.1005 [M+Cl]ꢀ.
Thymidine oxetane 8: Compound 7 (500 mg,1.55 mmol) was dissolved in
dry acetonitrile (30 mL). At 08C sodium hydride (46 mg,1.91 mmol) was
added followed by portionwise addition of a-1’-chloro-3’,5’-di-O-toluoyl-
ribofuranoside (756 mg,1.94 mmol). The mixture was allowed to warm to
room temperature and stirred for 4 h. After dilution with chloroform
(100 mL) the mixture was washed once with sat. sodium bicarbonate and
with brine,dried over sodium sulfate and concentrated in vacuo. The
crude product was purified by flash chromatography (silica gel,7.5%
ethyl acetate/toluene) to give the desired b-nucleoside
8 (290 mg,
27.7%). Additionally the a-anomer was obtained (260 mg,24.8%). Rf =
0.20 (10% ethyl acetate/toluene); m.p. 1698C; [a]2D6 = +52.1 (c=0.026
in chloroform); 1H NMR (300 MHz,CDCl 3,25 8C): d=7.81–7.72 (m,
4H),7.50–7.48 (m,2H),7.30–7.05 (m,12H),6.34 (dd,
9.2 Hz,1H),5.30 (d, 3J
(H,H)=6.5 Hz,1H); 4.63 (s,1H),4.25 (m,1H),
4.12 (dd, 3J(H,H)=3.6, 2J(H,H)=12.1 Hz,1H),3.58 (dd, 3J
(H,H)=3.0,
2J
(H,H)=12.1 Hz,1H),2.52 (s,3H),2.40–2.35 (m,1H),2.32 (s,3H),
2.30 (s,3H),2.09–1.99 (m,1H),1.56 ppm (s,3H);
13C NMR (75 MHz,
3J
A
AHCTREUNG
A
N
ACHTREUNG
Experimental Section
AHCTREUNG
General: Melting points are measured on a Büchi B-540 apparatus and
are uncorrected. 1H NMR spectra were recorded on Varian Mercury 200
(200 MHz),Bruker AMX 300 (300 MHz),Varian XL400 (400 MHz) and
Bruker AMX 600 spectrometers. The chemical shifts were referenced to
DMSO (d=2.50 ppm) in [D6]DMSO and CHCl3 (d=7.26 ppm) in
CDCl3. 13C NMR spectra were recorded on Bruker AMX 300 (75 MHz),
Varian XL400 (100 MHz) and Bruker AMX 600 (150 MHz) spectrome-
ters. The chemical shifts were referenced to DMSO (d=39.43 ppm) in
[D6]DMSO and CHCl3 (d=77.00 ppm) in CDCl3. 31P NMR spectra were
recorded on Varian Mercury 200 (81 MHz). Standard pulse sequences
were employed for 1H 2D NOESY, 1H,1H and 1H,13C correlation studies.
IR spectra were recorded with a Perkin-Elmer FT-IR spectrum 100
equipped with an ATR unit. Mass spectra and high-resolution mass spec-
tra were measured on Finnigan MAT TSQ 700,Finnigan MAT 95,Finni-
gan MAT 90,Finnigan LTQ FT-ICR and Bruker Autoflex II (MALDI-
TOF) mass spectrometers. Optical rotations were measured on a Perkin–
Elmer 241 polarimeter.
CDCl3,25 8C): d=170.0,166.0,165.7,151.9,144.3,143.9,142.2,137.2,
129.6 (4C),129.1 (4C),128.9,128.6 (2C),128.0 (2C),127.9,126.7,126.3,
126.2 (2C),126.1 (2C),103.2,90.7,85.2,80.7,76.9,74.2,63.5,61.7,35.3,
27.3,22.0,21.5 ppm; IR (neat): n˜ = 3438,3064,3035,2990,2924,1722,
1686,1611,1448,1410,1373,1271,1201,1179,1101,1019,970,866,843,
752,705 cm ꢀ1; HRMS (ESI+): m/z: calcd for C40H38N2O823Na1: 674.2526;
found 674.2513 [M+Na]+.
Deprotected thymidine oxetane 9: Potassium carbonate (141 mg,
1.02 mmol) was added to a solution of 8 (276 mg,0.41 mmol) in dry
methanol (20 mL). The resulting solution was stirred for 3 h at room tem-
perature. After neutralization with Amberlite IR-120 resin (2.000 g) the
solvent was removed under reduced pressure. The crude product was pu-
rified by flash chromatography (silica gel,2% methanol/chloroform) to
give 9 (151 mg,84%) as a compact white foam. Rf =0.31 (10% metha-
nol/chloroform); m.p. 668C; [a]D25
1H NMR (300 MHz,CDCl 3,25 8C): d=7.62–7.52 (m,4H),7.40–7.29 (m,
6H),7.30–7.05 (m,12H),6.29 (dd, 3J
(H,H)=6.0 Hz,8.3 Hz,1H),4.63 (s,
1H),4.30 (m,1H),3.79 (m,1H),3.34 (dd, (H,H)=
3J(H,H)=3.1, 2J
12.2 Hz,1H),3.20 (dd, (H,H)=12.2 Hz,1H),2.61 (s,
3J(H,H)=3.0, 2J
3H),2.40–2.35 (m,1H),2.06 (ddd, (H,H)=13.5 Hz,
3J(H,H)=2.7,6.0, 2J
(H,H)=6.7,8.2, 2J
= +2.96 (c=0.009 in chloroform);
AHCTREUNG
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. All reactions requiring dry
conditions were carried out under nitrogen atmosphere in dried glass-
ware.
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
1H),1.98 (ddd, 3J
C
G
3-Methylthymine (6):[34] Sodium hydride (1.293 g,56.26 mmol) was added
to a stirred solution of 4-thiothymine (4; 4.000 g,28.14 mmol) in dry
DMF (400 mL). Methyl iodide (5.26 mL,84.40 mmol) was added drop-
wise and the resulting mixture was stirred at ambient temperature for
18 h. After removal of the solvent under reduced pressure,the residue
was triturated with chloroform and filtered. The filtrate was concentrated
under reduced pressure and the remaining solid was purified by flash
chromatography (silica gel,1% methanol/chloroform) to give the inter-
mediate (2S)-methyl-3-methylthymine (5; 3.125 g,65%) as a white solid.
This was dissolved in 2n HCl (200 mL) and heated to 808C for 8 h. The
solvent was removed under reduced pressure and the residue extracted
with CHCl3 (300 mL). The solution was concentrated in vacuo to give 6
(2.360 g,93%) as a white solid. All spectroscopic data were in agreement
with those given in ref. [34].
128.7,128.5,128.3 (2C),128.1 (2C),126.5 (2C),126.1 (2C),91.3,86.2,
85.5,76.8,71.4,62.4,62.3,39.4,27.1,22.6 ppm; IR (neat):
n˜ = 3022,
2927,1718,1672,1463,1448,1417,1374,1293,1202,1149,1095,1052,
1020,918,876,750,706,653,596 cm
ꢀ1; HRMS (ESIꢀ): m/z: calcd for
C24H26N2O635Cl1: 473.1479; found 473.1452 [M+Cl]ꢀ.
DMT-protected oxetane 10: Compound 9 (110 mg,0.25 mmol) was co-
evaporated twice with dry pyridine (1 mL) and dissolved in pyridine
(2 mL). After addition of molecular sieves 4 Š the solution was stirred
for 4 h. 4,4’-Dimethoxytritylchloride (102 mg,0.30 mmol) was added and
the mixture was stirred for 4 h at room temperature. The solvent was re-
moved in vacuo. The residue was purified by flash chromatography
(silica gel,1% methanol/chloroform + trace pyridine) to give compound
10 (156 mg,84%) as a yellowish film. Rf =0.18 (5% methanol/chloro-
form); m.p. 848C (decomp); 1H NMR (600 MHz,CDCl 3,25 8C): d=7.59
Thymine oxetane 7: In a Pyrex vessel (l
> 340 nm) benzophenone
(m,1H),7.41–7.32 (m,5H),7.24–7.10 (m,10H),6.94–6.92 (m,3H),6.73
(6.137 g,33.68 mmol) was added to a solution of 3-methylthymine ( 6;
2.360 g,16.84 mmol) in dry acetonitrile (800 mL). The resulting mixture
was degassed by sparging with argon for 20 min. The solution was irradi-
ated at 108C under an argon atmosphere for 2 h using a medium pressure
mercury lamp (750 W). The solvent was removed under reduced pres-
sure. The residue was purified by flash chromatography (silica gel,1%
methanol/chloroform) to give 7 as a white solid (2.270 g,44%). Rf =0.36
3
(dd, J
A
G
2
1H),4.01 (m,1H),3.77 (m,1H),3.71 (s,6H),3.10 (dd,
3J
(H,H)=4.7, J-
A
A
N
ACHTREUNG
(s,3H),2.21 (brs,1H),2.12–2.08 (m,1H),1.72–1.68 (m,1H),1.59 ppm
(s,3H); 13C NMR (150 MHz,CDCl ,25 8C): d=169.7,158.6,151.0,149.7,
3
144.4,143.6,138.5,136.0,135.6,135.4,130.0 (2C),129.9 (2C),128.1 (2C),
6474
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
Chem. Eur. J. 2006, 12,6469 – 6477

Excess Electron Transfer in DNA
FULL PAPER
128.0 (2C),127.9 (2C),127.8 (4C),127.6,127.0,125.9,125.4,123.8,113.2
(4C),91.2,86.7,85.5,84.0,73.2,63.7,61.4,55.2 (2C),38.9,27.1,
phases were extracted three times with ethyl acetate (50 mL). The com-
bined organic phases were dried over magnesium sulfate,filtered and the
solvent was removed under reduced pressure. The crude product was pu-
rified by flash chromatography (silica gel,50% ethyl acetate/hexanes) to
give 15 (1.420 g,86%) as a white solid. Rf =0.18 (50% ethyl acetate/hex-
anes); m.p. 2318C; 1H NMR (400 MHz,[D 6]DMSO,25 8C): d=10.48 (s,
22.7 ppm; IR (neat): n˜ = 3058,2927,1719,1677,1607,1580,1508,1444,
1413,1371,1293,1247,1200,1175,1147,1068,1051,1030,1002,951,916,
876,827,800,791,746,726,706 cm
ꢀ1;HRMS (ESI+): m/z: calcd for
C45H44N2O623Na1: 763.2995; found 763.2983 [M+Na]+.
1H),8.55 (t, 3J
4.26 (d, 3J
(H,H)=16.6 Hz,1H),3.96–3.85 (m,2H),3.61 (d,
16.6 Hz,1H),3.15–3.12 (m,1H),1.59 ppm (s,3H);
13C NMR (100 MHz,
ACHTREUNG
Oxetane phosphoramidite 2: Compound 10 (130 mg,0.17 mmol) was dis-
solved in degassed tetrahydrofuran (1 mL). After addition of diisopropy-
lethylamine (130 mL,0.74 mmol) the solution was cooled to ꢀ788C and
b-cyanoethyltetraisopropylphosphordiamidite (77 mL,0.29 mmol) was
added dropwise. The solution was allowed to warm to room temperature
and stirred for 4 h. The solvent was removed under reduced pressure.
The residue was purified by flash chromatography (deactivated silica gel,
1% methanol/chloroform + trace pyridine) to give 2 (110 mg,67%) as a
A
ACHTREUNG
CDCl3,25 8C): d=169.6,167.0,151.1,144.3,139.4,128.3 (2C),128.0
(2C),127.5,127.4,125.4 (2C),124.9 (2C),90.6,80.7,76.0,73.0,65.2,48.1,
27.9,23.0 ppm; IR (neat): n˜ = 3366,3224,3062,2872,1724,1693,1669,
1533,1492,1448,1411,1388,1369,1290,1237,1216,980,785,750,
700 cmꢀ1; MS (ESI+): m/z: 426.1 [M+Na]+,404.1 [ M+H]+; HRMS
(ESI+): m/z: calcd for C23H21N3O423Na1: 426.1430; found 426.1423
[M+Na]+.
1
yellowish film. Rf =0.29 (2% methanol/chloroform); H NMR (600 MHz,
CDCl3,25 8C): d=7.54–7.53 (m,1H),7.43–7.20 (m,15H),6.99–6.92 (m,
3J
(H,H)=5.6 Hz,6.4 Hz,1H),4.43 (s,
A
TBDMS-protected oxetane deoxyuridine conjugate 16: Compound 15
(600 mg,1.50 mmol) and DIEA (0.72 mL,4.14 mmol) were dissolved in
dry DMF (4 mL). In a second flask cuprous iodide (57 mg,0.30 mmol),
2’-deoxy-3’,5’-di-O-TBDMS-5-iodouridine (728 mg,1.25 mmol) and [Pd-
3H),6.81–6.77 (m,4H),6.10 (dd,
1H),4.23 (m,1H),4.12 (m,1H),3.72 (s,6H),3.71–3.51 (m,5H),2.96
3
2
ACHTREUNG
(dd, J
(H,H)=5.0, J
(H,H)=9.9 Hz,1H),2.69 (m,1H),2.62 (s,3H),2.58
3
2
ACHTREUNG
(t, J
(H,H)=6.2 Hz,2H),2.29 (dd, 3J
A
(H,H)=13.2 Hz,1H),
A
1.64 (s,3H),1.26–0.96 ppm (m,12H);
,
3
Both solutions were degassed by several freeze/thaw cycles then com-
bined. The resulting mixture was stirred for 12 h at room temperature.
The solution was diluted with ethyl acetate (50 mL) then washed with sa-
turated aqueous ammonium chloride (30 mL) and brine (20 mL). The
combined aqueous phases were extracted once with ethyl acetate
(30 mL). The combined organic phases were dried over magnesium sul-
fate,filtered and the solvent was removed under reduced pressure. The
crude product was purified by flash chromatography (silica gel,40% hex-
anes/ethyl acetate) to give 16 (706 mg,66%) as a yellowish solid. Rf =
0.13 (50% ethyl acetate/hexanes); m.p. 1478C (decomp); 1H NMR
(600 MHz,CDCl 3,25 8C): d=10.00 (brs,1H),8.40 (brs,1H),7.89 (s,
258C): d=169.8,158.6,151.2,144.5,143.7,138.7,135.8,135.5,130.1 (2C),
130.0 (2C),128.1 (2C),128.0 (2C),127.9 (2C),127.8 (2C),127.6,127.5,
126.9,125.9 (2C),125.4 (2C),117.5,113.1 (2C),113.0 (2C),91.2,86.4,
85.9,84.6,74.5,74.3,63.2,61.0,58.1,55.2 (2C),43.3,43.2,37.5,29.7,27.1,
24.6,24.5 (2C),22.6,20.3 ppm;
150.1,149.3 ppm; IR (neat): n˜ = 3042,2950,2922,1710,1672,1605,1574,
31P NMR (81 MHz,CDCl 3,25 8C): d=
1504,1444,1405,1362,1293,1247,1193,1131,1060,1047,1028,998,947,
912,870,821,794,791,742,720,702 cm
ꢀ1;HRMS (ESI+): m/z: calcd for
C45H61N4O923Na1P1: 963.4073; found 963.4059 [M+Na]+.
[33]
2-Thymine acetic acid benzyl ester(12)
:
Thymine 11 (5.000 g,
39.64 mmol) and potassium carbonate (5.480 g,39.65 mmol) were dis-
solved in dry DMF (120 mL). Benzyl bromoacetate (5.48 mL,
36.00 mmol) was added dropwise and the resulting mixture was stirred
for 20 h at room temperature. The suspension was filtered and the sol-
vent was removed in vacuo. The residue was treated with water
(38.5 mL) and conc. HCl (1.6 mL) and stirred for 30 min. The solid was
filtered off and dried under high vacuum,then crystallized from metha-
nol to give 12 (7.300 g,68%) as a white powder. 1H NMR (200 MHz,
[D6]DMSO,25 8C): d=11.40 (s,1H),7.52 (s,1H),7.36 (m,5H),5.18 (s,
2H),4.54 (s,2H),1.75 ppm (s,3H).
1H),7.58–7.51 (m,1H),7.29–7.09 (m,10H),6.12 (t,
3J
(H,H)=6.6 Hz,
A
1H),4.80 (s,1H),4.42 (d, (H,H)=16.3 Hz,1H),4.29–4.25 (m,1H),
3J
AHCTREUNG
4.08–3.92 (m,2H),3.86–3.82 (m,1H),3.79–3.74 (m,1H),3.66–3.61 (m,
1H),3.37–3.31 (m,1H),2.22–2.17 (m,1H),1.92–1.85 (m,1H),1.61 (s,
3H),0.81 (s,9H),0.77 (s,9H),0.01 (s,3H),0.00 (s,3H),
ꢀ0.04 (s,3H),
ꢀ0.04 ppm (s,3H); 13C NMR (150 MHz,CDCl 3,25 8C): d=169.7,167.2,
162.8,151.8,149.1,143.8,143.0,138.7,128.6 (2C),128.4 (2C),128.1,
127.9,126.0 (2C),125.3 (2C),99.0,91.1,89.3,88.4,85.9,76.4,74.7,72.1,
65.9,62.8,48.7,42.0,29.9,25.9 (3C),25.7 (3C),23.3,18.4,18.0,
ꢀ4.6,
ꢀ4.9, ꢀ5.5, ꢀ5.5 ppm; IR (neat): n˜ = 3207,3065,2952,2929,2856,1682,
1532,1463,1448,1279,1253,1205,1104,1029,968,833,777,750,
701 cmꢀ1; MS (FAB+): m/z: 880.7 [M+Na]+,858.8 [ M+H]+,536.5,514.6;
HRMS (ESI+): m/z: calcd for C44H60N5O9Si2: 858.3930; found 858.3923
[M+H]+.
Thymine acetic acid benzyl esterbenzophenone oxetane (13) :[33] Com-
pound 12 (7.000 g,25.50 mmol) was dissolved in dry acetonitrile
(650 mL) under sonication and benzophenone (9.320 g,51.90 mmol) was
added. The solution was transferred to a pyrex vessel and degassed by
sparging with argon for 30 min. Then the solution was irradiated under
argon atmosphere for 6 h with a medium pressure Hg lamp (750 W). The
solvent was removed under reduced pressure and the crude product was
purified by flash chromatography (silica gel,25% ethyl acetate/hexanes)
DMT-protected oxetane deoxyuridine conjugate 18: In a polypropylene
tube 16 (234 mg,0.27 mmol) was dissolved in EtOAc (12 mL). HF·pyri-
dine complex (Fluka,70% HF,0.1 mL,3.85 mmol) and pyridine (0.1 mL,
1.02 mmol) were added and the resulting solution was shaken for 24 h at
room temperature. The resulting suspension was treated with methoxytri-
methylsilane (0.6 mL,4.35 mmol,to destroy the excess HF) and shaken
for another two hours. The solid was collected by centrifugation and
washed twice with cold EtOAc to give crude 17. Crude 17 was dissolved
in pyridine (8 mL) and,after molecular sieves 4 Š and a catalytic amount
of 4-DMAP were added,stirred for 12 h at room temperature. 44, ’-Dime-
thoxytritylchloride (102 mg,0.30 mmol) was added and the mixture was
stirred for another 36 h. After removal of the solvent in vacuo the crude
product was purified by flash chromatography (silica gel,0.5% pyridine/
6% methanol/dichloromethane) to give 18 (103 mg,41%) as a colourless
oil. Rf =0.15 (silica gel,0.5% pyridine/5% methanol/dichloromethane);
1H NMR (600 MHz,[D 6]DMSO,25 8C): d=11.67 (s,1H),10.47 (s,1H),
8.57 (brs,1H),7.91 (s,1H),7.41–7.33 (m,8H),7.32–7.20 (m,11H),6.92–
6.87 (m,4H),6.12–6.07 (m,1H),5.33 (s,1H),4.77 (s,1H),4.34–4.24 (m,
2H),4.03–3.97 (m,1H),3.96–3.90 (m,2H),3.73 (s,6H),3.63–3.57 (m,
1H),3.28–3.23 (m,1H); 3.11–3.06 (m,1H),2.30–2.24 (m,1H),2.21–2.15
to give 13 (6.600 g,57%) as
[D6]DMSO,25 8C): d=10.48 (s,1H),7.20 (m,15H),5.21–4.97 (m,2H),
4.97 (s,1H),4.28 (d, 3J(H,H)=17.6 Hz,1H),4.08 (d, 3J
(H,H)=17.4 Hz,
1H),1.50 ppm (s,3H); MS (70 eV): m/z: 456.2 [M +].
a
white solid.
1H NMR (200 MHz,
A
ACHTREUNG
Thymine acetic acid benzophenone oxetane (14):[33] 13 (1.000 g,
2.20 mmol) and 10% Pd/C (100 mg,0.09 mmol) were suspended in ethyl
acetate (20 mL). The suspension was stirred under H2 atmosphere for 3 h
at atmospheric pressure. After filtration through a pad of Celite the sol-
vent was removed under reduced pressure to give 14 (790 mg,98%) as a
white solid. 1H NMR (200 MHz,[D 6]DMSO,25 8C): d=12.95 (brs,1H),
10.43 (s,1H),7.52 (s,1H),7.23 (m,10H),4.88 (s,1H),4.24 (d,
(H,H)=17.6 Hz,1H),3.85 (d, 3J
(H,H)=17.6 Hz,1H),1.57 ppm (s,3H).
Oxetane alkyne 15: HOBt (682 mg,5.04 mmol) and TBTU (1.610 g,
4.98 mmol) were added successively to solution of 14 (1.500 g,
3J-
A
ACHTREUNG
a
4.10 mmol) in dry DMF (30 mL) and the resulting mixture was stirred for
10 min at room temperature. Propargylamine (0.53 mL,8.21 mmol) and
triethylamine (3.4 mL,217.75 mmol) were added and the solution was
stirred for another 30 min. After dilution with ethyl acetate (150 mL) it
was washed three times with brine (50 mL). The combined aqueous
(m,1H),1.60 ppm (s,3H);
13C NMR (150 MHz,[D 6]DMSO,25 8C): d=
169.7,166.9,161.4,158.0 (2C),151.1,149.2,144.7,144.3,143.3,139.4,
135.5,135.1,129.6 (2C),129.5 (2C),128.3 (2C),128.0 (2C),127.8 (2C),
Chem. Eur. J. 2006, 12,6469 – 6477
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
6475

T. Carell et al.
127.6,127.5,127.4 (2C),126.5,125.4 (2C),124.9 (2C),113.1 (4C),98.1,
90.6,89.0,85.7 (2C),85.0,76.1,74.3,70.3,65.2,63.7,54.9 (2C),48.1,40.0,
28.7,23.0 ppm; IR (neat): n˜ = 3407,3057,2818,2254,1698,1541,1508,
1465,1448,1282,1249,1178,1049,1023,1002,822,759,702 cm
(ESI+): m/z: 954.3 [M+Na]+,303.1; HRMS (ESI +): m/z: calcd for
C53H49N5O1123Na1: 954.3326; found 954.3329 [M+Na]+.
mass spectrometer using 3-hydroxypicolinic acid as matrix substance and
measuring in negative polarity mode. Table 1 shows the calculated and
experimentally determined molecular masses for the investigated oligo-
nucleotides.
ꢀ1; MS
Table 1. Molecular masses of the synthesized DNA hairpins (matrix: 3-
hydroxypicolinic acid,negative polarity mode).
Oxetane building block 3: Compound 18 (103 mg,0.11 mmol) and diiso-
propylammoniumtetrazolate (10 mg,0.055 mmol) were dissolved in de-
gassed dichloromethane (2 mL). To the solution was added b-cyanoethyl-
tetraisopropylphosphordiamidite (44 mg,0.15 mmol). The solution was
stirred for 12 h at room temperature. The solvent was removed in vacuo.
The crude product was purified by flash chromatography (deactivated
Oligonucleotide
m/z calcd [MꢀH]ꢀ
m/z found [MꢀH]ꢀ
2a
2b
2c
2d
3a
3b
3c
3d
5535.1
5538.1
6152.3
6156.2
5726.2
5729.2
6343.3
6347.3
5537.4
5542.7
6155.4
6159.9
5729.8
5732.8
6348.4
6351.9
silica gel,0.5% pyridine/5% methanol/dichloromethane) to give
3
(72 mg,58%) as a colourless oil. Rf =0.26 (silica gel,0.5% pyridine/5%
methanol/dichloromethane); 1H NMR (600 MHz,[D 6]benzene,25 8C):
d=10.90 (brs,1H),8.84–8.79 (m,1H),8.08 (s,1H),7.50–7.18 (m,20H),
6.94–6.88 (m,4H),6.32–6.22 (m,1H),4.94 (s,1H),4.57–4.50 (m,1H),
4.36–4.25 (m,1H),4.21–4.02 (m,3H),3.94–3.20 (m,8H),3.72 (s,6H),
2.99–2.93 (m,1H),2.87–2.78 (m,1H),2.71–2.63 (m,1H),1.67 (s,3H),
1.22–1.01 ppm (m,12H); 13C NMR (150 MHz,[D 6]benzene,25 8C): d=
170.1,167.7,163.3,159.3 (2C),152.7,149.9,145.1,143.2 (2C),140.0,
136.2,136.0,130.6 (2C),129.8 (2C),128.9 (2C),128.7 (2C),128.5,128.5
(2C),128.1,127.9 (2C),127.4,126.7 (2C),125.9 (2C),119.9,113.9 (4C),
99.7,91.5,90.0,87.4,86.3,86.2,77.1,75.1,73.5,66.8,63.6,58.6,55.1 (2C),
Melting point experiments: UV melting points were measured on a Cary
100 UV/Vis spectrometer using 1 mL quartz cuvettes with 1 cm path
length. The samples contained 150 mm NaCl,10 m m Tris-HCl (pH 7.4)
and 3 mm of the oligonucleotide. For every strand five temperature cycles
from 858C to 08C were recorded,the average melting point was calculat-
ed computationally using Microcal Origin.
49.3,43.5,43.4,40.4,30.1,25.8,24.6 (2C),24.5 (2C),23.6 ppm;
31P NMR
CD experiments: CD spectra were recorded on a Jasco J-810 spectropo-
larimeter equipped with a Peltier temperature controller using 2 mL
quartz cuvettes with 1 cm pathlength. The samples contained 150 mm
NaCl,10 m m Tris-HCl (pH 7.4) and 3 mm of the oligonucleotide.
(81 MHz,C 6D6,25 8C): d=149.6,149.5 ppm; IR (neat): n˜ = 3198,3062,
2980,2837,1693,1609,1510,1464,1448,1281,1249,1207,1179,1079,
1030,1003,977,828,754,703 cm
ꢀ1; MS (ESIꢀ): m/z: 1193.4 [M+NO3]ꢀ,
1166.4 [M+Cl]ꢀ,1130.5
[
MꢀH]ꢀ; HRMS (ESIꢀ): m/z: calcd for
Irradiation experiments: In a typical irradiation experiment a 20 mm solu-
tion of the oligonucleotide containing 150 mm NaCl and 10 mm Tris-HCl
(pH 7.4,total volume 150 mL) was transferred to a 1 mL quartz fluores-
cence cuvette sealed with a rubber septum. The solution was degassed by
sparging with argon for 20 min. For reduction of the flavin two different
procedures were used. For one part of the experiments 15 mL of an alka-
line solution of sodium dithionite (87 mg dithionite in 5 g water + 75 mL
5n NaOH,pH ~8) was added. Alternatively an EDTA-mediated photo-
reduction was used. For this purpose 80 mL EDTA 0.2m was added and
the solution pre-irradiated under anaerobic conditions for 1 minute with
white light to photoreduce the flavin (monitored by fluorescence spectro-
scopy). After reduction we further irradiated the samples at 208C with a
Thermo Oriel 1000 W Xe-lamp,equipped with a cooled 340 nm cut-off
filter. For solvated electron quenching experiments the solution was satu-
C62H65N7O12P1: 1130.4434; found 1130.4505 [MꢀH]ꢀ.
X-ray crystallographic study of compound 8: A single crystal suitable for
X-ray analysis was obtained by slow crystallisation from chloroform at
ambient temperature.
X-ray data: Empirical formula: C40H38N2O8; M=674.72 gmolꢀ1; crystal
size: (0.33”0.43”0.53) mm3 as colourless plate; monoclinic; space group
P21(6); unit cell dimensions: a=10.855(2), b=14.361(3), c=11.353(2) Š;
b=100.52(3)8; V=1740.0(6) Š3; Z=2, 1=1.288 gcmꢀ1
;
m
ACHTREUNG
=
AHCTREUNG
Data collection and analysis: NONIUS-CAD-4-Diffractometer, l=
0.71073 Š, T=22(2)8C, w-2V scan, V range: 2.78–25.578; 7030 reflections
collected,3315 independent reflections,2752 observed reflections ( I>
2sI).
rated with N2O prior to irradiation,a slow stream of N O was maintained
2
throughout the experiment and 3 mL 20 mm d-mannitol was added to
capture putative OH radicals. For the analysis 10 mL aliquots were re-
moved from the assay solution after defined time intervals,aerated for
30 min and analysed. Analysis of the data was performed by HPLC (3 m
C18-reversed phase columns by Macherey-Nagel and 0.1m NHEt3OAc in
water: acetonitrile as eluent) and MALDI-TOF-MS.
Structure solution and refinement: Solution with SHELXS-90-program
system;[41] refinement of 451 variables with SHELXL-97[42] converged at
R1
C
G
U
0.177 and ꢀ0.146 eŠ
.
CCDC-289066 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
DNA synthesis and purification: Solid-phase DNA synthesis was carried
out on a Perseptive Biosystems Expedite 8900 Synthesizer using Ultra-
mild bases and reagents (Glen Research) and following standard phos-
phoramidite protocols. Coupling times for building blocks 2 and 3 were
increased to 10 and 15 min,respectively. Oligonucleotides comprising
building block 2 were cleaved from the solid support and deprotected
using conc. ammonia/ethanol 3:1 for 24 h at room temperature. For hair-
pins containing 3 cleavage and deprotection was carried out with conc.
ammonia for 3 h at 308C due to the base lability of the amide moiety.
Analytical and preparative HPLC was performed with a Merck LaChrom
system using 3 m or 5 m C18-reversed phase columns by Macherey-Nagel
and 0.1m NHEt3OAc in water: acetonitrile as eluent. After concentration
in vacuo the strands were desalted on Waters SepPak-C18 cartridges and
concentrated again. The final DNA concentration was estimated by UV
absorption measured on a Cary 100 UV/Vis spectrometer following
standard procedures. The strands were further characterized by MALDI-
TOF-MS. MALDI mass spectra were recorded on a Bruker Autoflex II
Acknowledgements
We thank the Volkswagen Foundation and the Deutsche Forschungsge-
meinschaft (Research group: Hybrid Compounds) for generous support.
S.B. and U.H. thank the Stiftung Stipendien-Fonds des Verbandes der
Chemischen Industrie e.V. for a Fonds fellowship. We are grateful to Dr.
K. Polborn for the crystal structure analysis of compound 8,to J. Gierlich
for the molecular modeling and to C. Nolte for help with the preparation
of compound 3.
[1] B. Giese, Annu. Rev. Biochem. 2002, 71,51–70.
[2] S. Delaney,J. K. Barton, J. Org. Chem. 2003, 68,6475–6483.
[3] G. B. Schuster,U. Landman, Top. Curr. Chem. 2004, 236,139–161.
[4] M. A. OꢁNeill,J. K. Barton, Top. Curr. Chem. 2004, 236,67–115.
[5] F. D. Lewis,M. R. Wasielewski, Top. Curr. Chem. 2004, 236,45–65.
6476
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
Chem. Eur. J. 2006, 12,6469 – 6477

Excess Electron Transfer in DNA
FULL PAPER
[6] B. Giese, Top. Curr. Chem. 2004, 236,27–44.
[26] M. G. Friedel,M. K. Cichon,T. Carell, Org. Biomol. Chem. 2005, 3,
1937–1941.
[27] Q. H. Song,H. Xiaoming,X. Zhixiu,Z. Xiang,G. Qingxiang,
Bioorg. Chem. 2003, 32,357–366.
[28] T. Stafforst,U. Diederichsen, Chem. Commun. 2005,3430–3432.
[29] S. Encinas,N. Belmadoui,N. J. Climent,S. Gil,M. A. Miranda,
Chem. Res. Toxicol. 2004, 17,857–862.
[7] A. Joy,G. B. Schuster, Chem. Commun. 2005,2778–2784.
[8] B. Giese,M. Spichty, ChemPhysChem 2000, 1,195–198.
[9] B. Giese,J. Amaudrut,A.-K. Kçhler,M. Spormann,S. Wessely,
Nature 2001, 412,318–320.
[10] H.-A. Wagenknecht, Angew. Chem. 2003, 115,2558–2565; Angew.
Chem. Int. Ed. 2003, 42,2454–2460.
[11] C. Behrens,T. Carell, Top. Curr. Chem 2004, 236,187–204.
[12] B. Giese,S. Wessely,M. Spormann,U. Lindemann,E. Meggers,
M. E. Michel-Beyerle, Angew. Chem. 1999, 111,1050–1052; Angew.
Chem. Int. Ed. 1999, 38,996–998.
[30] M. A. Miranda,M. A. Izquierdo, J. Am. Chem. Soc. 2002, 124,
6532–6533.
[31] M. A. Miranda,M. A. Izquierdo, Chem. Commun. 2003,364–365.
[32] Q. H. Song,H.-B. Wang,W.-J. Tang,Q.-X. Guo,S.-Q. Yu,
Org.
[13] B. Giese, Acc. Chem. Res. 2000, 33,631–636.
Biomol. Chem. 2006, 4,291–298.
[14] A. Manetto,S. Breeger,C. Chatgilialoglu,T. Carell,
Angew. Chem.
[33] C. Behrens,M. Ober,T. Carell, Eur. J. Org. Chem. 2002,3281–3289.
[34] T. Itahara,Y. Fujii,M. Tada, J. Org. Chem. 1988, 53,3421–3424.
2005, 117,325–328; Angew. Chem. Int. Ed. 2006, 45,318–321.
[15] T. Ito,S. E. Rokita, J. Am. Chem. Soc. 2003, 125,11480–11481.
[16] T. Ito,S. E. Rokita, Angew. Chem. 2004, 116,1875–1878; Angew.
Chem. Int. Ed. 2004, 43,1875–1878.
[35] C. R. Cantor,M. M. Warshaw,H. Shapiro,
1059–1077.
Biopolymers 1970, 9,
[36] C. Behrens,L. T. Burgdorf,A. Schwçgler,T. Carell,
Angew. Chem.
[17] P. Kaden,E. Mayer-Enthart,A. Trifonov,T. Fiebig,H.-A. Wagen-
knecht, Angew. Chem. 2005, 117,1662–1666; Angew. Chem. Int. Ed.
2005, 44,1636–1639.
2002, 114,1841–1844; Angew. Chem. Int. Ed. 2002, 41,1763–1766.
[37] R. Traber,H. E. A. Kramer,P. Hemmerich, Biochemistry 1982, 21,
1687–1693.
[18] C. Wagner,H.-A. Wagenknecht, Chem. Eur. J. 2005, 11,1871–1876.
[19] A. M. Brun,A. Harriman, J. Am. Chem. Soc. 1994, 116,10383–
10393.
[20] A. Schwçgler,L. T. Burgdorf,T. Carell, Angew. Chem. 2000, 112,
4082–4085; Angew. Chem. Int. Ed. 2000, 39,3918–3920.
[21] G. Prakash,D. E. Falvey, J. Am. Chem. Soc. 1995, 117,11375–
11376.
[38] T. Todo,H. Takemori,H. Ryo,M. Ihara,T. Matsunaga,O. Nikaido,
K. Sato,T. Nomura, Nature 1993, 361,371–374.
[39] S.-T. Kim,K. Malhotra,C. A. Smith,J.-S. Taylor,A. Sancar,
Chem. 1994, 269,8535–8540.
J. Biol.
[40] A. Sancar, Chem. Rev. 2003, 103,2203–2238.
[41] G. M. Sheldrick,ShelXS-90,Program for the Solution of Crystal
Structures,University of Gçttingen,Gçttingen (Germany), 1990.
[42] G. M. Sheldrick,ShelXL-97,Program for the Refinement of Crystal
Structures,University of Gçttingen,Gçttingen (Germany), 1997.
[22] A. Joseph,G. Prakash,D. E. Falvey, J. Am. Chem. Soc. 2000, 122,
11219–11225.
[23] A. Joseph,D. E. Falvey, J. Am. Chem. Soc. 2001, 123,3145–3146.
[24] A. Joseph,D. E. Falvey, Photochem. Photobiol. Sci. 2002, 1,632–
635.
[25] M. K. Cichon,S. Arnold,T. Carell, Angew. Chem. 2002, 114,793–
796; Angew. Chem. Int. Ed. 2002, 41,767–770.
Received: January 17,2006
Revised: May 29,2006
Published online: July 10,2006
Chem. Eur. J. 2006, 12,6469 – 6477
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
6477